Biochem. J. (2011) 439, 349–374 (Printed in Great Britain)
349
doi:10.1042/BJ20110949
REVIEW ARTICLE
Molecular mechanisms of endolysosomal Ca2 + signalling in health and
disease
Anthony J. MORGAN*1 , Frances M. PLATT*, Emyr LLOYD-EVANS† and Antony GALIONE*1
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, U.K., and †School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, U.K.
Endosomes, lysosomes and lysosome-related organelles are
emerging as important Ca2 + storage cellular compartments with
a central role in intracellular Ca2 + signalling. Endocytosis at
the plasma membrane forms endosomal vesicles which mature
to late endosomes and culminate in lysosomal biogenesis.
During this process, acquisition of different ion channels and
transporters progressively changes the endolysosomal luminal
ionic environment (e.g. pH and Ca2 + ) to regulate enzyme
activities, membrane fusion/fission and organellar ion fluxes, and
defects in these can result in disease. In the present review we
focus on the physiology of the inter-related transport mechanisms
of Ca2 + and H + across endolysosomal membranes. In particular,
we discuss the role of the Ca2 + -mobilizing messenger NAADP
(nicotinic acid adenine dinucleotide phosphate) as a major
regulator of Ca2 + release from endolysosomes, and the recent
discovery of an endolysosomal channel family, the TPCs (twopore channels), as its principal intracellular targets. Recent
molecular studies of endolysosomal Ca2 + physiology and its
regulation by NAADP-gated TPCs are providing exciting new
insights into the mechanisms of Ca2 + -signal initiation that control
a wide range of cellular processes and play a role in disease. These
developments underscore a new central role for the endolysosomal
system in cellular Ca2 + regulation and signalling.
INTRODUCTION
Discovery of the lysosome
Although proton transport has been the most extensively studied
ion movement across the membranes of the endolysosomal
system, recent functional and biochemical studies have indicated
a plethora of ion-transport mechanisms in endosomes and
lysosomes which may play important roles in the functioning
of these organelles.
The recognition that the organelles of the endolysosomal system contain appreciable amounts of Ca2 + [1,2]
(Supplementary Table S1 at http://www.BiochemJ.org/bj/439/
bj4390349add.htm), together with roles for this ion in regulating
their fusion and trafficking, has prompted intense study of
Ca2 + -transport mechanisms in these organelles [3–9]. Interest
in endolysosomes as Ca2 + -storage organelles has intensified with
the discovery that NAADP (nicotinic acid adenine dinucleotide
phosphate), a Ca2 + -mobilizing messenger first discovered to
evoke Ca2 + signals in sea urchin eggs, does so in many cases
by activating Ca2 + -release mechanisms on acidic stores with
characteristics of lysosomes [10].
In the present review, we highlight the role of the endolysosomal
system as an acidic Ca2 + -storage organelle, with a particular focus
on recent evidence that it is the target of the Ca2 + -mobilizing
messenger NAADP which regulates Ca2 + release and ion fluxes
across endolysosomal membranes, placing these organelles at
centre stage for regulating intracellular Ca2 + signalling and
homoeostasis in health and disease.
The lysosome is a highly specialized acidic intracellular organelle
that mediates a complex set of functions in eukaryotic cells [11].
The concept of the lysosome can be traced to the pioneering
studies by Metchnikoff (reviewed in [12]). He was the ‘father’
of innate immunity whose contribution was recognized by the
1908 Nobel Prize in Physiology or Medicine (along with Paul
Ehrlich) for his seminal studies of phagocytosis [12]. He found
that particles fed to simple unicellular organisms and mammalian
phagocytes were digested in an acidic compartment rich in
digestive enzymes. In 1907, using simple pH-sensitive dyes,
he reported that when peritoneal macrophages from the guinea
pig were fed with bacteria, acidic reactions took place within
phagosomes [13]. It was many years later that the general
relevance of this finding to non-phagocytic cells was appreciated
and the ubiquitous catabolic organelle (the lysosome) proposed
and identified.
In the 1950s Christian de Duve was studying the intracellular
localization of acid phosphatase and identified a sedimentable
particle surrounded by a continuous membrane that was enriched
in hydrolases. Significantly, this particle was distinct from peroxisomes (which he also discovered) and mitochondria [14,15].
Electron microscopy studies revealed an organelle with highly
heterogeneous electron-dense contents enclosed within a single
limiting membrane that was termed at the time ‘pericanalicular
dense bodies’ [16]. De Duve proposed the term lysosome to
Key words: ATPase, calcium store, endolysosome, nicotinic acid
adenine dinucleotide phosphate (NAADP), pH, two-pore channel
(TPC).
Abbreviations used: ACA, autoinhibited Ca2 + -ATPase; AtTPC1, Arabidopsis thaliana TPC1; cADPR, cyclic adenosine diphosphate ribose; CaM,
calmodulin; CAX, Ca2 + /H + exchanger; CICR, Ca2 + -induced Ca2 + release; ClC, Cl − channel; ER, endoplasmic reticulum; FCCP, carbonyl cyanide
p -trifluoromethoxyphenylhydrazone; HEK, human embryonic kidney; IP3 , inositol 1,4,5-trisphosphate; IP3 R, IP3 receptor; LAMP, lysosomal-associated
membrane protein; MLIV, mucolipidosis type IV; NAADP, nicotinic acid adenine dinucleotide phosphate; NAADP-AM, NAADP-acetoxymethyl ester; NCKX,
Na + /Ca2 + -K + exchanger; NCX, Na + /Ca2 + exchanger; NHE, Na + /H + exchanger; NPC, Niemann–Pick type C; pHL , luminal pH; PKC, protein kinase C;
PIP2 , PI(3,5)P2 ; PMCA, plasma membrane Ca2 + -ATPase; Po , mean open probability; RNAi, RNA interference; RyR, ryanodine receptor; SERCA, sarcoendoplasmic reticulum Ca2 + -ATPase; shRNA, short hairpin RNA; siRNA, small interfering RNA; SPCA, secretory pathway Ca2 + -ATPase; SR, sarcoplasmic
reticulum; tBHQ, tert-butylhydroquinone; TMD, transmembrane domain; TPC, two-pore channel; HsTPC2, human TPC2; Trp, transient receptor potential;
TRPML1, mucolipin 1; SpTPC, sea urchin TPC; V-ATPase, vacuolar-type H + -ATPase.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
350
A. J. Morgan and others
be applied to this specialized organelle that was the cellular
repository of mature acid hydrolases. Reviews by de Duve detail
the experimental path that led to the discovery of the lysosome,
for which he was awarded the Nobel Prize for Physiology or
Medicine in 1974 along with Albert Claude and George Palade
for their collective discoveries of the structural and functional
organization of the cell [15,17]. We now know that the lysosome
is the terminal organelle in the endocytic pathway and plays a
complex role in multiple aspects of cellular homoeostasis [11,18].
The next major discovery (1963) was that genetic deficiencies
in lysosomal enzymes were responsible for a group of inherited
metabolic disorders termed ‘storage diseases’. The seminal work
by Hers [19] revealed that the disease described in 1932 by Pompe
(termed Pompe disease or generalized glycogenosis) was due to a
defect in lysosomal α-glucosidase involved in lysosomal glycogen
catabolism. During the process of autophagy, glycogen in the
cytoplasm enters the lysosomal system when autophagosomes
fuse with lysosomes and this enzyme is responsible for lysosomal
glycogen catabolism. The concept of lysosomal storage diseases
was born and over 50 disorders have now been identified, the
majority of which are due to lysosomal enzyme deficiencies,
but they can also be caused by defects in lysosomal membrane
proteins [20]. Indeed, the study of these diseases has contributed
numerous insights into the fundamental functions of lysosomes
and this knowledge is now being applied to the development of
therapies for these devastating disorders.
Lysosomes and lysosome-related organelles
All eukaryotic cells contain lysosomes (termed vacuoles in
plants and fungi), with the exception of some highly specialized
cells such as mammalian erythrocytes, which lack multiple
organelles including lysosomes. Lysosomes degrade exogenous
and endogenous macromolecules derived from biosynthetic and
endocytic pathways, and catabolize cytosolic components that are
obtained from the autophagic pathway [11,18].
Following the discovery and definition of the lysosomal
compartment subsequent studies identified several specialized
organelles to be lysosome-related. This included melanosomes
(in melanocytes), lytic granules (in natural killer and cytotoxic
T lymphocytes of the immune system), the MHCII compartment
(antigen-presenting cells), alpha granules of platelets and dense
core granules from various haemopoietic cell lineages (e.g.
basophils and neutrophils) [21,22]. In many of these cell
types conventional lysosomes are present to mediate basic
catabolic functions, whereas the lysosome-related organelles
mediate specialist functions appropriate for the cell type.
Lysosome-related organelles are believed to share common
origins in terms of their evolution and biogenesis, although we
still have an incomplete knowledge of how they are formed
and how specialized proteins are sorted to them [21]. The
definition of these organelles is that they have a specialized
function unrelated to macromolecule catabolism, share common
hallmarks with lysosomes and differ from the endocine/exocrine
granules, which lack lysosomal features [21]. The other
salient feature/definition of lysosome-related organelles is that
they respond to physiological stimuli rather than functioning
constitutively in the way that classical lysosomes do. Secretory
lysosomes are another interesting subgroup of lysosome-related
organelles, which are essentially classical lysosomes with
additional proteins present such as Rab27a. Indeed, they combine
many features of lysosomes with secretory granules [23]. An
interesting fact that has emerged is that a subgroup of lysosomes
in non-secretory cells can fuse with the plasma membrane and
mediate repair following plasma membrane damage [24].
c The Authors Journal compilation c 2011 Biochemical Society
Lysosomal integrity and enzyme targeting
The identification of the lysosome as a digestive compartment
involved in macromolecular catabolism defined the first functional
role of this compartment. One paradox was how the limiting
membrane of the lysosome remains intact when in contact with
multiple catabolic hydrolases. It became clear that the lysosomal
membrane is protected by a complex and substantial glycocalyx
on its luminal leaflet, due to the presence of heavily glycosylated
integral membrane proteins. These are termed LAMPs (lysosomal-associated membrane proteins) and include LAMP1, LAMP-2 and LAMP-3 (CD63). Evidence has emerged
that processing of sialic acid residues on LAMP-1 regulates
lysosomal exocytosis [25]. The other membrane proteins of the
lysosome are involved in transport of catabolites generated in
the lysosome to feed into metabolic recycling and signalling
pathways, or play roles in ion transport and pH maintenance.
Another question the identification of the lysosome raised
was how were the lysosomal hydrolases that function in this
compartment targeted to this organelle. The breakthrough
came when the mannose 6-phosphate-targeting system was
discovered, which most but not all soluble hydrolases utilize
[26,27]. Alternative targeting systems include LIMP-2 and
sortilin [27,28]. Membrane proteins of the lysosome are targeted
via motifs in their cytoplasmic domains, which are typically
dileucine- or tyrosine-based [27].
The operational definition of lysosomes that has emerged after
decades of research is an organelle containing mature acidic
hydrolases and LAMPs coupled with a lack of mannose 6phosphate receptors. We are now in an era where understanding
how the lysosome and lysosome-related organelles are regulated
and interface with other cellular systems is a major focus
of current research. Recent progress has been made in the
area of Ca2 + regulation in the lysosome, informed in part by
the study of lysosomal storage diseases, and this is the subject of
the present review.
HOW DO ACIDIC STORES REGULATE pHL (luminal pH)?
Given that the luminal pH (pHL ) and membrane potential are
absolutely critical for endolysosomal Ca2+ homoeostasis, we shall
first discuss what factors influence these fundamental properties.
Although the lumen of the major ER (endoplasmic reticulum)
Ca2 + store is close to neutral pH [29–31], the lumen of the
acidic organelle family is in the range 4.5–6.5 (Figure 1) [32,33].
How is this distinguishing acidity maintained? Multiple important
physical factors impinge upon the steady-state pHL of small
acidic vesicles that include, but are not restricted to, membrane
capacitance (via its effect on ψ), the luminal H + -buffering
capacity (approximately 40–60 mM/pH [34,35]), the Donnan
potential and ion-transport pathways [36]. Focussing on the
latter, acidification is the net result of the effect of multiple ion
transporters (Figure 2) discussed below.
H + translocation
Naturally, the uptake of H + into the lumen of the organelle
requires energy either from ATP (or PPi ) or by exploiting the
gradients of other ions. The former represents families of H +
pumps, the latter families of H + exchangers.
H + pumps
V-ATPase (vacuolar-type H + -ATPase). Now understood and
scrutinized at a detailed molecular level, the V-ATPase is the
ubiquitous primary route that drives H + into the lumen against
the electrochemical gradient at the expense of ATP (with a
Endolysosomal Ca2 + signalling in health and disease
351
Figure 1 Interrelationship between archetypal acidic organelles and the
factors that set their pH
Time progresses from left to right, pHL is reflected in the intensity of the organelle shading. The
leak of H + is greatest in the earlier pathway and is the most important factor that sets the pHL
of each compartment. In addition, the organelle membrane potential (ψ) inhibits acidification
in early vesicles, but less so in mature vesicles (whereas the pH gradient pH inhibits
proportionally more in later vesicles). Endo, endosome; Lyso, lysosome; MVB, multivesicular
body; TGN, trans -Golgi network.
stoichiometry of 2–4 H + per ATP) [37– 41]. In translocating H + ,
the pump is electrogenic and will therefore generate a lumenpositive membrane potential [38].
Weighing in at a hefty 910 kDa and comprised of 14 subunits,
this impressive rotary machine is meta-organized into ATPhydrolysing (V1 ) and the transmembrane H + -translocating (V0 )
domains, thereby resembling and functioning in much the same
way as does its cousin the mitochondrial F1 F0 ATP synthase
[37,40]. Although virtually all cell types express isoforms of VATPase, the precise subunit composition is often tissue-specific
and is a means of controlling subcellular targeting [42]. For
example, whereas the majority is found in the endolysosomal
system, a small fraction is targeted to the Golgi and plasma
membrane [37,40].
As befits such a key player, the V-ATPase is regulated in
disparate ways including its subunit complement, subcellular
location, reversible dissociation of its V1 and V0 domains (often
as a function of metabolic status [43]), the efficiency of coupling
to ATP hydrolysis, cytosolic pH [43], and regulatory proteins
[38,42,44,45] including protein kinases PKA (protein kinase A),
PKC (protein kinase C) and SOS (salt overly sensitive) [46–48].
Central to many studies investigating organellar pH (and
Ca2 + ) have been the potent and specific V-ATPase inhibitors,
the macrolides bafilomycin A1 and concanamycin [49], that
bind to the V0 subunit in most species [37]. Bafilomycin A1
is astonishingly selective (up to several million-fold) for the VATPase over other ATPases (e.g. F1 F0 , Na + /K + and Ca2 + ) [50],
but there are isolated reports of off-site actions [51].
H + -PPase and H + P-ATPase. Some plants utilize a smaller Ptype ATPase (P3A or the ‘autoinhibited H + -ATPase’) to regulate
vacuole pH [52] (important for flower petal colour [53]). By
contrast, the H + -PPases (or vacuolar proton pyrophosphatases VPPases) pump protons at the expense of pyrophosphate hydrolysis.
They are absent from mammalian cells and have been studied
mainly in plants and lower organisms, where there are hundreds
of homologous sequences available [54].
H + exchangers
Complementing the H + pumps are a retinue of H + exchangers
(antiporters) that modulate pHL by coupling H + translocation to
Figure 2
Transport proteins that affect pHL
On the left-hand hemisphere are the factors that promote acidification: H + pumping by the
V-ATPase establishes the pH and the inside-positive ψ; outward cation channels and Cl −
uptake (via ClC) exemplify the charge compensation for H + ; the polyanionic matrix (high
molecular mass and membrane impermeable) establishes an inside-negative Donnan potential
that contributes to H + uptake. On the right-hand hemisphere are the factors that inhibit
acidification: the inside-positive ψ inhibits further H + uptake; ill-defined H + -leak pathways
dissipate pH to different extents in different organelles; the electrogenic NKA reinforces the
inside-positive ψ, particularly in earlier compartments; the NHEs are electroneutral and are
thought to dissipate the pH when acting in reverse mode (K + may substitute for Na + ); ClC
proteins are electrogenic Cl − /H + exchangers that both inhibit and promote acidification (H +
extrusion dissipates the pH, but the net negative charge uptake facilitates the V-ATPase by
dissipating the inhibitory ψ).
cation (or anion) movements; as such, they are normally expected
to dissipate the organellar pH and thereby contribute to setting
the pHL point [55,56] (Figure 2).
Cation exchangers. As is typical for the acidic vesicle field,
far more is known about the cation/H + exchangers in plants
and lower organisms, where detailed molecular analyses of a
plethora of H + antiporters for Na + , K + and Ca2 + abound ([57–
60] and references therein; Ca2 + antiporters are discussed below).
By contrast, in the mammalian world our view of cation/H +
exchange is pretty much restricted to those electroneutral NHE
(Na + /H + exchanger) isoforms that are located principally on
intracellular organelles (Table 1) including exocytotic granules,
endosomes and the trans-Golgi network (NHE6–9) in contrast
with the plasma membrane (NHE1–5) [55,56,63]. That said,
vesicle trafficking events (exocytosis/endocytosis) do blur the
delineation of the two groups, but importantly there are no (or
few) NHEs in later acidic organelles.
Functionally, NHE isoforms are thought to inhibit acidification.
A ‘reverse mode’ of action for the internal NHE7–9 would
extrude H + and dissipate the pH gradient, at least experimentally
[55,56]. This is energetically most feasible when NHEs transport
K + (as they can instead of Na + ) considering the permissive
luminal/cytosolic [K + ] ratio (Supplementary Tables S1 and S2
at http://www.BiochemJ.org/bj/439/bj4390349add.htm).
Physiologically, organellar NHEs can be necessary for vacuole
fusion [64], vesicular trafficking in development [65], metal/salt
tolerance [57] and cytosolic pH regulation [57]. Moreover, NHE8
regulates late-endosomal morphology and function [63].
c The Authors Journal compilation c 2011 Biochemical Society
++
x
+
+
+++
+++
+++
++
2,3§
3,6,7
√
H + leak
Exchangers
Inhibition
x
1,2,4,7,8,9
4,5
√
3,6,9
3,4,5
√
V-ATPase
Cl −
K+
NHE
ClC‡
Na + /K + ATPase
?
Pump
Counterions
Stimulation
*Although counterion fluxes are probably present, their contribution to pHL regulation is minimal when compared with H + leak [72].
†NHE1 is present in phagosomes, but is probably not active.
‡ClCs are notionally listed under ‘inhibitory’ exchangers since they expel H + from the organelle; however, they are clearly ‘stimulatory’ when considered as a charge shunt for the V-ATPase (see the main text).
§Controversy surrounds the ClC-3 localization to insulin granules [318,319]. Resting pH values were: Golgi, 6.2–6.7; early endosome, 6.9; recycling endosome, 6.4; late endosome, 5.7–5.9; lysosome, 4.0–4.7; secretory vesicle, 5.2–6.3; phagosome, 5.0
(Supplementary Table S2 at http://www.BiochemJ.org/bj/439/bj4390349add.htm).
[34,37,84,87,307–309]
[34,71,87,310,311]
[35,74,79,82,83,95,98,308,312]
[55,307]
[66,308,313–316]
[76,77,307,317]
[33,70,79,98]
7
Phagosome
√
√
√
*
1†
Secretory vesicle
√
√
√
Lysosome
√
√
√
√
√
√
Late endosome
Recycling endosome
√
√
√
Early endosome
√
√
√
Golgi
√
√
√*
*
7,8
Transporter
Class
Effect on acidification
Organellar distribution of ion-transport mechanisms that affect pHL
Table 1
References
A. J. Morgan and others
√
A tick ( ) signifies the presence of transport, a blank field signifies that no reports have been recorded and x signifies that it is demonstrably absent. The counterion rows refer to the importance of ion transport without necessarily identifying the transporter protein
concerned. In the exchangers row, the numbers refer to the specific isoforms detected, e.g. 7 = NHE7.
352
c The Authors Journal compilation c 2011 Biochemical Society
Anion exchangers. Cl − conductance has long featured as a
charge shunt for the V-ATPase (see below) and this was shown
subsequently to be carried by the ClC (Cl − channel) family [66].
However, it later emerged that whereas the plasma membrane
ClCs are indeed Cl − channels (ClC-1, -2 and -K) those found on
intracellular organelles are actually Cl − antiporters (coupled to
H + ) (ClC-3–7) (Table 1 and Figure 2) [66]. Unlike NHEs, ClCs
are present in all acidic compartments from recycling endosome
to lysosome [66] and exchanger loss culminates in a variety
of pathologies such as renal Dent’s disease (ClC-5) [66,67],
neurodegeneration (ClC-3, -6 and -7) [66] and osteopetrosis [68].
Membrane potential, counter ions and pHL
By definition, H + pumps are electrogenic establishing a lumenpositive membrane potential (ψ) and H + gradient (pH) and,
eventually, the protonmotive force (a function of both) would
oppose further acidification [32,33]. At chemical equilibrium, VATPase would theoretically lower pHL to ∼ 2.6 [33,69,70], but
this is not reached because the ψ offers ‘more resistance’ to the
H + pumps than does pH [69] requiring ∼ 65 % of the overall
free energy [36]. If the build-up of lumen-positive charge is a
brake on acidification, then ψ must be partially dissipated in
order to unfetter the pump. How can this be achieved?
Counterions
Charge neutralization (Figure 2) comes in the form of both
anion co-transport and cation counter-transport. ClCs demand our
attention as they are electrogenic (2Cl − :1H + ) and voltage-gated
carriers [66,71]. That is, their recorded currents reflect the net
negative charge uptake which is thought to be a major counter
ion for organellar acidification as knock-down (or mutation) of
the ClC proteins testifies [66,71]. Indeed, the fact that they are
Cl − /H + exchangers is almost incidental: mutants that are pure
Cl − channels are just as good at maintaining endolysosomal
pHL [67,68]. The notion of other families of Cl − channels
contributing to charge compensation has been quite polemic, with
the CFTR (cystic fibrosis transmembrane conductance regulator)
protein representing one hotly debated candidate [72], whereas
the organellar CLICs (chloride intracellular channels) appear
promising [73].
However, many contend that cation counter-transport is also
fundamental for acidification [74,75], even in the absence of
Cl − channels [35], and the endolysosomal lumen environment
probably contain sufficient K + to support this (Supplementary
Tables S1 and S2). The relative contributions (and routes)
of anion and cation fluxes are not fully understood (and not
mutually exclusive), but, theoretically, cation fluxes dominate
anion fluxes at least in newly formed vesicles [32]. As others
have astutely observed [35], the osmotic overhead that Cl − cotransport presents (net uptake of HCl drives water movement)
might favour cation exchange.
Na + /K + -ATPase and ψ
Finally, the organellar membrane potential can be shaped
substantially by the Na + /K + -ATPase. The Na + /K + -ATPase is an
electrogenic pump (3Na + :2K + ) that is abundant in intracellular
acidic vesicles (Table 1) where they profoundly affect pHL
via ψ. In the organellar membrane, they reinforce a lumenpositive membrane potential and therefore will be inhibitory to
H + pumping. This brake on acidification was first suggested
in early (recycling) endosomes [76], but has now been woven
into the fabric of pHL regulation across the endosomal spectrum
Endolysosomal Ca2 + signalling in health and disease
[77] and the intricacies and permissive conditions (such as
high luminal [K + ]) modelled in detail [32,36]. Presumably,
functional Na + /K + -ATPases are retained within the plasma
membrane/endosomal axis as a result of endosome recycling.
What is the resting ψ of acidic organelles?
For consistency, the plasma membrane and organellar membrane
potentials would be best defined as ψ = ψ Cyt − ψ Exo , where Exo
refers to the exoplasmic compartment (the extracellular space
or organelle lumen) and Cyt refers to the cytosol as proposed
previously [78]. Adopting this convention means that a cytosolnegative potential at the organelle membrane would also be
returned as a negative value. Nonetheless, the (understandable)
focus on the organelle lumen means that workers often prefer to
emphasize the lumen-positive ψ and consequently reverse the
sign [79,80]. Except for the calculations below, we shall adopt
the latter practice and refer to a lumen-positive potential as
+ XmV.
The membrane potential is comprised of two components, the
‘fixed charge’ Donnan potential (attributable to luminal anionic
macromolecules) and the diffusion potential (governed by ionic
gradients) [36]. The Donnan potential is lumen-negative, spanning
the −20 to −70 mV range [81,82], and not only provides
a driving force for H + entry into secretory granules [83–85]
and lysosomes [86], but also drives Cl − clearance from the
early endosomes [87]. This Donnan negative charge is initially
dominant in early endosomes, but is titrated as acidification
proceeds [87]. Similarly, neutralization of the Donnan potential
by the entry of other cations (Na + , K + and Ca2 + ) may also occur
[83].
Although the Donnan equilibrium can be a sizeable potential,
the diffusion potential is capable of dominating and reversing
it (especially in the more acidic organelles). Accordingly, there
are numerous studies that propose a lumen-positive resting
membrane potential from the distribution of radioactive ions
or fluorescent dyes in phagosomes [79], endosomes [34,74],
lysosomes [88–90], secretory granules [83,91,92], yolk platelets
[93] and vacuoles [75, 94]. Note that lumen-negative potentials
are usually attributable to ‘non-physiological’ conditions (for
example in the absence of counterions or ATP) in platelet alpha
granules [84] and liver lysosomes [95].
Reports that actually put hard numbers on ψ are less
abundant and are confusingly scattered from + 10 to + 100 mV
(Supplementary Table S2 at http://www.BiochemJ.org/bj/439/
bj4390349add.htm), but cluster around lower potentials such as
+ 30 mV. Recently lysosomal ψ was determined in intact single
cells as + 19 mV using FRET (fluorescence resonance energy
transfer)-based indicators [90]; realistically, a small ψ is the
more likely scenario when endolysosomes are highly permeable to
counterions that dissipate charge [70]. Only more in-depth surveys
will reveal whether this data spread constitutes a biological
variation (e.g. trends between organelles and/or cellular sources)
or a methodological one.
The good news for Ca2 + signalling is that this range of ψ is
highly favourable for Ca2 + release from acidic vesicles (more so
than the ER which has a smaller ψ) and is far from the Ca2 +
equilibrium potential of − 72 to − 110 mV (lumen-negative)2
where Ca2 + release could not occur.
353
How do different compartments maintain a characteristic pHL ?
It should be self-evident that the characteristic pH set point
(Table 1) is dictated primarily by the unique transporter protein
profile of each organelle, although other factors such as the
luminal H + -buffering capacity and organelle shape/size may also
influence it to a degree [36] (but see [32]) and so the final pHL
is determined chiefly by organelle-specific permutations of three
factors: ψ, pH and the H + pump/leak balance (see Table 1
for their differential distribution).
Protonmotive force
Given that H + pumping is more sensitive to ψ than to pH
[36,69], some suggest that their relative contribution along the
endolysosomal pathway is reciprocal with endosomes being
limited more by the ψ, whereas lysosomes are limited by the
pH [36,90] (Figure 1). In theory, the Na + /K + -ATPase is
the principal player that shapes ψ to strongly inhibit
acidification in the earlier compartments but not the later ones
(Table 1). In contrast, the counterion conductances (promoting
acidification) are ubiquitous throughout the endolysosomal
system and so would not be expected to differentially set the ψ.
Unfortunately, as we have seen, reliable determinations of ψ
across the endolysosomal system are sorely lacking (and seldom
within in the same cell type) so we do not know whether ψ
actually does vary across the endolysosomal system.
Pumps and leaks
For all the worthy discussion of the protonmotive force, the
principal determinant of the pHL set-point is the H + pump/leak
balance. The current model states that the ratio of H + pumps to
leaks is highest in the most acidic organelles; that is, the less acidic
organelles (e.g. endosomes) leak H + more than do the ‘tighter’
acidic ones (e.g. lysosomes) [33,70].
Surprisingly, rigorous quantification of the H + pump density
throughout the acidic system has still not been conducted [70],
but given the variations in V-ATPase assembly this is not a trivial
undertaking. What is more certain is that the H + -leak rate is
demonstrably different in different compartments as revealed by
V-ATPase inhibitors3 and is typified by the leaky Golgi and tighter
secretory granules [70]. The sea urchin egg provides another more
extreme example: the yolk platelets are acidic organelles believed
to be endolysosomal in origin [7], but they are extremely ‘tight’
vesicles with low permeability to H + (or indeed other ions) and
application of bafilomycin A1 fails to change pHL at all unless
combined with ionophores that enhance counterion and H + leaks
[7,93].
What pathway(s) underlies the H + leak? Probably not the VATPase itself [69] and the involvement of NHEs is equivocal
[55,63,97]. In the Golgi, it was concluded to be a hitherto
uncharacterized H + conductance [98].
In summary, H + leakiness is the primary factor that
determines the characteristic pHL set-point of each compartment.
Nevertheless, ψ also makes a substantial contribution to pHL
regulation in earlier compartments and the concerted action of
both brings the pHL to the appropriate level.
2
Calculated using E Ca = (RT/2F)·ln([Ca2 + ]o /[Ca2 + ]i ), where R is the gas constant, T is temperature and F is the Faraday constant. The free luminal ER
[Ca2 + ]o = 30–600 μM and cytosolic [Ca2 + ]i = 0.1 μM. At 20 ◦ C, RT/2F = 12.63 mV.
3
By definition, the steady-state pHL reflects an exact balance between the rate of H + uptake (pumps) and H + loss (leaks) and therefore, the rate of
H + leak is unmasked when the V-ATPase is inhibited by bafilomycin A1 or concanamycin
c The Authors Journal compilation c 2011 Biochemical Society
354
A. J. Morgan and others
Ca2 + UPTAKE INTO ACIDIC Ca2 + STORES
For an organelle to be a long term Ca2 + store, mechanisms for
Ca2 + uptake and its luminal sequestration must be present to
maintain stored Ca2 + at a sufficiently high concentration. As is
well known, Ca2 + filling in the neutral ER is effected primarily
by members of the SERCA (sarco-endoplasmic reticulum Ca2 + ATPase) family, comprised of three pump isoforms (plus splice
variants), each with its own unique tissue distribution [99]. In
stark contrast, the route(s) of Ca2 + uptake into acidic stores is not
as well understood, at least in the animal kingdom.
Golgi
Although the least acidic, the Golgi is the best studied Ca2 +
store with a family of Ca2 + pumps called the SPCAs (secretory
pathway Ca2 + ATPases) mediating Ca2 + uptake. SPCAs are Ptype Ca2 + pumps sensitive to inhibition by vanadate, but relatively
insensitive to the SERCA inhibitor thapsigargin (except at very
high concentrations) [100]. Mutations of the SPCA gene result
in Hailey–Hailey disease of the skin [100]. In the trans-Golgi
network, the SPCA is reported to be the only Ca2 + pump required
[101], although others have suggested that additional pumps are
also present [100].
To the best of our knowledge, the SPCAs are unique to the
Golgi meaning that other acidic compartments accumulate Ca2 +
via different Ca2 + pumps and/or exchangers. Unsurprisingly, we
understand less about acidic Ca2 + -store refilling in mammals than
we do in primitive life forms (yeast, plants and protists) where a
higher affinity Ca2 + pump co-exists with a lower affinity (highcapacity) Ca2 + exchanger (Figure 3).
Yeast, plant and protist vacuoles
In yeast and plants, the clearance of Ca2 + from the cytosol is
dominated by the internal compartments such as the massive
acidic vacuole (occupying 80 % of the cell volume) which
then becomes a major Ca2 + -storing organelle [102]. Thankfully,
a wealth of genetic and functional data illuminate these more
primitive organisms and we will briefly discuss the two transport
pathways.
Vacuolar Ca2 + -ATPases
Vacuolar Ca2 + -ATPases are P-type ATPases (i.e. their reaction
cycle involves a phosphorylated enzyme intermediate [103])
which are, perhaps surprisingly for organellar pumps, related
to mammalian PMCAs (plasma membrane Ca2 + -ATPases) in
terms of sequence motifs and topology, but which differ in their
subcellular localization and finer properties [102]. For instance,
the affinity of the PMCA for Ca2 + is high (0.2–0.5 μM) [103],
but the affinity of the vacuolar Ca2 + -ATPases is somewhat lower
(0.3–4.3 μM) [102,104].
Intriguingly, their regulation is very much species-specific.
Plant Ca2 + -ATPases emulate their mammalian cousins: PMCAs
are activated by CaM (calmodulin) displacing an autoinhibitory
domain [103] and a similar mechanism exists for the plant
vacuolar enzymes [for which reason they are known as
ACAs (autoinhibited Ca2 + -ATPases)] [102]. Consequently, CaM
activation lowers the K m and increases the V max values and thus
the pump responds efficiently and removes cytosolic Ca2 + [102].
By contrast, yeast and slime mould Ca2 + -ATPases lack
the autoinhibitory domain [105,106] and so pump activity is
controlled chronically at the transcriptional level (but still in a
Ca2 + -dependent fashion) [105]. Finally, some pumps (such as the
c The Authors Journal compilation c 2011 Biochemical Society
Figure 3
Ca2 + -filling mechanisms of acidic Ca2 + stores
Ca2 + uptake is fuelled by an ion gradient (exchangers) or ATP hydrolysis (pumps) or both. (A)
Plants express both a CAX family and ACAs. Both are activated by regulatory proteins (R, R’),
where R can be a CAX-interacting protein and R’ is CaM. (B) Yeast also express both pathways,
e.g. the vacuolar CAX (VCX1) and Pmc1. Neither family requires the regulatory proteins like
plants do. (C) Archetypal endolysosomal vesicles. The left-hand hemisphere depicts putative
Ca2 + -exchange modes, the upper mechanism shows pure Ca2 + /H + exchange and the lower
mechanism shows coupled transport, e.g. NHE acting with a NCX. Clearly, the NCKX family could
substitute for NCX. The right-hand hemisphere shows putative Ca2 + pumps (for simplicity,
with low or negligible thapsigargin sensitivity): a PMCA-like Ca2 + -ATPase that translocates
only 1 Ca2 + per cycle, with low vanadate sensitivity; and a SERCA3-like Ca2 + -ATPase that
translocates 2 Ca2 + per cycle, with high vanadate sensitivity and inhibitable by tBHQ. The
Ca2 + :H + stoichiometry is given where known; nH + is used when the number (n ) of H + per
Ca2 + is not clear.
ACAs) may also be regulated by phosphorylation (e.g. PKC) in
another parallel with PMCAs, although it is unclear whether this
is by direct phosphorylation of the pump itself [102].
Unfortunately, the pharmacology of vacuolar Ca2 + -ATPases is
not well developed because they are relatively insensitive to the
conventional SERCA inhibitors thapsigargin and cyclopiazonic
acid. This is not so surprising for a PMCA-related protein that
lacks the M3 helix Phe256 to which thapsigargin binds in SERCA
[103].
Vacuolar CAXs (Ca2 + /H + exchangers)
Hijacking the potential energy of the vacuolar H + gradient, CAXs
transport substantial amounts of Ca2 + into the lumen. Compared
with the pumps their Ca2 + affinity is dramatically lower (15–
25 μM), but this is more than compensated for by their V max value
which can be orders of magnitude greater than that of the Ca2 + ATPases [102]. Such proteins are referred to overall as the CAX
family and there are myriad members (six in Arabidopsis alone)
which exchange with a stoichiometry of 1 Ca2 + to 2 or 3 H +
[102].
Endolysosomal Ca2 + signalling in health and disease
Like the Ca2 + pumps, these low-affinity exchangers are also
tightly regulated, sometimes in a Ying-Yang relationship with the
sister Ca2 + -ATPases; for example, in yeast, whereas calcineurin
up-regulates the Pmc1 Ca2 + pump, it inhibits the CAX (Vcx1),
presumably to prevent Ca2 + overload of the vacuole (although
how it actually inhibits is incompletely understood) [105].
Interestingly, neither the pump nor the exchanger in yeast possess
autoinhibitory domains.
By contrast, an autoinhibitory domain motif is present in many
CAX proteins in plants, as it is in their ACA pumps [102].
However, this CAX version of the domain rather discriminates
against CaM (to which it is insensitive) in favour of other
activating proteins such as novel CAX-interacting proteins,
immunophilins and kinases [102].
Endolysosomal Ca2 + uptake
That the endolysosomes of higher organisms are gaining
acceptance as Ca2 + stores bearing Ca2 + channels makes it all the
more imperative to understand the mechanisms by which they fill,
but unfortunately our understanding is woefully incomplete when
compared with the vacuolar field. It has long been known that
acidic organelles are packed with Ca2 + (Supplementary Tables
S1 and S2), but how it actually gets there is more hazy.
What we do know is that Ca2 + filling of acidic stores is dependent upon the protonmotive force because manoeuvres that dissipate it usually empty the store(s) and/or prevent refilling: the abrogation of Ca2 + storage has been shown on numerous occasions by
direct alkalinization of the lumen with membrane-permeant bases
such as NH4 Cl or with protonophores (nigericin or monensin)
[10,94,107–111]. Alternatively, the H + gradient can be indirectly
collapsed by inhibiting the V-ATPase with bafilomycin A1 or concanamycin (thereby depending on a sufficient H + leak [10,93])
and likewise this can prevent Ca2 + storage [1,2,10,112–118].
Δψ or ΔpH? As discussed above, the protonmotive force is
comprised of two components, ψ and pH, so are we right
to assume that these agents primarily affect Ca2 + uptake via
pH effects and ignore ψ? Given that the V-ATPase is the
principal generator of the lumen-positive ψ, its inhibition results
in vesicle depolarization [79,82–84,89,90,95] and similarly,
simple H + -translocating ionophores [e.g. FCCP (carbonyl
cyanide p-trifluoromethoxyphenylhydrazone)] also depolarize the
organelles [83,89,92,95,119]. In stark contrast, electroneutral
cation-exchanging protonophores (nigericin or monensin) or
NH4 Cl do not depolarize (and, if anything, hyperpolarize)
[89,90,120,121]. In other words, these agents differentially affect
ψ (but commonly increase pHL ) affirming that the pH is, after
all, the major component of the protonmotive force governing
Ca2 + uptake. This also makes sense considering that Ca2 + uptake
would otherwise be favoured upon vesicle depolarization by VATPase inhibitors or protonophores.
Ca2 + leaks. Irrespective of the organelle (acidic or neutral),
inhibition of the Ca2 + -uptake mechanism(s) in stores that are
already replete with Ca2 + will only empty the store if Ca2 +
leaks out (at steady-state, this Ca2 + leak is precisely balanced
by the Ca2 + uptake). Hence in the ER, SERCA inhibition by
thapsigargin unmasks the Ca2 + leak, evokes a ‘Ca2 + release’ and
the stores empty. By the same token, dissipating pH in acidic
stores could inhibit Ca2 + uptake, but will only deplete them
of Ca2 + if there is a sufficient Ca2 + leak. For many cell types,
acidic stores are indeed ‘Ca2 + leaky’ and collapsing pH causes
a detectable Ca2 + release and/or inhibition of Ca2 + signals
[2,112,114–117,122–128], but this need not be the case. In the
355
sea urchin egg, bafilomycin A1 does not readily deplete acidic
stores of Ca2 + [10] (in part because they are not very H + leaky
[93], but their Ca2 + leak may also be small).
Endolysosomal Ca2 + /H + exchange
Simple exchangers. By analogy with the vacuolar systems
described above, the effect of bafilomycin A1, nigericin etc. is explained most simply by the presence of a Ca2+ /H+ exchanger (Figure 3), the reasoning being that if you abrogate the H + gradient
then the driving force for Ca2 + uptake is eliminated. However,
this has almost become dogma when the supporting evidence is
often circumstantial. For instance, addition of high concentrations
of Ca2 + to the ‘cytosolic’ face of the acidic vesicle increase
pHL , consistent with driving a Ca2+ /H+ exchanger in reverse, as
exemplified by sea urchin egg acidic vesicles [93,107], vacuoles
[94], protist acidocalcisomes [129], synaptic vesicles [130],
fungal spheroplasts [131] and endosomes [132]. Considering
there are other explanations for the Ca2 + -induced alkalinization,
e.g. H + -pump inhibition [75], it is reassuring when the converse
is observed, i.e. a pH-driven Ca2 + accumulation [94,130,132].
So, is the H + gradient thermodynamically capable of generating the observed levels of luminal Ca2 + (free Ca2 + ∼ 600 μM,
total Ca2 + 4–14 mM; Supplementary Tables S1 and S2)? By
analogy with plasma membrane exchanger thermodynamics
[133], the equilibrium relationships between pHL , ψ and
luminal Ca2 + filling can be calculated for an acidic store
exchanger (Figure 4). Electroneutral transport (1 Ca2 + :2 H + ) is,
of course, unaffected by ψ and a pHL of 5.0 could generate a
25000-fold Ca2 + gradient (2.5 mM luminal Ca2 + ). In contrast, the
same pH and an electrogenic 1 Ca2 + :1 H + could only generate
a paltry ∼ 50-fold gradient (assuming + 30 mV, lumen-positive
ψ). The vacuolar (CAX) forms suitably utilize 2–3 H + per
Ca2 + [102] and the endolysosomal system would require a similar
stoichiometry on thermodynamic grounds alone.
Assuming that Ca2 + /H + exchange does occur across the
acidic store membrane, what proteins might be responsible? It is
frustrating that CAX proteins are absent from higher vertebrates
[102] so they would have to belong to an entirely new family.
Alternatively, Ca2 + /H + exchange may not be mediated by a single
transporter, but rather by ‘nested’ exchangers acting in series and
one plausible scenario is Na + /Ca2 + exchange coupled to Na + /H +
exchange (Figure 3).
Coupled exchangers. For coupled transporters to work there
are several prerequisites. First, the H + gradient must drive Na +
uptake into the lumen (e.g. by a reverse-mode NHE) to establish a
Na + gradient that is sufficient, in turn, to drive Ca2 + uptake (e.g.
by an Na + /Ca2 + exchanger; Figure 3). Indeed, Supplementary
Tables S1 and S2 suggest that the [Na + ] within acidic vesicles
appropriately exceeds that of the cytosol (clearly, a Na + /K + ATPase would also reinforce Na + accumulation). In principle,
K + could substitute for Na + , but the luminal/cytosolic [K + ] ratio
appears less favourable for driving Ca2 + uptake (Supplementary
Tables S1 and S2).
Secondly, Na + (or K + ) must leak promptly from the lumen
when the V-ATPase is inhibited in order to dissipate the Na +
(K + ) gradient and hence the driving force for Ca2 + uptake. The
leak of monovalent cations is not particularly well studied, but
reducing the pH with FCCP or NH4 Cl has been reported to
lower lysosomal K+ [111,134] or Na+ [111] hinting at a degree of
leakiness consonant with the model.
Practically speaking, Na + /Ca2 + exchange has been implicated
in secretory vesicles [135,136] and genomically speaking there
is great scope for coupled transport. To compliment the NHEs,
c The Authors Journal compilation c 2011 Biochemical Society
356
A. J. Morgan and others
exchangers) [137,138]. All are electrogenic [137,139] and some
might be found on intracellular organelles (but reports are few)
[137]. Significantly, isoforms of NCKX have been suggested to
contribute to Ca2 + filling of (secretory) chromaffin granules [140]
and melanosomes [141] and, needless to say, endocytotic retrieval
of plasma membrane forms of any of these proteins could recruit
them for work in the endolysosomal system.
As tempting as the hypothesis is, there remains a problem
with the coupled transport scheme as it stands. Its pH-sensitive
component is the NHE, but these are not expressed throughout the
entire endolysosomal system (nor for that matter are the Na + /K + ATPases) (Table 1). How then would later compartments like
lysosomes fill with Ca2 + in a pH- and Na + -dependent manner?
Clearly, these are issues that must be addressed in the future.
Endolysosomal Ca2 + pumps
SERCA inhibitors. Ever since SERCA inhibitors became widely
available, they have been tested against organellar Ca2 + uptake
mechanisms. However, vacuolar Ca2 + pumps show greater
homology with the PMCA pumps that are insensitive to
thapsigargin and cyclopiazonic acid, and there is no reason
to suspect that an endolysosomal pump would be any different.
The literature, however, is confusing because there are reports
of thapsigargin-sensitive and -insensitive acidic Ca2 + stores. For
example, a thapsigargin-insensitive Ca2 + uptake mechanism may
exist in egg yolk platelets [10,142], slime mould vacuoles [143]
and the acidic stores of HEK (human embryonic kidney)-293
cells [144], corneal endothelium [109], insect S2 cells [111]
and pancreatic β cells [114,145–147]. By contrast, thapsigarginsensitivity has been reported for the secretory granules of mouse
pancreatic β cells [148], and acidic stores of MDCK (Madin–
Darby canine kidney) cells [149] and lobster lysosomes (albeit
with a low thapsigargin-sensitivity and Zn2 + as substrate) [150].
Whether these are biological or methodological differences is
presently unclear. Nonetheless, the sheer volume of reports does
lend more support to a thapsigargin-insensitive route.
Figure 4
Relationship between Ca2 + filling and pHL
(A) Thermodynamic constraints on Ca2 + /H + exchange. The equation shown was used to
generate the graphs where subscripts Lum and Cyt refer to the organelle lumen and cytosol
respectively, n is the number of H + exchanged per Ca2 + , F is the Faraday constant, ψ is
the membrane potential across the organellar membrane (ψ = ψ Cyt − ψ Lum ), R is the gas
constant and T is the temperature (310K); the cytosolic pH and [Ca2 + ] were assumed to be 7.2
and 0.1 μM respectively. The graph describes Ca2 + filling as a function of the luminal pH (pHL )
at different H + stoichiometries of Ca2 + /H + exchange (n = 0.5–3); ψ was set at − 30 mV
(lumen-positive). The broken lines indicate a zero Ca2 + gradient (grey, lumen/cytosol = 1),
and the Ca2 + gradient in endosomes and lysosomes assuming a luminal [Ca2 + ] of 30 μM
and 600 μM respectively. (B) Similarly, Ca2 + filling as a function of the ψ; pHL was set at
5.0 and the inset schematics indicate the charge orientation on an acidic vesicle. (C) Taking the
values in Supplementary Table S1 (at http://www.BiochemJ.org/bj/439/bj4390349add.htm), the
empirically determined relationship between pHL and the free [Ca2 + ]L is plotted across different
organelles. EE, early endosome; LE, late endosome; Lyso, lysosome; Phago, phagosome; SG,
secretory granule. Ca2 + and pH are paired from the same study (and/or the same cellular
source) where possible.
there are three families of CaCAs (Ca2 + /cation antiporters)
in higher organisms, namely NCX (Na + /Ca2 + exchangers),
NCKXs (Na + /Ca2 + -K + exchangers)4 and CCX (Ca2 + /cation
Thapsigargin-insensitive Ca2 + -ATPases. If stores are unaffected by thapsigargin, this could either mean that: (i) there are
no Ca2 + pumps at all (just exchangers) or (ii) that there are
thapsigargin-insensitive Ca2 + pumps. One way of distinguishing
between these two possibilities is to test other Ca2 + -ATPase
inhibitors, and two informative ones have been vanadate and
tBHQ (tert-butylhydroquinone).
Ca2 + -ATPases are P-type pumps and as such are blocked by
vanadate (resembling the γ phosphate of ATP at the active site)
[151] (note that Na + /K + -ATPases are also inhibited by vanadate
[76], but the V-ATPase is unaffected [89,119]). Importantly,
vanadate also inhibits thapsigargin-insensitive Ca2 + -ATPases,
and so we can test for other pumps. Being membrane-impermeant,
the use of vanadate is restricted to broken cell preparations,
but nonetheless there are numerous reports of its inhibiting
Ca2 + uptake into the slime mould vacuoles [104,143], synaptic
vesicles [152], Golgi [153], protist acidocalcisomes [129],
reticulocyte endosomes [154], and parotid or pancreatic acidic
stores [155,156]. Using a slightly different readout, vanadate also
affected the Ca2 + -ATPase activity of rat liver lysosomes [157] and
neurosecretory vesicles [119,158]. Taken together, these results
are consistent with the presence of a P-type Ca2 + -ATPase on some
acidic Ca2 + stores.
4
Also referred to as K + -dependent Na + /Ca2 + exchange since the counter-flow of Na + and K + down their concentration gradients drives Ca2 +
extrusion in a stoichiometry of 4 Na + :(1 Ca2 + + 1 K + ) compared with NCX stoichiometry of 3 Na + :1 Ca2 + .
c The Authors Journal compilation c 2011 Biochemical Society
Endolysosomal Ca2 + signalling in health and disease
Although the above is compelling evidence for Ca2 + pumps,
not all preparations exhibit a vanadate sensitivity, and resistance
to inhibition has been seen with fungal stores [131], egg
yolk platelets [10], melanin granules [159] and neutrophil
endosomes [160]. Assuming that the non-trivial issues of vanadate
polymerization and preparation [158] were adequately overcome,
this implies that P-type Ca2 + -ATPases are absent from particular
organelles.
More recently, the use of another inhibitor, tBHQ, has led
some to conclude that the Ca2 + -ATPase on NAADP-sensitive
acidic vesicles is a SERCA3-like pump [161,162]. According to
this model tBHQ is selectively blocking the SERCA3 isoform,
but not the SERCA2b on neutral stores (whereas the converse
is true for thapsigargin which exhibits a modest selectivity for
SERCA2b) [163]. Indeed, the results are in accordance with the
known pharmacology [164,165] and subcellular distribution of
SERCA3 in platelets [166].
The scheme is intriguing, so is this a platelet peculiarity or a
universal model? In support of universality: (i) SERCA3 does
exhibit a low (or undetectable) sensitivity to thapsigargin and
a low Ca2 + affinity [151] (compare with plants and yeast);
(ii) tBHQ does preferentially inhibit acidic store Ca2 + -ATPases
in other systems [143]; or (iii) of the canonical SERCAs,
SERCA3 exhibits a 6–30-fold greater sensitivity to vanadate
(IC50 ∼ 10 μM) [151], which is in reasonable agreement with
the vanadate sensitivity of some Ca2 + -ATPases on other stores
(IC50 1–30 μM) [104,143,152,153]. On the negative side: (i)
other acidic store Ca2 + -ATPases are substantially less sensitive
to vanadate (IC50 200–320 μM) [119,157]; and (ii) it is difficult
to envisage this being a universal model for Ca2 + uptake into
endolysosomal compartments when SERCA3 expression is, as far
as we know, restricted to very few tissues [167], even accounting
for the six splice variants of SERCA3a–f [168]. For the moment
we should perhaps be cautious about extrapolating the platelet
work to other cell types at least until the SERCA3 expression
pattern is better defined.
In conclusion, there is compelling evidence for Ca2+ pumps on
many types of acidic Ca2+ store, although their molecular nature
remains uncertain (with the possible exception of platelet alpha
granules). However, the fact that other acidic Ca2+ stores are
apparently resistant to Ca2+ inhibition suggests that pumps
are absent from certain preparations and highlights the differences
between acidic vesicle classes.
Ca2 + -ATPases and pH. An often overlooked fact is that
for P-type Ca2 + -ATPases, ATP hydrolysis not only drives
the translocation of Ca2 + across a membrane (against its
electrochemical gradient), but also transports H + in the opposite
direction [103,169]. The ramifications of this obligate exchange
mechanism should be obvious. First, whether the pump is
electrogenic or not will depend on the stoichiometry (a matter of
debate, some suggest it is electroneutral [103,169]); secondly, and
perhaps more important for this discussion, the pH will affect
Ca2 + -ATPase activity. At the plasma membrane or neutral ER,
the pH is negligible and can ostensibly be ignored by resident
pumps, but a Ca2 + -ATPase on an acidic vesicle is a very different
matter. Could the potential energy of the H + gradient help drive
Ca2 + uptake as has been suggested in lower organisms [104,170]?
357
Similar to analyses in yeast [170], one can calculate5 that
the Ca2 + electrochemical driving force (G) across a lysosome
might be expected to be of the order of − 28 to − 42 kJ/mol. In
comparison, the G of ATP hydrolysis in vivo is of the order
of − 52 to − 64 kJ/mol [36]. A vacuolar/PMCA-type Ca2 +
pump displays a stoichiometry of 1 ATP:1 Ca2 + [103], so ATP
hydrolysis itself should be more than sufficient to translocate
Ca2 + . However, the SERCA family translocate 2 Ca2 + per cycle
[103] which doubles the energy requirement ( − 56 to − 84 kJ/
mol). In other words, this is close to (or exceeds) the energy
from a single ATP molecule which may not be competent to fuel
the pumping of 2 Ca2 + into the lumen of lysosomes (depending
on the membrane potential), and therefore the H + gradient
could provide the energetic shortfall6 . That is, the intrinsic H + exchange activity of SERCA is not just an epiphenomenon, but is
probably an essential requirement for Ca2 + pumping into acidic
compartments, and this may be pertinent to the platelet SERCA3
story.
This is not mere nit-picking because it has a bearing
on interpreting the effect of altering pH on Ca2 + signals.
Classically, dissipating the pH gradient with protonophore or
bafilomycin A1 is expected to inhibit Ca2 + /H + exchange, but
what would be the effect on a Ca2 + pump in light of the above?
On the one hand, pump activity might be severely inhibited by
eliminating the H + gradient (the thermodynamic ‘top up’). On
the other hand, the store becomes more ER-like (little ψ) which
presents a lower energy obstacle for Ca2 + pumping7 .
The upshot of collapsing the H + gradient may be that the
(formerly) ‘acidic’ store may not empty (or might even increase
their Ca2 + filling) and the involvement of an acidic Ca2 + store
may be erroneously dismissed. For example, it is one inventive
explanation for an apparently bafilomycin A1-insensitive NAADP
response (e.g. [171]). Indeed, these considerations remind us
that the relationship between luminal [H + ] and [Ca2 + ] need not
be the reciprocal one that it is often suggested (i.e. the more acidic,
the greater the Ca2 + filling), and this complexity is borne out by
looking at the endolysosomal system as a whole (Figure 4).
One or two uptake pathways?
For a given endolysosomal organelle, does Ca2 + /H + exchange
and a Ca2 + pump exist simultaneously, as is the case for
plants and yeast vacuoles? In these organisms, the diversity of the
Ca2 + affinity (and capacity) of the two pathways is commensurate
with their different roles (e.g. store priming compared with cytosol
clearance), and this would equally make sense in other organisms.
Indeed, multiple Ca2 + uptake pathways do seem to co-exist in
secretory granules [152] and pancreatic acidic stores [156], but
we will have to wait and see if this is ubiquitous.
Endocytosis and Ca2 + filling
One final point is that processes such as endocytosis and
phagocytosis may also contribute to Ca2 + filling of acidic vesicles.
Having engulfed extracellular fluid, the earliest phago- or endosomal structures would contain millimolar [Ca2 + ]. Nonetheless,
the luminal free [Ca2 + ] is lowered with some rapidity (in minutes)
5
From the Nernst equation G Ca = 2F ψ + RT ln(Cacyt /CaLum ) where T = 310 K (37 ◦ C), the cytosolic [Ca2 + ] (Cacyt ) = 0.1 μM, luminal [Ca2 + ]
(CaLum ) = 600 μM (Supplementary Table S2). When ψ = − 0.03 V (cytosol-negative, lumen-positive), G = − 28 kJ/mol, when ψ = − 0.1 V,
G = − 42 kJ/mol.
6
The protonmotive force = 17 kJ/mol if G = 2.3RT pH – F ψ, and assuming pHL = 4.8, cytosolic pH = 7.2 and ψ = − 0.03 V (cytosol-negative,
lumen-positive) at 310 K.
7
Note that SERCA on neutral stores would not have to contend with a substantial ψ and simply removing this component reduces the G Ca overhead
to − 44 kJ/mol for 2 Ca2 + which is amply covered by ATP hydrolysis.
c The Authors Journal compilation c 2011 Biochemical Society
358
A. J. Morgan and others
to the micromolar range [172–174]. Assuming that the Ca2 + buffering capacity of the lumen will, if anything, be decreased as
these vesicles acidify, this most probably reflects a real decrease
in total Ca2 + due to extrusion from the vesicle. Will a modest 30–
300-fold gradient still be sufficient for endosomes to act as Ca2 +
stores? Circumstantial evidence suggests that they can. They’ve
been implicated in endocytosis in pancreatic Ca2 + signalling [175]
and TPCs (two-pore channels) (see below) are indeed found
on endosomes [123,127,128], but, again, this awaits empirical
determination.
Conclusion. All this serves to illustrate that the Ca2 + -filling
pathways of acidic stores are still ill-defined in systems other than
plants and yeast. There is patchy (but consistent) evidence for
Ca2+ /H+ exchangers and/or Ca2 + -ATPases, and thermodynamics
impose constraints on their stoichiometry and modes of function.
Luminal Ca2 + buffers
Once Ca2 + is translocated into any Ca2 + store, only a small
fraction of the total Ca2 + is free, the remainder is bound
(to help reduce the Ca2 + electrochemical gradient and thereby
facilitate Ca2 + storage). Within the ER, Ca2 + binds to the ERselective proteins calsequestrin, calreticulin and calnexin [176–
178], which possess highly acidic domains that bind Ca2 + with an
appropriately low affinity yet high capacity (∼ 500 μM and 20–
50 mol/mol respectively [176,177]), ideal properties to reversibly
sequester 50–90 % of the ER Ca2 + [179,180].
Acidic Ca2 + stores emulate this precept and chelate substantial
quantities of Ca2 + , as a comparison of Supplementary Tables
S1 and S2 attests (note the orders of magnitude difference in the
total and free [Ca2 + ], up to 99.9 % buffered [136]). What chemical
groups could bind Ca2 + in such an acidic environment? Intuitively,
one would expect that Ca2 + chelation might occur through anionic
groups such as phosphate, sulfate and carboxylate (recall that S
and P are abundant in acidic vesicles; Supplementary Table S1).
The side-chain carboxylate on proteins (i.e. aspartate/glutamate)
has a pK a value of ∼ 4, sulfate conjugates <1, and polyprotic
phosphate extends over 2–12. Therefore, despite the aggressively
low pHL of 4.5–5.0, these groups will all be anionic (to some
extent) and available to form complexes with Ca2 + ions (albeit
with a lower affinity than at neutrality owing to H + competition).
What molecules provide these groups in situ is more open
to debate with a paucity of information available, but work
has implicated several classes of molecules, in increasing
size: (i) small organic acids such as oxalic acid that are
abundant and Ca2 + -binding in vacuoles (to the point of
forming crystals) [102,105]; (ii) polyanionic matrixes that come
in several forms, including polyphosphates (acidocalcisomes
[181], vacuoles [102], lysosome-related organelles [182]), and
glycosaminoglycans such as heparin [183]; and (iii) proteins,
either binding Ca2 + through specific metal-binding motifs (e.g.
melanin [184], toposome [7]) or non-specifically via extensive
decoration by glycosylation (e.g. mucin [185]). It is worth
recalling that it is these larger molecular mass molecules that
contribute to the Donnan potential mentioned above, as well as to
the pHL buffering capacity [34,35].
Ca2 + RELEASE MECHANISMS IN THE ENDOLYSOSOMAL SYSTEM
The study of Ca2 + release from acidic stores has been galvanized
by the finding that in many cell types NAADP appears to trigger
directly Ca2 + release from acidic organelles. We will initially
describe the studies leading to NAADP as a Ca2 + -mobilizing
messenger, and how the dissection of its mechanism of action has
c The Authors Journal compilation c 2011 Biochemical Society
led to the discovery of a novel class of endolysosomal channels
in animal cells, the TPCs, whose functional characterization
is beginning to enhance our understanding of Ca2 + -release
mechanisms from the endolysosomal system.
NAADP-evoked Ca2 + release
In a landmark paper, Lee and colleagues [186] discovered
that two pyridine nucleotide metabolites released Ca2 + from
homogenates prepared from sea urchin eggs. The sea urchin
egg had been one of the earliest systems in which the then
newly discovered Ca2 + -mobilizing messenger IP3 (inositol 1,4,5trisphosphate) had been shown to be active in releasing Ca2 +
from intracellular stores [187]. Previously, it had been shown that
such egg homogenates displayed a robust response to IP3 which
releases Ca2 + from non-mitochondrial stores in this preparation
[188]. Cell fractionation showed that the IP3 -sensitive stores
correlated with the distribution of ER markers. An important
finding was that following IP3 -evoked Ca2 + release, homogenates
remained refractory to a further challenge with IP3 for some
hours. Pyridine nucleotides were known to undergo dramatic
changes at fertilization in the sea urchin egg [189], prompting
Lee and colleagues to investigate their possible role in regulating
vesicular Ca2 + transport processes in their newly defined cellfree system [186]. In egg homogenates rendered refractory to IP3 ,
the pyridine nucleotides NAD + and NADP + both caused Ca2 +
release, but apparently by distinct mechanisms which varied in
several important respects. First, whereas NADP + gave a rapid
response, NAD + did so but after a latency of several seconds.
Secondly, although, like IP3 , NAD + and NADP + showed the
property of homologous desensitization, they did not affect the
ability of each other to evoke a response. Thirdly, homogenate
fractionation indicated that NAD + and NADP + released Ca2 +
from different populations of vesicles.
Further analyses of these three key observations led to several
important discoveries in Ca2 + signalling. First, was the chemical
identification of the Ca2 + -mobilizing molecules themselves. The
effect of NAD + was ascribed to a novel cyclic metabolite cADPR
(cyclic adenosine diphosphate ribose) [190]. The conversion of
NAD + into cADPR by enzymes termed ADP-ribosyl cyclases
was shown to account for the latency between NAD + addition and
the initiation of Ca2 + release [191]. The rapid Ca2 + -mobilizing
effects of NADP + was due to the contamination of commercial
sources of the compound by the related analogue NAADP [192].
The homologous self-desensitization of the three Ca2 + -release
mechanisms activated by IP3 , cADPR and NAADP suggested
that they activated distinct Ca2 + -release mechanisms. The rapid
kinetics of the responses to each Ca2 + -mobilizing agent was
indicative of activation of three separate Ca2 + -release channels.
In the late 1980s, two related families of Ca2 + -release channel
from the sarco-endoplasmic reticulum had been identified, IP3 Rs
(IP3 receptors) activated by IP3 [193] and RyRs (ryanodine
receptors) [194] which could be opened by increases in
cytoplasmic Ca2 + , so called CICR (Ca2 + -induced Ca2 + release)
[195,196]. An important finding was that cADPR triggered
Ca2 + release through the activation of RyRs [197], and with the
cognate receptor for IP3 identified [193], this left the question
of the nature and identity of the NAADP-gated Ca2 + release
channel. Pharmacological studies showed that NAADP-evoked
Ca2 + release was not affected by blockers of IP3 Rs or RyRs, but
sensitive to VGCC (voltage-gated Ca2 + channel) blockers [198].
The other major finding was that NAADP released Ca2 + from
a population of vesicles distinct from those sensitive to IP3 and
cADPR. The depletion of endoplasmic stores by the SERCA
Endolysosomal Ca2 + signalling in health and disease
359
pump inhibitor thapsigargin abolished Ca2 + release by both IP3 or
cADPR, but the Ca2 + -mobilizing effect of NAADP still persisted
[142], even in intact eggs [199]. In addition, it was found in
stratified sea urchin eggs, that NAADP mobilized Ca2 + from
an area of the egg which was distinct from that sensitive to IP3
or cADPR [200]. The identification of the principal NAADPsensitive store followed in an important study in 2002, where
NAADP was demonstrated to release Ca2 + from organelles
identified as reserve granules, acidic lysosomal-related organelles
[10]. This study for the first time linked NAADP-evoked Ca2 +
release with acidic Ca2 + stores which has now been strengthened
by studies in cell types from a variety of organisms including
many from vertebrate systems [201]. In addition, NAADP may
also trigger Ca2 + influx at the plasma membrane, but it is not clear
whether this is a direct effect on plasma membrane channels or a
consequence of Ca2 + release from internal stores.
The trigger hypothesis: juxta-organellar Ca2 + signalling
In many cells, NAADP evokes sizeable Ca2 + signals [202]. In
a minority that includes sea urchin eggs, this may be explained
by a preponderance of acidic Ca2 + stores [7,10], but in most
cells lysosomes and lysosomal-related organelles constitute a
much smaller cellular volume than the ER, and consequently
mobilization of Ca2 + stores in lysosomes might be expected to
produce only small and localized cytoplasmic Ca2 + signals [148].
One explanation of this apparent paradox has been advanced
in the formulation of the ‘trigger hypothesis’ to explain Ca2 +
mobilization by NAADP [204].
Early analysis of NAADP-induced Ca2 + release from
homogenates of sea urchin egg [142,205] or brain [206],
indicated that the NAADP-sensitive Ca2 + release mechanism
was not modulated by cytosolic Ca2 + (or Ca2 + surrogate ions).
Nevertheless, NAADP can initiate regenerative global Ca2 +
signals (waves and oscillations) and this is because ‘trigger’ Ca2 +
provided by NAADP is subsequently amplified by recruitment of
IP3 Rs and RyRs that exhibit the characteristic property of CICR
[199,207]. This ‘channel chatter’ [208] may occur by two modes:
most obviously by the trigger Ca2 + stimulating ER channels via
the CICR, but also by trigger Ca2 + being sequestered into and
‘priming’ the ER (which sensitizes ER channels from the luminal
face) (Figure 5).
Such communication between Ca2 + -storing organelles
demands close appositions as typified by the interactions between
SR (sarcoplasmic reticulum)/ER and mitochondria [209] that is
cemented physically by mitofusins acting to tether the organelles
together [210]. Is there an analogous juxtaposition of acidic
organelles and ER? Lysosome/SR junctions have certainly been
observed in vascular smooth muscle cells which are the site of
initiation of NAADP and agonist-evoked Ca2 + signals [117,211]
and sea urchin egg yolk platelets are cradled by proximate
ER [7,212]. Whether this is ubiquitous and requires specialized
tethering proteins remain to be shown.
Modulation of pHL by NAADP
Given that NAADP appears to mobilize Ca2 + from acidic stores
in sea urchin eggs, the effect of NAADP on organellar luminal pH
was investigated in egg homogenates and intact sea urchin eggs
[93,213]. Employing acridine orange or Lysosensor dyes as pH
indicators, NAADP was found to evoke an alkalinization of acidic
stores [93,213]. This effect was highly specific in that neither IP3
or cADPR affected pHL , and the effects of NAADP were blocked
by prior desensitization of the NAADP-sensitive Ca2 + release
Figure 5
Trigger hypothesis of NAADP-induced Ca2 + release
Schematic diagram of a global Ca2 + transient induced by a stimulus depicted in two components:
first the small phase [AS (acidic stores); red] followed by the subsequent large regenerative spike
(from the ER; green). ‘Trigger’ Ca2 + is released from acidic Ca2 + stores by NAADP to gives a
globally small (but locally high) [Ca2 + ] (first phase). There are two modes of recruiting the ER
Ca2 + -release channels, CICR (upper scheme) and store priming (lower scheme). Trigger Ca2 +
acts at the cytosolic face of ER channels (IP3 Rs or RyRs) to sensitize them by CICR and evoke a
global (green) Ca2 + spike. Alternatively, trigger Ca2 + is taken up into the ER by SERCA action
and acts to luminally sensitize ER channels and thereby evoke a global Ca2 + spike (green).
mechanism [93]. This is not peculiar to eggs because NAADPdependent alkalinization has been recapitulated in pancreatic
acinar cells [214]. The mechanism by which NAADP modulates
pHL is unclear (see [7] for discussion), but it has been proposed to
be secondary to Ca2 + release from acidic stores. Given that pHL
is important for endolysosomal function, including membrane
fusion and luminal enzyme activities [215], and NAADP receptor
channel gating (see below) it may be of major physiological
importance.
The search for the NAADP receptor
Since the discovery of the Ca2 + -mobilizing properties of NAADP,
there has been intense effort to identify the mechanism by which
this molecule evokes Ca2 + signals in cells. A number of candidate
ion channels have been implicated [9], for which there are various
degrees of evidence, both for and against.
RyRs
Although there is substantial evidence that RyRs are the principal
effectors of cADPR-induced Ca2 + release from the ER, a number
of studies have also implicated RyRs in NAADP-evoked Ca2 +
release [116,216]. In many cells, NAADP-evoked Ca2 + release
is certainly sensitive to inhibition by RyR blockers, and in many
c The Authors Journal compilation c 2011 Biochemical Society
360
A. J. Morgan and others
cases this may be a manifestation of the trigger hypothesis outlined
above, where NAADP recruits ER stores via CICR [117,211].
However, in a number of studies, NAADP has been proposed to
directly activate RyRs on the ER [217,218], their major site of
subcellular localization, or RyRs on acidic stores, for which there
is some evidence [146].
Extensive studies by Dammermann and Guse [216] in Jurkat
cells have characterized a robust Ca2 + -mobilizing response
to microinjection of NAADP, which has the characteristic
bell-shaped concentration–response curve observed widely in
mammalian cells. However, this response is blocked by RyR
inhibition, RNAi (RNA interference)-based knockdown of RyR
expression, and is apparently insensitive to agents that interfere
with Ca2 + storage by acidic organelles [216]. Similarly, in
pancreatic acinar cells, NAADP releases Ca2 + from the nuclear
envelope, a contiguous Ca2 + store with the ER, which again is
blocked by RyR inhibitors, but not by agents affecting acidic store
Ca2 + storage [116].
Direct evidence for NAADP regulation of RyRs has come
from studies on purified RyRs incorporated into lipid bilayers,
whereby NAADP at nanomolar concentrations was found to
activate the skeletal muscle RyR1 isoform with conductances
typical for authentic RyRs [218]. Such conclusions are based on
the purity of the incorporated protein fractions. However, other
bilayer studies of purified RyRs have failed to show significant
activation by NAADP [219,220], and heterologous expression
of RyRs in HEK-293 cells enhances cADPR, but not NAADPevoked Ca2 + release [221]. Furthermore, NAADP mobilizes Ca2 +
in cells lacking RyRs, but requiring TPC expression [127,221].
indirectly affect TPCs (see the Acidic Ca2 + Store Pathology
section for further discussion).
TRPM2
TRPM2, a member of the melastatin family of Trp channels,
is a channel with enzyme activity termed a chanzyme [234].
It is regulated by ADP-ribose and the channel expresses ADP
pyrophosphatase activity associated with its Nudix hydrolase
domain [235]. Although it is expressed primarily at the plasma
membrane, a recent report has shown localization to lysosomes
[236]. Furthermore, the channel is sensitive to NAADP. However,
the concentrations of NAADP required to modulate channel
gating are in the high micromolar concentration range, very
different from the nanomolar concentrations of this most potent
of Ca2 + messengers that have routinely been found to be effective
in most cells [237].
P2X4
Whereas both metabotropic (P2Y) and ionotropic (P2X) families
of purinoceptors are well-studied cell-surface receptors for ATP
and other nucleotides, P2X4 has been found to be expressed in
lysosomes and targeting motifs identified [238]. Topologically,
nucleotide-binding sites would be predicted to be luminal
rather than cytoplasmic, requiring intra-organellar synthesis of
ligands or transport into the lysosome. However, there are no
reports that NAADP can modulate this channel, although high
concentrations of NAADP may interact with P2Y receptors at the
plasma-membrane-activating phospholipase C-linked signalling
pathways.
TRPML1 (mucolipin 1)
Genetic analysis of the lysosomal storage disease MLIV
(mucolipidosis type IV) uniquely identified mutations in
a gene encoding a putative ion channel [222], whereas
other storage diseases are due to defects in enzymes or
transporters. The protein encoded by this gene, TRPML1, has
homologies with ion channels of the Trp (transient receptor
potential) family [223]. Two homologues mucolipin-2 and
mucolipin-3 were subsequently identified [224]. TRPML1 was
found as expected to localize to lysosomes, and lysosometargeting motifs were identified [225,226]. TRPML1 is a
cation channel with evidence that it is permeant to H+ ,
Ca2+ and Fe2+ , among others [227–229]. As the first ion
channel definitively localized to lysosomes, it was an obvious
candidate for mediating NAADP-evoked Ca2 + release from
acidic stores. The first study of TRPML1 to examine the possible link with NAADP failed to find any modulation of the
channel by NAADP, and neither did overexpression enhance
[32 P]NAADP binding to membranes from overexpressing cells
[225]. However, two subsequent reports from Li and colleagues
based on single channel recordings implicated TRPML1 as targets
for NAADP [230,231]. Nonetheless, a recent study involving both
overexpression of all three isoforms of mucolipin as well as RNAi,
failed to confirm these findings [232] and ruled out direct gating
of mucolipins by NAADP. Therefore it may be highly relevant
that TPCs co-immunoprecipitate with TRPML1 [232] and may
explain why purified TRPML1 complexes generate convincing
NAADP responses in bilayers [218,219]. Instead, TRPML1 may
be modulated physiologically by PIP2 [PtdIns(3,5)P2 ] [233].
In conclusion, on balance, the case for TRPML1 as being an
obligatory component of the NAADP-gated Ca2 + -release channel
is not supported. As an endolysosomal ion channel, TRPML1
could nonetheless influence luminal [Ca2 + ], pHL or ψ to
c The Authors Journal compilation c 2011 Biochemical Society
TPCs
A recent addition to the network of ion channels regulating
lysosomal function is a small family of proteins termed TPCs
encoded by TPCN genes (Figure 6) which have been the subject
of recent reviews [3,5,8,9,239,240]. The founding member of this
family TPC1 was cloned in 2000 by screening a rat kidney cDNA
library for sequences based on known voltage-gated channels,
the superfamily of which TPC is a member [241]. α (poreforming) subunits of voltage-gated sodium channels contain four
homologous domain repeats of 6TMD (transmembrane domain)
architecture, whereas α subunits of K+ channels contain only one
domain and tetrameric assemblies are required to form functional
channels [242]. In contrast, a full sequence of a protein was
identified with only two domains of 6TMD and thus named
TPC1. These channels thus appear to represent an evolutionary
intermediate form between putative voltage-gated Ca2 + 6TMD
channel from the bacterium Bacillus halodurans [243], and
24TMD four-domain channels typified by mammalian voltagegated Ca2 + or Na+ channels, generated by gene duplication during
evolution. In the S4 domain (TMD1), repeats of charged amino
acids are partially conserved with those in the voltage-sensor
region of mammalian voltage-gated channels, suggesting that the
TPC1 protein may possess a degree of voltage-sensitivity.
The initial description of the TPC1 protein was confined to
mammalian tissue distribution by Northern blot analysis, and
mRNA transcripts were found to be distributed across most
rat tissues, with particularly high signals in kidney and liver
[241]. Immunostaining of kidney tubules with a polyclonal
antibody raised against the TPC1 recombinant protein showed
that the protein showed a polarized expression in renal tubules
with more prominent staining in the apical domain [241].
Endolysosomal Ca2 + signalling in health and disease
361
in sequence, exhibit similarities and differences in structure and
function [248]. Whether plant TPC1 channels are also gated by
NAADP has not been demonstrated, but in the only report of
NAADP-evoked Ca2 + release in plants to date, microsomal rather
than vacuolar fractions appeared to comprise the major NAADPsensitive Ca2 + stores [249].
Figure 6
Predicted structure of TPCs
Schematic diagram showing the predicted secondary structure and domain structure of TPCs.
Putative voltage-sensing cationic amino acids are shown by ‘ + ’ in the S4 domain. Published
inactivating point mutations in the predicted pore region are shown as L273 (HsTPC1) or N257
and E643 (mouse TPC2) where the numbering corresponds with the residues in the given
species. Mutation of the D454 residue in plant TPC (green) suggests that it is a component
of a putative luminal Ca2 + -sensing domain, according to the fou2 mutant. EF-hands (green)
are present in plants and may confer an additional sensitivity of plant TPCs to cytosolic Ca2 + .
For comparison, four-pore voltage-gated Ca2 + channels (Cav1–3) and single-pore mucolipin
(TRPML1–3) and TRPM2 channels are also shown. Putative glycosylation sites immediately
precede the pore loop of the second domain only for human TPC2 or both domains for human
TPC1.
However, expression of rat TPC1 cRNA in Xenopus oocytes
failed to generate any voltage-dependent plasma membrane
currents significantly different from background [241]. In
this study, the authors noted that a distantly related protein
with approximately 25 % homology, had been deposited in
the Arabidopsis database, and a study subsequently followed
describing an initial characterization of AtTPC1 (Arabidopsis
thaliana TPC1) [244].
AtTPC1 was found to be expressed in most Arabidopsis tissues
and initially it was thought to be a plasma membrane channel.
Employing aequorin as a Ca2 + probe, AtTPC1 expression was
found, in part, to mediate Ca2 + signals elicited by osmotic
shock with sucrose solutions [245]. Modulation of membrane
potential by genetic manipulation of H + -transport proteins was
also found to affect the response. Finally, in yeast the expression of
AtTPC1 was found to rescue Ca2 + transport processes in mutant
strains. The authors concluded that AtTPC1 is a voltage-sensitive
Ca2 + -permeable transporter. In an important study, Sanders and
colleagues showed that AtTPC1 was a vacuolar channel, and that
it mediates the well-characterized SV (slow vacuolar) current
important in stomatal movements and germination [246]. In
contrast with animal TPCs, the plant TPC1 proteins have two
cytoplasmic EF-hands in the loop between the two 6TMDs
(Figure 6) which may be more physiologically relevant to
its regulation than its voltage-sensitivity conferred by charged
residues in the S4 segments. This domain allows the channel to
function as a CICR effector, which may be its major role in plants.
For 8 years, the plant TPC1 was the only TPC protein to be
studied electrophysiologically, revealing an additional regulation
by luminal Ca2 + (increases in Ca2 + decrease the channel open
probability) an effect that is reduced in the D454N (fou) mutation
in a putative luminal loop (Figure 6) of the TPC1 channel [247].
Thus plant and animal TPCs, although only weakly homologous
Identification of animal TPCs as NAADP-gated channels. In
another landmark study, major candidates for NAADP-gated
Ca2 + -release channels were identified as TPCs [127]. The strength
of the candidature of TPCs as possible mediators of NAADPinduced Ca2 + release was based on two properties of these
channels. The first was their localization to the endolysosomal
system, indeed inspection of the human TPC2 sequence revealed
a putative dileucine lysosomal-targeting motif. The second was
their homology with voltage-gated cation channels, a possibility
given that dihydropyridines and other cation channel blockers
inhibit NAADP-stimulated Ca2 + release [198]. Subsequently,
other groups confirmed that TPCs could be gated by NAADP
[123,144].
TPC structure. The homology of TPCs with voltage-gated
cation channels has led to the prediction of their transmembrane
arrangement (Figure 6). Each of the two repeated 6TMD regions
contain a putative pore-forming region between the putative S5
and S6 membrane-spanning sequences, and this prediction is
consistent with topology studies mapping trypsin and antibody
access to fluorescent protein tags at different sites along TPCs
[250].
Like other lysosomal proteins, TPCs are glycosylated
[128,144,250], probably luminally (Figure 6), affording them
protection in a highly acidic environment (see above) and
giving rise to different TPC electrophoretic mobilities (apparent
molecular masses ranging from 80–100 kDa depending on the
degree of glycosylation). Such TPC glycosylation may well
regulate their sensitivity to activation by NAADP as judged by
mutation of glycosylation sites near the putative pore [250].
Given their domain structure, TPCs might be predicted to form
dimeric functional channels. Indeed, co-immunoprecipitation
studies have indicated that TPC2 homodimerization may occur
[144], but how the TPC family assembles must be examined in
greater detail.
Localization of TPCs. There are three isoforms of TPCs
expressed in most animals, an exception being the primate and
rodent genomes which either completely lack TPC3 or contain a
pseudogene [251]. The three TPCs are equally distant from each
other (see below), and from plant TPC1, with approximately 30 %
conserved amino acid identity in the conserved transmembrane
regions [127]. Heterologous expression of human and mouse
TPC1 and TPC2, and chicken TPC3 in HEK-293 cells showed
that although all three isoforms localize to components of the
endolysosomal system, there are differences [127]. TPC2 appears
to localize predominantly to lysosomes and late endosomes,
whereas TPC1 is more endosomal. TPC3 may be largely in
recycling endosomes (Figure 7). A polyclonal antibody raised
against the HsTPC2 (human TPC2) sequence, showed that
endogenous TPC2 protein co-localizes with the lysosomal marker
LAMP2 in HEK-293 cells. There have been no reports of
endogenous localization of TPCs to the ER or plasma membrane.
Both the N-terminal sequence and certain leucine residues appear
to be important for HsTPC2 targeting to lysosomes, since deletion
or changes to these residues results in their appearance at the
plasma membrane [125].
c The Authors Journal compilation c 2011 Biochemical Society
362
Figure 7
A. J. Morgan and others
TPC distribution throughout the endolysosomal system
Relative density of the three TPC isoforms is represented by the density of the gradients shown
below. Summary based on the data with recombinant human and sea urchin TPCs. Endo,
endosome; Lyso, lysosome; MVB, multivesicular body; PM, plasma membrane.
TPCs and NAADP-evoked Ca2 + release. Several vertebrate and
invertebrate TPCs have been cloned and their Ca2 + -releasing
properties and sensitivity to NAADP has been examined by
heterologous expression in cell lines. Three broadly consistent
reports appeared in 2009, each supporting the notion that TPCs
are mediators of Ca2 + release from endolysosomal stores. The
first report was a comprehensive study which encompassed all
three vertebrate TPC isoforms [127]. Introduction of NAADP
or caged NAADP into control HEK-293 cells through a patch
pipette elicited only a small Ca2 + response. However, in
cells overexpressing lysosomal HsTPC2 channels, NAADP or
photolysis of caged NAADP, induced a large biphasic Ca2 +
release [127]. The first phase was proposed to be due to direct
NAADP-evoked Ca2 + release from acidic stores since it was
blocked by bafilomycin. Following this, a second larger Ca2 +
release was found to be heparin-sensitive and probably due to
amplification through the recruitment of IP3 Rs on ER stores, and
nicely consistent with the trigger hypothesis for NAADP action.
Conversely, NAADP responses were abolished in cells treated
with siRNA (small interfering RNA) to TPC2, and also in
pancreatic β cells derived from TPC2-knockout mice [127]. Thus
it was proposed that TPCs are likely to be excellent candidates
for the elusive NAADP-gated Ca2 + -release channels on acidic
stores. In a subsequent independent study, expression of TPC2
was similarly found to evoke Ca2 + release from lysosomal Ca2 +
stores, although under the conditions of this study, amplification
of the Ca2 + response by recruitment of ER Ca2 + stores was not
apparent [144].
Subsequently, Ca2 + responses to microinjected NAADP in the
breast cancer SKB3 cell line were shown to be affected by altering
TPC expression. TPC1 shRNA (short hairpin RNA) reduced both
the endogenous expression and Ca2 + responses, whereas TPC1
overexpression enhanced the sensitivity of NAADP-induced Ca2 +
release [123]. Since bafilomycin and ryanodine both abolished
NAADP responses, the authors invoked an NAADP trigger
hypothesis implicating RyRs on the ER. In summary, these
three studies support the idea that TPCs are likely to be the
targets for NAADP action at acidic stores, but differences
in details, especially with regard to juxta-organellar coupling
between different classes of Ca2 + -release channel, are likely to
be due to cell-specific differences, and variations in the levels of
heterologous expression.
In view of this, it was thus important to assess properties of
endogenous TPC proteins with regard to their interactions with
NAADP. Because of the prior extensive studies in the sea urchin
c The Authors Journal compilation c 2011 Biochemical Society
egg, TPCs were immunopurified from solubilized membrane
fractions from these cells. Remarkably, [32 P]NAADP binding with
these immunoprecipitates exhibited all of the hallmark features
of binding to intact membranes. Binding was high affinity (K d
∼ 1 nM), showed a high degree of selectivity over NADP + ,
and was irreversible in the presence of high concentrations of
K + ions [128]. In terms of Ca2 + signals, pre-treatment with
subthreshold concentrations of NAADP inactivated NAADPinduced Ca2 + release as occurs with bona fide sea urchin egg
NAADP receptors [128]; moreover, heterologous expression of
SpTPC (sea urchin TPC) 1 and SpTPC2 in HEK-293 cells
enhanced Ca2 + release in response to patch-applied NAADP or
the membrane-permeant NAADP-AM (NAADP-acetoxymethyl
ester). In contrast, overexpression of SpTPC3, failed to enhance
NAADP responses, and acted in a dominant-negative fashion,
and abolished the enhanced response to NAADP in cells
co-expressed with TPC2 [128]. However, in a separate study of
SpTPCs, heterologous expression in SKB3 cells demonstrated
that all three SpTPCs could mediate NAADP-evoked Ca2 + release
[124].
TPC properties. The localization of TPCs to the endolysosomal
system presents difficulties in their study by electrophysiological
techniques to examine their channel properties. However, three
complimentary approaches have now been reported, all examining
HsTPC2 channel properties, all with various degrees of strengths
and weaknesses (Figure 9). However, useful parameters from
these comparative approaches have been obtained, giving deeper
insights into the nature of these proteins and their regulation.
The first is patching of purified lysosomes [80,252]. Using the
planar patch-clamp technique (Figure 9), currents were recorded
from single lysosomes swollen with the small molecule, vacuolin,
prepared from cells overexpressing TPC2 (or wild-type controls).
This approach has the advantage of examining the properties of
channels in their native membrane, although it is not clear whether
vacuolin affects channel activity or whether other lysosomal
channels (or other organelles) confound the preparation. Only
in the TPC2-overexpressing cells were NAADP-evoked whole
lysosomal currents detected, and the current was abolished
by mutations in the proposed pore loop 1 (S5–S6) (N257A)
suggesting a role in channel gating [80]. The current was selective
for Ca2 + over K + by a factor of >1000, but mutations in E643A
in the pore loop of the second putative S5–S6 domain changed the
ion selectivity in favour of monovalent cations [80]. The currents
were modulated by intraluminal pH, with currents only observed
at acidic pH [80].
In a traditional approach much used to study IP3 and RyRs,
immunopurified TPC2 was incorporated into artificial planar lipid
bilayer membranes (Figure 9). In contrast with whole organelle
electrophysiology, this approach may give an indication of TPC
properties uncontaminated by the presence of other channels
that exist in cellular membranes (depending on the purity). This
study gave the first insight into single-channel characteristics of
TPC2 channels, and details of the molecular mechanisms by
which NAADP may evoke Ca2 + release by TPC2 activation
[220]. The single-channel conductance was approximately 300 pS
for K + , but only 15 pS for Ca2 + [220]. NAADP increased Po
(mean open probability), and was dramatically sensitized by
increasing luminal Ca2 + concentrations (EC50 for NAADP of
5 nM at 200 μM Ca2 + , in the range reported for lysosomal Ca2 +
content [1,2]).
Channel activity was reported at both acidic and neutral pH,
but at acidic pH, the characteristic bell-shaped concentration–
response curve was observed with high concentrations
(millimolar) of NAADP failing to open the channels, in line with
Endolysosomal Ca2 + signalling in health and disease
363
In all three studies, current–voltage relationships were virtually
ohmic (perhaps a slight inward rectification) suggesting that the
channel is not voltage-gated in spite of modest conservation
of charged amino acid residues in the putative S4 domains.
That the membrane potential across lysosomes (Supplementary
Table S2) is much more modest than that across the plasma
membrane also mitigates against a major role for voltage in
this channel’s regulation [90]. The major conclusions from these
studies are that the TPC2 protein functions as a cation-selective
channel, is permeant to Ca2 + , modulated by cytoplasmic NAADP
at physiologically relevant concentrations, and is also highly
dependent on luminal Ca2 + and pH, the latter consistent with
its lysosomal localization. The possible lack of sensitivity to
cytoplasmic Ca2 + is in marked contrast with IP3 Rs and RyRs,
and consistent with its role as a trigger of local rather than global
Ca2 + release.
PHYSIOLOGICAL ROLES OF Ca2 + RELEASE FROM
ENDOLYSOSOMAL Ca2 + STORES
Figure 8
gating
Working model of the effect of luminal [Ca2 + ] and pH on TPC
Schematic diagram depicting the time course of Ca2 + release via TPC. An activating
concentration of NAADP is added at the dotted line, and the luminal [Ca2 + ] ([Ca2 + ]L ), pHL
and open probability (po ) is shown in the middle traces. The model is derived from the data
with HsTPC2 [220]. NAADP binding is reversible at acidic pHL , but irreversible at alkaline pHL
(upper trace). Stores replete with [Ca2 + ]L have a greater sensitivity to NAADP than empty ones
(indicated by the density of the gradient bar). Lower cartoons represent a single acidic vesicle
with a TPC channel. The intensity and colour of the lumen represents vesicle alkalinization by
NAADP, as seen in sea urchin egg stores (red is acidic, green is relatively alkaline), Ca2 + ions
are shown by the yellow spheres.
mammalian cell NAADP-induced Ca2 + release [204]. In addition,
pH appears to regulate the reversibility of NAADP effects on
channel activity. Alkalinization of the lumen, as shown to be
induced by NAADP, would promote an increase in channel Po ,
but also the irreversibility of NAADP effects on the channel.
This latter effect could conceivably be related to the profound
desensitization properties of NAADP-sensitive mechanisms
widely observed. Closure of channels would allow both the reacidification, which would promote NAADP dissociation ready
for a future round of activation by NAADP, and refilling of Ca2 +
stores would sensitize the channel to another round of NAADP
activation (Figure 8). Finally, validation of TPC2 as a target for
the selective inhibitor of NAADP-evoked Ca2 + release Ned-19,
was achieved by showing that it blocked the channels and their
activation by NAADP, although interesting at low concentrations
it may activate the channels [220]. This study highlighted the
complex interactions between cytoplasmic NAADP, and pHL and
luminal Ca2 + concentrations in dictating the activity of these
channels and presumably Ca2 + release from lysosomes.
In a third approach, TPC2 was mutated and the N-terminus
deleted resulting in its mistargeting to the plasma membrane,
where it is more amenable to study by patch-clamp electrode
techniques (Figure 9) [125]. Although this approach may raise a
few concerns about changes in the channel structure or non-native
location, it does provide elegant support for the trigger hypothesis
(Figure 5). In this setup, the plasma membrane form is uncoupled
from the CICR via ER Ca2 + channels and it conducts currents
across the plasma membrane that are appropriately sensitive to
NAADP and Ned-19 [125]. Single-channel currents (Cs + as a
carrier) were ∼ 130 pS and, again, mutations in putative poreforming loops abolished this current [125].
Although there had been sporadic reports of agonist-evoked Ca2 +
release from acidic stores, it has been NAADP that has galvanized
this field over the last decade. The identification of acidic Ca2 +
stores and TPCs as NAADP targets has provided chemical and
molecular tools by which to study the physiological roles of Ca2 +
release from the endolysosomal system. This new component of
the Ca2 + toolkit provides new ways of controlling how and where
Ca2 + signals are generated.
Agonist-mediated NAADP signalling
A growing number of receptors have now been implicated as
coupled to NAADP-mediated Ca2 + signalling [4]. Until now, four
approaches of various directness have been employed to implicate
NAADP in receptor-mediated Ca2 + -signalling pathways. These
include inhibition of agonist-evoked Ca2 + signals by selfinactivation of NAADP [204], use of the selective membranepermeant NAADP antagonist Ned-19 [253,254], measurement of
cellular NAADP levels following agonist stimulation [255] and
more indirectly by ablation of Ca2 + storage by acidic stores with,
for example, GPN (glycyl-L- phenylalanine-naphthylamide) or
bafilomycin. The pathways leading to NAADP synthesis need
clarification, but several reports in different cell types have
implicated CD38 [118,214,256,257], an ADP-ribosyl cyclase
enzyme shown previously to catalyse the synthesis of NAADP
from NADP in vitro.
The characteristic ‘Ca2 + signature’ response evoked by a given
cell-surface receptor may, in part, be the product of ‘mixing and
matching’ the three Ca2 + -mobilizing messengers and cognate
Ca2 + stores [114,258]. This then allows Ca2 + signals to be
fine-tuned and in turn specify how the cell will respond. Thus
receptors previously ascribed to couple to IP3 production may
also link to NAADP-dependent pathways [254]. In view of the
trigger hypothesis, NAADP may act to provide co-agonist Ca2 +
at the juxta-organellar junctions to sensitize IP3 Rs when cellular
IP3 levels are also elevated. Similar arguments can be advanced
for NAADP/cADPR combinations which would recruit RyRs,
as found for endothelin-1-evoked Ca2 + signals in rat pulmonary
arteriolar smooth muscle cells [117].
Alteration in Ca2 + release from lysosomes may be important
in several lysosomal disorders as discussed below, but also in
other pathological processes, which may result in alterations
in agonist-mediated Ca2 + signalling. Enlargement of lysosomes
by treatment with cathepsin inhibitors, modulates the responses
c The Authors Journal compilation c 2011 Biochemical Society
364
Figure 9
A. J. Morgan and others
Methods for recording TPC channel activity
TPC channels are depicted in yellow and current flow is indicated by yellow arrows. Enriched TPCs can be monitored in lipid bilayers and monitored at the single-channel level (Bilayer). Single
purified lysosomes can be patched using chip-based technology (Organelle Patching). TPCs inserted into the plasma membrane by point mutation (or exocytosis?) can be recorded via conventional
microelectrode electrophysiology (PM targeting and whole cell patch).
to both NAADP and glutamate, and such aberrant Ca2 + signalling
may be of relevance in the pathophysiology of neurodegeneration
[112]. In an accumulating body of work examining aberrant
Ca2 + signalling in pancreatic acinar cells linked to premature
intracellular trypsin activation as occurs in acute pancreatitis,
Petersen et al. [259] have found that bile acids, and alcohol
and metabolites may disproportionately evoke Ca2 + release from
acidic stores over the ER.
Regulation of plasma membrane excitability by NAADP
It has long been known that Ca2 + release from intracellular
stores by NAADP may modulate plasma membrane channels
and membrane excitability. NAADP was found to depolarize
the plasma membrane of invertebrate eggs and evoke Ca2 +
influx [260,261], and may be important in mediating the fast
block to polyspermy at fertilization, a property not shared by
either IP3 or cADPR [261]. NAADP activates membrane Ca2 + activated currents in pancreatic acinar cells [204], important for
fluid secretion, whereas in excitable cells it can trigger changes
in membrane potential [115,127]. In many cases, these effects
are secondary to Ca2 + release from acidic stores which may or
may not involve amplification by ER Ca2 + -release mechanisms,
although direct action at plasma membrane channels has also been
proposed [262]. Importantly, it was found that NAADP activates
plasma membrane currents in mouse pancreatic β cells, effects
that are abolished in cells from TPC2-knockout mice [127]. Thus
Ca2 + release from acidic stores proximal to the plasma membrane
may be important determinants of plasma membrane excitability,
and since such TPC-expressing organelles are dynamic [127],
they can be targeted to different subcellular sites to initiate various
Ca2 + -dependent cellular responses.
Endolysosomal trafficking
The localization of TPCs to the endolysosomal system, and in
particular TPC2 to lysosomes, has prompted an investigation of
c The Authors Journal compilation c 2011 Biochemical Society
the possible role for these channels in endolysosomal trafficking
and lysosome biogenesis. Ca2 + release and luminal Ca2 + in
endolysosomal vesicles has been proposed to play a key role
in vesicular fusion mechanisms in the endolysosomal system
[6,263–265]. TPCs thus are an attractive mediator for local Ca2 +
signals regulating both luminal and juxta-vesicular Ca2 + levels in
the endolysosomal system. Indeed, overexpression of TPC1 and
TPC2 cause enlarged lysosomal structures to form, consistent with
enhanced endolysosomal Ca2 + release promoting fusion, and also
changing endocytosis and trafficking of material from the plasma
membrane [128]. Importantly, these effects were rescued by the
NAADP antagonist Ned-19 fully implicating the NAADP/TPC
axis in these processes [128]. NAADP-mediated Ca2 + signalling
may be affected in pathological processes affecting lysosomes,
the most prominent being lysosomal storage diseases. These are
discussed below.
TPC-dependent Ca2 + signalling
With the identification of TPCs as targets for NAADP, cells
from TPC-knockout mice and treatment with TPC siRNAs, have
offered a new approach to identify stimuli and physiological
processes coupled to NAADP/TPCs.
In mouse detrusor smooth muscle, muscarinic receptor
activation induces contraction by releasing Ca2 + from both
acidic and SR stores [266]. In permeabilized detrusor muscle
strips, NAADP evoked contraction with the characteristic
bell-shaped curve, but had no effect in muscle derived
from TPC2-knockout mice [266]. Consequently, muscarinic
receptor activation contracted muscle from TPC2-knockout mice
exclusively through the SR and confirms acetylcholine signals
through NAADP/TPCs [266].
In a recent study, histamine-evoked Ca2 + release via the H1
receptor in human endothelial cells was suggested to be linked to
NAADP signalling by pharmacological studies, and accordingly,
the secretion of von Willebrand’s Factor was significantly reduced
by siRNA constructs for both TPC1 and TPC2 [267].
Endolysosomal Ca2 + signalling in health and disease
With the realization that there are multiple messengers for Ca2 +
mobilization, an obvious question is whether activation of distinct
mechanisms control specific cellular responses. One fundamental
cellular process regulated by Ca2 + , cell differentiation, has
been selectively linked to Ca2 + release by NAADP from acidic
stores. In skeletal muscle, differentiation was affected by Ned-19,
bafilomycin or siRNA to TPC2 (but was less sensitive to blocking
IP3 Rs or RyRs) [268]. Similarly, liposomal delivery of NAADP
(but not IP3 or cADPR) induced neurite extension [269]. These
studies underscore the role of Ca2 + release from acidic stores to
mediate specific cellular responses that cannot be substituted by
ER-mediated Ca2 + release.
Finally, in a genome-wide association study it was
reported that SNPs (single nucleotide polymorphisms) in
the hsTPC2 gene were associated with pigmentation [270].
Since melanosomes are lysosome-related organelles, changes
in the ion-transport processes across the membrane of these
melanin-storing organelles might impair pigment storage
giving rise to associations with particular hair and skin
pigmentation.
Why different TPCs?
Differential distribution
Obvious questions are why are there different TPCs and in what
ways are they similar and different? First, the primary sequences
of the three isoforms are quite different from one another (within
a given species only ∼ 20 % identical, ∼ 35 % similar), but
each orthologue is quite well conserved across species (40–
90 % identical, 55–93 % similar) [123,127,128]. This hints at
specific conserved functions for each isoform. Accordingly, the
subcellular distribution throughout the endolysosomal system is
exquisitely isoform specific (Figure 7), with TPC1 especially
exhibiting a far broader, more nebulous, distribution in the
endosomal compartments, whereas TPC2 is restricted to late
endosomes/lysosomes (Figure 7). The paucity of reliable
antibodies means that the subcellular localization of endogenous
TPCs has only occasionally been investigated with TPC1 found
apically in rat kidney tubules [241] and TPC2 in lysosomes
in HEK-293 cells [127], whereas TPC3 is localized in cortical
puncta in sea urchin eggs (agreeing with heterologous expression
of SpTPC1–3 in echinoderm oocytes) [128].
As to tissue distribution, Northern blot analyses revealed that
both TPC1 [241] and TPC2 [127] appear to be expressed widely,
the corollary being that multiple TPCs may be co-expressed
within individual cells [123,128,266]. Although early days, it
seems that TPC1 dominates TPC2 expression, at least in human
endothelial cells (real-time PCR analysis revealed a transcript
ratio of 9:1 [267]), skeletal muscle [268], SKBR3 and PC12
cells, and sea urchin eggs [123]. To add to the complexity, their
weakly overlapping subcellular distribution may well allow some
heterodimerization in selected subcompartments (compare with
TPC2 homodimerization [144]). This could potentially generate
an even broader spectrum of TPC assemblies and this crucially
awaits experimental clarification.
Properties of TPC isoforms
Despite their divergence, the NAADP-binding properties of
individual TPCs seem well conserved, at least for endogenous
SpTPC1 and SpTPC3 immunoprecipitates [128] and for
heterologously expressed HsTPC2 [127]. Translating this into
Ca2 + release, there are manifest differences in the pattern
365
mediated by TPC isoforms. Compared with TPC2, TPC1 is a
less-efficient ‘trigger’ and couples weakly to ER Ca2 + release in
some cell types. Activation of heterologously expressed human
TPC1 in HEK-293 cells evoked only a local non-propagating
Ca2 + response [127] and similarly, the sea urchin orthologue
SpTPC1 recruited IP3 Rs, but was delayed ∼ 100 s compared with
SpTPC2 [128]. A working hypothesis is that endosomal TPC1 is
not so closely apposed to the ER as is the late endosome/lysosome
TPC2 and therefore the kinetics of Ca2 + accumulation at the juxtaorganellar interface is suboptimal with TPC1.
In contrast, expression of HsTPC1 [123] or SpTPCs [124] in
a breast cancer cell line did not reveal any obvious ER-coupling
deficiencies when microinjected with NAADP and all gave robust
Ca2 + responses. Why this seems to differ with the other studies
is unclear, but may be due to experimental differences. SKBR3
cells may inherently couple more efficiently (due to a different cell
architecture or different amplifiers, RyRs in this case) or the rapid
NAADP delivery by injection might be too fast to reveal coupling
delays (the previous studies used slower cytosolic dialysis).
The story for SpTPC3, however, is less clear, with reports of
overexpression of this isoform inhibiting [128] or potentiating
[124] NAADP-evoked Ca2 + signals. How can there be such
differences and how could TPC3 be inhibiting at all? The differences may not be so surprising when considering that the sea
urchin genome is highly prone to polymorphisms [128] and so
the respective sequences need to be scrutinized for potentially
informative mutations that alter function. How some TPC3
sequences act ostensibly as ‘dominant–negatives’ (with either
exogenous or endogenous TPC1/2) is less clear. If pHL is not
affected by TPC3 [128], then it is possible that TPC3/TPC2
heterodimerize to form dysfunctional channels (e.g. if SpTPC3
mediated the characteristic self-inactivation of sea urchin egg
NAADP receptors at sub-threshold [NAADP]). Further work is
required to examine the relevance of these observations to other
TPC3 orthologues and polymorphisms.
TPC isoform physiology
Finally, what physiological processes require TPCs? All three
sea urchin egg isoforms are cortical in eggs/oocytes [128]
and, circumstantially, in the right place to deliver Ca2 + for
exocytosis of the fertilization envelope and for the pHL ASH (a
term coined to describe the selective alkalinization of cortical
acidic vesicles) [213]. In mammalian cells, although TPC2 may
be the minority isoform, its abundance belies its importance,
and knockout of TPC2 in the mouse model abolishes NAADPinduced responses such as Ca2 + -dependent plasma membrane
currents in mouse pancreatic β cells [127] and bladder muscle
contraction [266]. Similarly, in studies of skeletal muscle
differentiation, TPC2 siRNA was more effective than TPC1
siRNA in inhibiting differentiation of myoblasts [268]. Although
of unknown physiological significance, heterologously expressed
TPC2 forms complex with TRPML1 (and TRPML3 to a lesser
extent), whereas TPC1 does not [232].
However, TPC1 shRNA profoundly reduced NAADP-induced
Ca2 + release in SKBR3 cells, in spite of the additional presence
of TPC2 [123]. Moreover, overexpression of SpTPC1 or SpTPC2
had similar effects upon endolysosomal trafficking [128].
On balance, the pre-eminence of TPC2 thus far might simply
be a function of coincidence (the few systems studied are more
TPC2-dependent), a global disruption of heterodimerization, or
a real possibility that TPC1 is less crucial (or efficient) for Ca2 +
release. Only more studies will illuminate this most important of
issues.
c The Authors Journal compilation c 2011 Biochemical Society
366
A. J. Morgan and others
ACIDIC Ca2 + STORE PATHOLOGY
Potential role in infection?
Many intracellular pathogens enter macrophages through
phagocytosis and need to avoid fusion with lysosomes if they
are to survive and replicate. Although the intracellular habitat
is potentially hostile (the acidic and hydrolase-rich lysosomes
will rapidly degrade bacteria) significant survival advantages are
associated with the intracellular environment, including reduced
exposure to immune surveillance and a rich supply of nutrients
provided by the host cell.
We now know of many human pathogens that have evolved
mechanisms to manipulate the endocytic pathway to their
advantage, including escaping from the phagosome into the cytosol (e.g. Listeria and Shigella species), manipulating the
phagosome membrane to hide the nature of the compartment
to prevent fusion with lysosomes (e.g. Legionella) and blocking
phagosome–lysosome fusion (e.g. pathogenic Mycobateria).
These mechanisms have been reviewed recently in [271].
However, in light of our new knowledge about acidic store Ca2 +
regulation, we speculate that micro-organisms may have evolved
mechanisms to subvert these processes for their own ends. This
would potentially prevent phagosome–lysosome fusion and would
appear to be an excellent evolutionary target and a future direction
for research.
Lysosomal storage diseases
Diseases that result from defects in any aspect of lysosomal
homoeostasis are termed lysosomal storage disorders. Impaired
lysosomal function leads to the accumulation (‘storage’) of
undegraded macromolecules in the late endocytic/lysosomal
system [20]. Currently approximately 50 disorders are known and
the majority are inherited as autosomal recessive traits [20]. They
occur at a collective frequency of 1:5000 live births and are the
most frequent cause of neurodegeneration in infants and children
[272]. Recent studies [resulting from the surveillance program
to monitor vCJD (variant Creutzfeldt–Jakob disease) cases] have
also highlighted that lysosomal disorders are a common cause of
progressive intellectual and neurological deterioration in children
in the U.K. [273].
Lysosomal disorders were identified clinically over a century
ago with the biochemical nature of storage elucidated from the
1960s onwards [20]. The advent of molecular biology led to the
identification of the causative gene defects. What has emerged
is that the majority of these disorders result from defects in
lysosomal hydrolase function in line with Hers original prediction
following his seminal studies on the glycogen storage disorder
Pompe disease [19].
However, not all lysosomal disorders result from enzyme
deficiencies and a significant subset are caused by mutations
in lysosomal membrane proteins [274]. We currently know of
two lysosomal storage diseases that involve defective acidic
store Ca2 + regulation [NPC (Niemann–Pick type C) and MLIV]
and interestingly these diseases have opposing defects. NPC is
characterised by reduced NAADP-induced Ca2 + release leading
to impaired late endosome/lysosome fusion, whereas in MLIV
there is increased Ca2 + release leading to enhanced endocytic
pathway fusion. The current status of our understanding of the
mechanism leading to dysregulated acidic store Ca2 + in these
two diseases is discussed below.
NPC
NPC presents as a progressive neurodegenerative disease and
patients typically die in childhood/adolescence [275], although
c The Authors Journal compilation c 2011 Biochemical Society
more chronic forms exist and usually present in young adults.
Cerebellar atrophy and a profound yet selective loss of Purkinje
neurons is characteristic, which leads to ataxia [276]. Additional
symptoms include learning difficulties, psychiatric symptoms,
epilepsy, speech loss, vertical gaze palsy and respiratory
dysfunction [277]. Neurofibrillar tangles similar to those found in
Alzheimer’s disease are present and dementia is a characteristic
symptom, suggesting potential pathophysiological convergence
between these two disorders [278].
NPC disease is one of the most complex lysosomal
disorders with multiple lipid species stored including cholesterol,
sphingomyelin, sphingosine and glycosphingoipids [279]. A clue
to the cellular pathogenesis of this disorder is that it has a unique
late endosome/lysosome fusion defect, suggestive of altered Ca2 +
homoeostasis. The storage bodies in NPC visualized by electron
microscopy have a heterogeneous morphology, presumably
reflecting the diverse range of lipids stored (Figure 10). This is
in contrast with the much more homogeneous inclusion bodies
observed in diseases such as Sandhoff disease where a single
major ganglioside species is stored as the result of a lysosomal
hydrolase defect (β-hexosaminidase; Figure 10).
NPC is unusual in that it is caused by mutations in either of two
independent genes termed NPC1 and NPC2 [280], responsible for
95 % and 5 % of clinical cases respectively [275]. Although the
NPC1 and NPC2 genes were cloned over a decade ago, the precise
functions of the proteins they encode and how they interact still
remains controversial [279].
NPC1 is a 13TMD protein of the limiting membrane of late
endosomes/lysosomes, whereas NPC2 is a soluble cholesterolbinding protein (previously known as HE1 due to its high levels
in human epididymal fluid) [275]. There are currently two main
theories in the field as to the function of the NPC pathway: (i)
that it transports cholesterol out of late endosomes/lysosomes
[281,282] or (ii) it transports non-sterol cargo(s) out of the acidic
compartment and is potentially regulated by cholesterol [2,283–
285].
In support of the second hypothesis, we identified NPC1 as a
candidate lysosomal sphingosine transporter [2]. When NPC1 is
inactivated the first metabolite to accumulate is sphingosine [2]
and exogenous addition of sphingosine to healthy cells induces
NPC cellular phenotypes [2]. Sphingosine storage also has a
profound effect on cells as it causes a dramatic reduction in
luminal acidic store Ca2 + content [2]. Indeed, the Ca2 + levels
in the acidic compartment are reduced to 30 % of the wild-type,
which translates into reduced Ca2 + release in response to NAADP
[2] (Figure 10). A drop in the luminal [Ca2 + ] not only reduces the
electrochemical driving force for Ca2 + release, but also may lower
the NAADP sensitivity of TPCs [220]. This defect in luminal Ca2 +
content in NPC was not due to altered acidic store pH, but due to
impaired store filling [2].
It is currently not known if sphingosine storage directly or
indirectly affects the enigmatic protein(s) involved in acidic store
Ca2 + filling, but the central role for defective lysosomal Ca2 +
regulation in the pathogenesis of this disease came from two lines
of experimental evidence. First, NPC disease phenotypes could
be induced in healthy cells by chelating luminal lysosomal Ca2 + ,
and secondly, NPC cells could be corrected by elevating cytosolic
Ca2 + using the weak SERCA inhibitor curcumin to compensate
for lack of adequate Ca2 + release from acidic stores [2]. Indeed,
curcumin treatment of a mouse model of NPC1 also resulted in
clinical benefit suggesting that mild elevation of cytosolic Ca2 +
may be a novel therapeutic approach to treat NPC disease in
patients [2]. We believe that the low luminal lysosomal Ca2 +
levels in NPC disease explains the block in late endosome–
lysosome fusion in this disorder. Fusion and vesicular release
Endolysosomal Ca2 + signalling in health and disease
Figure 10
367
Comparative morphology of storage bodies in diseases with and without aberrant endolysosomal Ca2 +
Schematic diagram conveying endolysosomal Ca2 + release and filling in each disease. The upward arrow indicates an increase, the downward arrow a decrease and a question mark signifies
currently unknown. Electron micrographs (A–F). (A and B) Cytoplasmic storage bodies in CNS (central nervous system) neurons from the mouse model of Sandhoff disease (GM2 gangliosidosis)
at low and high magnification. The storage in these mice is dominated by GM2 and GA2 ganglioside and results in relatively uniform storage body morphology with concentric circular membrane
inclusions (very similar to those seen in Tay–Sachs disease) or sheets of stacked membranes (termed zebra bodies). This disease has normal lysosomal Ca2 + regulation but has an ER Ca2 + defect
due to reduced SERCA activity [306]. By contrast the fusion and trafficking defects in the endocytic pathway in NPC (C and D) resulting from reduced acidic store luminal Ca2 + and Ca2 + release
leading to highly heterogeneous storage bodies in patient-derived fibroblasts, that range from empty vacuoles to lipid-rich membranous cytoplasmic bodies with diverse morphology. A similar
situation is seen in MLIV patient fibroblasts (E and F) where the opposite Ca2 + defect (enhanced Ca2 + release) results in inappropriate fusion leading to multiple mixed morphology storage bodies.
Although both NPC and MLIV exhibit complex storage bodies they differ morphologically from one another. Scale bar, 0.5 μm.
is a Ca2 + -dependent process [265] and the Ca2 + that facilitates
these processes is derived from the lysosomal compartment itself
[2]. The failure to release sufficient Ca2 + in this disease therefore
leads to a block in the normal trafficking and fusion essential
for the correct functioning of the endosomal/lysosomal system,
causing the secondary storage of cholesterol, glycosphingolipids
and sphingomyelin [2].
MLIV
Just as NPC is a disease that involves an endolysosomal Ca2 +
defect so too does MLIV, but via a different mechanism with
very different consequences. MLIV is a rare neurodegenerative
lysosomal storage disease caused by mutations in the Mcoln1
gene encoding TRPML1 [223,286–288]. MLIV patients have
progressive early developmental decline, increased blood gastrin
levels and neuronal loss followed by a period of stabilization that
lasts several decades [289].
TRPML1 was recently uncovered as a major gene product
involved in a network of lysosomal genes believed to regulate
lysosomal generation and function [290] and MLIV cells accumulate a unique combination of sphingolipids, phospholipids,
glycosaminoglycans and autofluorescent lipofuscin within the
endolysosomal system [291–294]. This storage profile probably
reflects alterations in endocytosis and lipid trafficking [225,295].
It has been suggested that retarded recycling of lipids out of the
endocytic system results from either enhanced late endosome–
lysosome fusion or defective fission [225,295–297]. Since ionic
changes (pH and Ca2 + ) are known to modulate fission and fusion,
are either affected in MLIV? It is germane that TRPML1 is a
lysosomal transmembrane protein that is an inwardly rectifying
channel permeable to multiple ions including Ca2 + , Fe2 + , Na + ,
K + and H + [229,298].
Several groups have monitored lysosomal pH in MLIV cells,
but there is no consensus as to whether there is a decrease, increase
or no change when compared with normal cells [225,299,300].
However, lysosomal enzyme activity in situ is unaltered [299]
suggesting no major alteration in pH. Further evidence against
a change in pH in MLIV lysosomes is that treatment of MLIV
cells with low concentrations of bafilomycin does not improve
lysosomal storage in MLIV fibroblasts [301].
One possible explanation for these disparate pH measurements
is that they are measuring pH in different organelles. In control
cells a hybrid compartment [265] is transient and undetectable
using these pH measurement techniques so the reading reflects
true lysosomes. However, in MLIV cells the hybrid organelle
persists and its higher pH skews the ‘lysosomal’ pH values
[6,222]. As a consequence it is not possible to conclude whether
pH alterations play a central role in MLIV. Because of the
homology between TRPML1 and other TRP family members
[223], an alternative hypothesis to altered pH is altered Ca2 +
homoeostasis and enhanced fusion would be consistent with
enhanced Ca2 + release from acidic stores [227,228,264,302,
303].
As discussed above TRPML1 is unlikely to be an NAADP
receptor. Indeed, NAADP-mediated Ca2 + release is not reduced
in TRPML1-null MLIV patient fibroblasts, but is in fact elevated
thereby potentially explaining the aberrant fusion observed in
MLIV (K. Peterneva, K. Rietdorf, A. M. Lewis, A. Galione,
G. C. Churchill, F. M. Platt and E. Lloyd-Evans, unpublished
work; Figure 10). Indeed, in terms of both Ca2 + homoeostasis
and defective endocytic pathway fusion, MLIV is the opposite
of NPC. The exact function of TRPML1 remains unknown,
but recent studies have highlighted some interesting modulators
of its function, including PIP2 and the Ca2 + -sensitive ALG-2
(apoptosis-linked gene-2) [233,304].
c The Authors Journal compilation c 2011 Biochemical Society
368
A. J. Morgan and others
It may be highly relevant that a related mucolipin family
member (TRPML3) was recently suggested to function as an
early endocytic Ca2 + -leak channel modulated by pH [305];
interestingly, TRPML3 impairment likewise resulted in enhanced
luminal Ca2 + levels and enhanced endosomal fusion [305].
Similarly, TRPML1 may also be a pH-modulated Ca2 + channel
[303], so whether it functions in a similar manner to TRPML3, but
at a later stage in the endocytic pathway, remains to be determined.
CONCLUSIONS
The property of high endolysosomal [H + ] important for the
well-established degradative function has been known for
more than a century, but endolysosomal cation and anion
channels/transporters have only emerged more recently as
fundamental regulators of organelle physiology. There is now
overwhelming evidence that the endolysosomal system also has
an appreciable Ca2 + storage capacity, with a reported luminal
Ca2 + concentration not dissimilar from that of the ER in the
case of lysosomes. Consequently, altered ion transport defects are
emerging as major contributors to certain pathologies within the
lysosomal storage disease family.
In the present review we have focused on mechanisms
of Ca2 + uptake and release from endolysosomal stores, and
in particular the NAADP/TPC pathway as a mediator of a
plethora of physiological signals including fertilization, secretion,
membrane trafficking, contractility and differentiation. Moreover,
the properties of TPCs may make them unique integrators of
converging signals from NAADP, luminal Ca2 + and pHL . This
convergence may then disseminate signals either locally (e.g.
endolysosomal trafficking) or globally (e.g. plasma membrane
excitability or regenerative Ca2 + signals by organellar cross-talk).
However, key questions still remain. Although NAADP is
now established as a Ca2 + -mobilizing messenger from the
endolysosomal system, we still do not know if there is a broad
sensitivity to this messenger or whether there is a subset of
organelles that are specialized in mediating NAADP-evoked Ca2 +
release. We know little about the molecular mechanism mediating
Ca2 + uptake and storage, and the study of the different TPC
isoforms is still in its infancy, but already hints at distinct roles.
More fundamentally, it is still unclear whether NAADP binds
directly to TPC proteins themselves or to accessory proteins
within the complex and we still do not understand the mechanism
of NAADP self-inactivation of the channel. Finally, aberrant
NAADP/TPC signalling and endolysosomal Ca2 + homoeostasis
may not only illuminate pathology, but also give clues as to which
cellular processes are under the control of endolysosomal Ca2 +
to offer unique drug targets.
ACKNOWLEDGEMENTS
We thank Professor Sergio Grinstein (University of Toronto, Toronto, Canada),
Dr Jon Pittman (University of Manchester, Manchester, U.K.), and Professor George
Ratcliffe and Dr Margarida Ruas (University of Oxford, Oxford, U.K.) for helpful comments.
We also thank Wendy Tynan for preparing the micrographs.
REFERENCES
1 Christensen, K. A., Myers, J. T. and Swanson, J. A. (2002) pH-dependent regulation of
lysosomal calcium in macrophages. J. Cell Sci. 115, 599–607
2 Lloyd-Evans, E., Morgan, A. J., He, X., Smith, D. A., Elliot-Smith, E., Sillence, D. J.,
Churchill, G. C., Schuchman, E. H., Galione, A. and Platt, F. M. (2008) Niemann-Pick
disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal
calcium. Nat. Med. 14, 1247–1255
c The Authors Journal compilation c 2011 Biochemical Society
3 Galione, A., Evans, A. M., Ma, J., Parrington, J., Arredouani, A., Cheng, X. and Zhu,
M. X. (2009) The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated
endolysosomal Ca2 + release channels. Pflügers Arch. 458, 869–876
4 Galione, A., Morgan, A. J., Arredouani, A., Davis, L. C., Rietdorf, K., Ruas, M. and
Parrington, J. (2010) NAADP as an intracellular messenger regulating lysosomal
calcium-release channels. Biochem. Soc. Trans. 38, 1424–1431
5 Patel, S. and Docampo, R. (2010) Acidic calcium stores open for business: expanding
the potential for intracellular Ca2 + signaling. Trends Cell Biol. 20, 277–286
6 Lloyd-Evans, E., Waller-Evans, H., Peterneva, K. and Platt, F. M. (2010) Endolysosomal
calcium regulation and disease. Biochem. Soc. Trans. 38, 1458–1464
7 Morgan, A. J. (2011) Sea urchin eggs in the acid reign. Cell Calcium 50, 147–156
8 Scott, C. C. and Gruenberg, J. (2011) Ion flux and the function of endosomes and
lysosomes: pH is just the start. BioEssays 33, 103–110
9 Guse, A. H. (2009) Second messenger signaling: multiple receptors for NAADP. Curr.
Biol. 19, R521–R523
10 Churchill, G. C., Okada, Y., Thomas, J. M., Genazzani, A. A., Patel, S. and Galione, A.
(2002) NAADP mobilizes Ca2 + from reserve granules, a lysosome-related organelle, in
sea urchin eggs. Cell 111, 703–708
11 Saftig, P. and Klumperman, J. (2009) Lysosome biogenesis and lysosomal membrane
proteins: trafficking meets function. Nat. Rev. Mol. Cell Biol. 10, 623–635
12 Tauber, A. I. (2003) Metchnikoff and the phagocytosis theory. Nat. Rev. Mol. Cell Biol. 4,
897–901
13 Metchnikoff, E. (1907) Immunity to Infective Disease. Cambridge University Press,
Cambridge
14 De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955)
Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver
tissue. Biochem. J. 60, 604–617
15 De Duve, C. and Wattiaux, R. (1966) Functions of lysosomes. Annu. Rev. Physiol. 28,
435–492
16 Baudhuin, P., Beaufay, H. and De Duve, C. (1965) Combined biochemical and
morphological study of particulate fractions from rat liver. Analysis of preparations
enriched in lysosomes or in particles containing urate oxidase, D-amino acid oxidase,
and catalase. J. Cell Biol. 26, 219–243
17 De Duve, C. (1963) The lysosome. Sci. Am. 208, 64–72
18 Maxfield, F. R. and Mukherjee, S. (2004) The endosomal-lysosomal system. In
Lysosomal Disorders of the Brain (Platt, F. M. and Walkley, S. U., eds), pp. 3–31, Oxford
University Press, Oxford
19 Hers, H. G. (1963) α-Glucosidase deficiency in generalized glycogen storage disease
(Pompe’s disease). Biochem. J. 86, 11–16
20 Platt, F. M. and Walkley, S. U. (2004) Lysosomal defects and storage. In Lysosomal
Disorders of the Brain (Platt, F. M. and Walkley, S. U., eds), pp. 32–49, Oxford
University Press, Oxford
21 Dell’Angelica, E. C., Mullins, C., Caplan, S. and Bonifacino, J. S. (2000)
Lysosome-related organelles. FASEB J. 14, 1265–1278
22 Raposo, G., Fevrier, B., Stoorvogel, W. and Marks, M. S. (2002) Lysosome-related
organelles: a view from immunity and pigmentation. Cell Struct. Funct. 27, 443–456
23 Griffiths, G. (2002) What’s special about secretory lysosomes? Semin. Cell Dev. Biol.
13, 279–284
24 Reddy, A., Caler, E. V. and Andrews, N. W. (2001) Plasma membrane repair is mediated
by Ca2 + -regulated exocytosis of lysosomes. Cell 106, 157–169
25 Yogalingam, G., Bonten, E. J., van de Vlekkert, D., Hu, H., Moshiach, S., Connell, S. A.
and d’Azzo, A. (2008) Neuraminidase 1 is a negative regulator of lysosomal exocytosis.
Dev. Cell 15, 74–86
26 Kornfeld, S. (1987) Trafficking of lysosomal enzymes. FASEB J. 1, 462–468
27 Braulke, T. and Bonifacino, J. S. (2009) Sorting of lysosomal proteins. Biochim.
Biophys. Acta 1793, 605–614
28 Reczek, D., Schwake, M., Schroder, J., Hughes, H., Blanz, J., Jin, X., Brondyk, W., Van
Patten, S., Edmunds, T. and Saftig, P. (2007) LIMP-2 is a receptor for lysosomal
mannose-6-phosphate-independent targeting of β-glucocerebrosidase. Cell 131,
770–783
29 Kneen, M., Farinas, J., Li, Y. and Verkman, A. S. (1998) Green fluorescent protein as a
noninvasive intracellular pH indicator. Biophys. J. 74, 1591–1599
30 Foyouzi-Youssefi, R., Arnaudeau, S., Borner, C., Kelley, W. L., Tschopp, J., Lew, D. P.,
Demaurex, N. and Krause, K. H. (2000) Bcl-2 decreases the free Ca2 + concentration
within the endoplasmic reticulum. Proc. Natl. Acad. Sci. U.S.A. 97, 5723–5728
31 Kim, J. H., Johannes, L., Goud, B., Antony, C., Lingwood, C. A., Daneman, R. and
Grinstein, S. (1998) Noninvasive measurement of the pH of the endoplasmic reticulum
at rest and during calcium release. Proc. Natl. Acad. Sci. U.S.A. 95, 2997–3002
32 Grabe, M. and Oster, G. (2001) Regulation of organelle acidity. J. Gen. Physiol. 117,
329–344
33 Demaurex, N. (2002) pH homeostasis of cellular organelles. News Physiol. Sci. 17, 1–5
Endolysosomal Ca2 + signalling in health and disease
34 Sonawane, N. D., Thiagarajah, J. R. and Verkman, A. S. (2002) Chloride concentration in
endosomes measured using a ratioable fluorescent Cl − indicator: evidence for chloride
accumulation during acidification. J. Biol. Chem. 277, 5506–5513
35 Steinberg, B. E., Huynh, K. K., Brodovitch, A., Jabs, S., Stauber, T., Jentsch, T. J. and
Grinstein, S. (2010) A cation counterflux supports lysosomal acidification. J. Cell Biol.
189, 1171–1186
36 Rybak, S. L., Lanni, F. and Murphy, R. F. (1997) Theoretical considerations on the role of
membrane potential in the regulation of endosomal pH. Biophys. J. 73, 674–687
37 Forgac, M. (2007) Vacuolar ATPases: rotary proton pumps in physiology and
pathophysiology. Nat. Rev. Mol. Cell Biol. 8, 917–929
38 Cipriano, D. J., Wang, Y., Bond, S., Hinton, A., Jefferies, K. C., Qi, J. and Forgac, M.
(2008) Structure and regulation of the vacuolar ATPases. Biochim. Biophys. Acta 1777,
599–604
39 Jefferies, K. C., Cipriano, D. J. and Forgac, M. (2008) Function, structure and regulation
of the vacuolar H + -ATPases. Arch. Biochem. Biophys. 476, 33–42
40 Marshansky, V. and Futai, M. (2008) The V-type H + -ATPase in vesicular trafficking:
targeting, regulation and function. Curr. Opin. Cell Biol. 20, 415–426
41 Kettner, C., Bertl, A., Obermeyer, G., Slayman, C. and Bihler, H. (2003)
Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio
and proton shunt. Biophys. J. 85, 3730–3738
42 Toei, M., Saum, R. and Forgac, M. (2010) Regulation and isoform function of the
V-ATPases. Biochemistry 49, 4715–4723
43 Dechant, R., Binda, M., Lee, S. S., Pelet, S., Winderickx, J. and Peter, M. (2010)
Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through
V-ATPase. EMBO J. 29, 2515–2526
44 Hurtado-Lorenzo, A., Skinner, M., El Annan, J., Futai, M., Sun-Wada, G. H., Bourgoin,
S., Casanova, J., Wildeman, A., Bechoua, S., Ausiello, D. A. et al. (2006) V-ATPase
interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative
pathway. Nat. Cell Biol. 8, 124–136
45 Diakov, T. T. and Kane, P. M. (2010) Regulation of vacuolar proton-translocating ATPase
activity and assembly by extracellular pH. J. Biol. Chem. 285, 23771–23778
46 Alzamora, R., Thali, R. F., Gong, F., Smolak, C., Li, H., Baty, C. J., Bertrand, C. A., Auchli,
Y., Brunisholz, R. A., Neumann, D. et al. (2010) PKA regulates vacuolar H + -ATPase
localization and activity via direct phosphorylation of the a subunit in kidney cells.
J. Biol. Chem. 285, 24676–24685
47 Nanda, A., Gukovskaya, A., Tseng, J. and Grinstein, S. (1992) Activation of vacuolar-type
proton pumps by protein kinase C. Role in neutrophil pH regulation. J. Biol. Chem. 267,
22740–22746
48 Silva, P. and Geros, H. (2009) Regulation by salt of vacuolar H + -ATPase and
H- pyrophosphatase activities and Na + /H + exchange. Plant Signaling Behav. 4,
718–726
49 Huss, M. and Wieczorek, H. (2009) Inhibitors of V-ATPases: old and new players. J. Exp.
Biol. 212, 341–346
50 Bowman, E. J., Siebers, A. and Altendorf, K. (1988) Bafilomycins: a class of inhibitors of
membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl.
Acad. Sci. U.S.A. 85, 7972–7976
51 Teplova, V. V., Tonshin, A. A., Grigoriev, P. A., Saris, N. E. and Salkinoja-Salonen, M. S.
(2007) Bafilomycin A1 is a potassium ionophore that impairs mitochondrial functions.
J. Bioenerg. Biomembr. 39, 321–329
52 Baxter, I., Tchieu, J., Sussman, M. R., Boutry, M., Palmgren, M. G., Gribskov, M., Harper,
J. F. and Axelsen, K. B. (2003) Genomic comparison of P-type ATPase ion pumps in
Arabidopsis and rice. Plant Physiol. 132, 618–628
53 Verweij, W., Spelt, C., Di Sansebastiano, G. P., Vermeer, J., Reale, L., Ferranti, F., Koes,
R. and Quattrocchio, F. (2008) An H + P-ATPase on the tonoplast determines vacuolar
pH and flower colour. Nat. Cell Biol. 10, 1456–1462
54 Serrano, A., Perez-Castineira, J. R., Baltscheffsky, M. and Baltscheffsky, H. (2007)
H + -PPases: yesterday, today and tomorrow. IUBMB Life 59, 76–83
55 Orlowski, J. and Grinstein, S. (2007) Emerging roles of alkali cation/proton exchangers
in organellar homeostasis. Curr. Opin. Cell Biol. 19, 483–492
56 Nakamura, N., Tanaka, S., Teko, Y., Mitsui, K. and Kanazawa, H. (2005) Four Na + /H +
exchanger isoforms are distributed to Golgi and post-Golgi compartments and are
involved in organelle pH regulation. J. Biol. Chem. 280, 1561–1572
57 Rodriguez-Rosales, M. P., Galvez, F. J., Huertas, R., Aranda, M. N., Baghour, M.,
Cagnac, O. and Venema, K. (2009) Plant NHX cation/proton antiporters. Plant Signaling
Behav. 4, 265–276
58 Shigaki, T., Rees, I., Nakhleh, L. and Hirschi, K. D. (2006) Identification of three distinct
phylogenetic groups of CAX cation/proton antiporters. J. Mol. Evolution 63, 815–825
59 Liu, H., Tang, R., Zhang, Y.U.E., Wang, C., Lv, Q., Gao, X., Li, W. and Zhang, H. (2010)
AtNHX3 is a vacuolar K + /H + antiporter required for low-potassium tolerance in
Arabidopsis thaliana . Plant, Cell Environ. 33, 1989–1999
60 Cagnac, O., Aranda-Sicilia, M. N., Leterrier, M., Rodriguez-Rosales, M. P. and Venema,
K. (2010) Vacuolar cation/H + antiporters of Saccharomyces cerevisiae . J. Biol. Chem.
285, 33914–33922
369
61 Rangel-Mata, F., Mendez-Marquez, R., Martinez-Cadena, G., Lopez-Godinez, J.,
Nishigaki, T., Darszon, A. and Garcia-Soto, J. (2007) Rho, Rho-kinase, and the actin
cytoskeleton regulate the Na+ -H + exchanger in sea urchin eggs. Biochem. Biophys.
Res. Commun. 352, 264–269
62 Moulin, P., Guiot, Y., Jonas, J. C., Rahier, J., Devuyst, O. and Henquin, J. C. (2007)
Identification and subcellular localization of the Na + /H + exchanger and a novel related
protein in the endocrine pancreas and adrenal medulla. J. Mol. Endocrinol. 38, 409–422
63 Lawrence, S. P., Bright, N. A., Luzio, J. P. and Bowers, K. (2010) The sodium/proton
exchanger NHE8 regulates late endosomal morphology and function. Mol. Biol. Cell 21,
3540–3551
64 Qiu, Q. S. and Fratti, R. A. (2010) The Na + /H + exchanger Nhx1p regulates the
initiation of Saccharomyces cerevisiae vacuole fusion. J. Cell Sci. 123, 3266–3275
65 Bassil, E., Ohto, M. A., Esumi, T., Tajima, H., Zhu, Z., Cagnac, O., Belmonte, M., Peleg,
Z., Yamaguchi, T. and Blumwald, E. (2011) The Arabidopsis intracellular Na + /H +
antiporters NHX5 and NHX6 are endosome associated and necessary for plant growth
and development. Plant Cell 23, 224–239
66 Jentsch, T. J. (2007) Chloride and the endosomal-lysosomal pathway: emerging roles of
CLC chloride transporters. J. Physiol. 578, 633–640
67 Novarino, G., Weinert, S., Rickheit, G. and Jentsch, T. J. (2010) Endosomal
chloride-proton exchange rather than chloride conductance is crucial for renal
endocytosis. Science 328, 1398–1401
68 Weinert, S., Jabs, S., Supanchart, C., Schweizer, M., Gimber, N., Richter, M., Rademann,
J., Stauber, T., Kornak, U. and Jentsch, T. J. (2010) Lysosomal pathology and
osteopetrosis upon loss of H + -driven lysosomal Cl − accumulation. Science 328,
1401–1403
69 Grabe, M., Wang, H. and Oster, G. (2000) The mechanochemistry of V-ATPase proton
pumps. Biophys. J. 78, 2798–2813
70 Paroutis, P., Touret, N. and Grinstein, S. (2004) The pH of the secretory pathway:
measurement, determinants, and regulation. Physiology 19, 207–215
71 Graves, A. R., Curran, P. K., Smith, C. L. and Mindell, J. A. (2008) The Cl − /H +
antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453,
788–792
72 Haggie, P. M. and Verkman, A. S. (2009) Unimpaired lysosomal acidification in
respiratory epithelial cells in cystic fibrosis. J. Biol. Chem. 284, 7681–7686
73 Edwards, J. C. and Kahl, C. R. (2010) Chloride channels of intracellular membranes.
FEBS Lett. 584, 2102–2111
74 Fuchs, R., Male, P. and Mellman, I. (1989) Acidification and ion permeabilities of highly
purified rat liver endosomes. J. Biol. Chem. 264, 2212–2220
75 Rodrigues, C. O., Scott, D. A. and Docampo, R. (1999) Presence of a vacuolar
H + -pyrophosphatase in promastigotes of Leishmania donovani and its localization to a
different compartment from the vacuolar H + -ATPase. Biochem. J. 340, 759–766
76 Fuchs, R., Schmid, S. and Mellman, I. (1989) A possible role for Na + ,K + -ATPase in
regulating ATP-dependent endosome acidification. Proc. Natl. Acad. Sci. U.S.A. 86,
539–543
77 Feldmann, T., Glukmann, V., Medvenev, E., Shpolansky, U., Galili, D., Lichtstein, D. and
Rosen, H. (2007) Role of endosomal Na + -K + -ATPase and cardiac steroids in the
regulation of endocytosis. Am. J. Physiol. Cell Physiol. 293, C885–C896
78 Bertl, A., Blumwald, E., Coronado, R., Eisenberg, R., Findlay, G., Gradmann, D., Hille, B.,
Kohler, K., Kolb, H. A., MacRobbie, E. et al. (1992) Electrical measurements on
endomembranes. Science 258, 873–874
79 Steinberg, B. E., Touret, N., Vargas-Caballero, M. and Grinstein, S. (2007) In situ
measurement of the electrical potential across the phagosomal membrane using FRET
and its contribution to the proton-motive force. Proc. Natl. Acad. Sci. U.S.A. 104,
9523–9528
80 Schieder, M., Rotzer, K., Bruggemann, A., Biel, M. and Wahl-Schott, C. A. (2010)
Characterization of two-pore channel 2 (TPCN2)-mediated Ca2 + currents in isolated
lysosomes. J. Biol. Chem. 285, 21219–21222
81 Cretin, H. (1982) The proton gradient across the vacuo-lysosomal membrane of lutoids
from the latex of Hevea brasiliensis . I. Further evidence for a proton-ATPase on the
vacuo-lysosomal membrane of intact lutoids. J. Membr. Biol. 65, 175–184
82 Van Dyke, R. W. and Belcher, J. D. (1994) Acidification of three types of liver endocytic
vesicles: similarities and differences. Am. J. Physiol. 266, C81–C94
83 Scherman, D., Nordmann, J. and Henry, J. P. (1982) Existence of an adenosine
5’-triphosphate dependent proton translocase in bovine neurosecretory granule
membrane. Biochemistry 21, 687–694
84 Grinstein, S. and Furuya, W. (1983) The electrochemical H + gradient of platelet
secretory alpha-granules. Contribution of a H + pump and a Donnan potential. J. Biol.
Chem. 258, 7876–7882
85 Lebel, D., Grondin, G. and Paquette, J. (1988) In vitro stability of pancreatic zymogen
granules: roles of pH and calcium. Biol. Cell 63, 343–353
86 Henning, R. (1975) pH gradient across the lysosomal membrane generated by selective
cation permeability and Donnan equilibrium. Biochim. Biophys. Acta 401, 307–316
c The Authors Journal compilation c 2011 Biochemical Society
370
A. J. Morgan and others
87 Sonawane, N. D. and Verkman, A. S. (2003) Determinants of [Cl − ] in recycling and late
endosomes and Golgi complex measured using fluorescent ligands. J. Cell Biol. 160,
1129–1138
88 Dell’Antone, P. (1984) Electrogenicity of the lysosomal proton pump. FEBS Lett. 168,
15–22
89 Harikumar, P. and Reeves, J. P. (1983) The lysosomal proton pump is electrogenic.
J. Biol. Chem. 258, 10403–10410
90 Koivusalo, M., Steinberg, B. E., Mason, D. and Grinstein, S. (2011) In situ measurement
of the electrical potential across the lysosomal membrane using FRET. Traffic 2, 972–982
91 Rottenberg, H. (1979) The measurement of membrane potential and pH in cells,
organelles, and vesicles. Methods Enzymol. 55, 547–569
92 Loh, Y. P., Tam, W. W. and Russell, J. T. (1984) Measurement of pH and membrane
potential in secretory vesicles isolated from bovine pituitary intermediate lobe. J. Biol.
Chem. 259, 8238–8245
93 Morgan, A. J. and Galione, A. (2007) NAADP induces pH changes in the lumen of acidic
Ca2 + stores. Biochem. J. 402, 301–310
94 Schumaker, K. S. and Sze, H. (1986) Calcium transport into the vacuole of oat roots.
Characterization of H + /Ca2 + exchange activity. J. Biol. Chem. 261, 12172–12178
95 Ohkuma, S., Moriyama, Y. and Takano, T. (1983) Electrogenic nature of lysosomal
proton pump as revealed with a cyanine dye. J. Biochem. 94, 1935–1943
96 Reference deleted
97 Ohgaki, R., Fukura, N., Matsushita, M., Mitsui, K. and Kanazawa, H. (2008) Cell surface
levels of organellar Na + /H + exchanger isoform 6 are regulated by interaction with
RACK1. J. Biol. Chem. 283, 4417–4429
98 Schapiro, F. B. and Grinstein, S. (2000) Determinants of the pH of the Golgi complex.
J. Biol. Chem. 275, 21025–21032
99 Laporte, R., Hui, A. and Laher, I. (2004) Pharmacological modulation of sarcoplasmic
reticulum function in smooth muscle. Pharmacol. Rev. 56, 439–513
100 Missiaen, L., Dode, L., Vanoevelen, J., Raeymaekers, L. and Wuytack, F. (2007) Calcium
in the Golgi apparatus. Cell Calcium 41, 405–416
101 Lissandron, V., Podini, P., Pizzo, P. and Pozzan, T. (2010) Unique characteristics of
Ca2 + homeostasis of the trans-Golgi compartment. Proc. Natl. Acad. Sci. U.S.A. 107,
9198–9203
102 Pittman, J. K. (2011) Vacuolar Ca2 + uptake. Cell Calcium 50, 139–146
103 Brini, M. and Carafoli, E. (2009) Calcium pumps in health and disease. Physiol. Rev. 89,
1341–1378
104 Rooney, E. K. and Gross, J. D. (1992) ATP-driven Ca2 + /H + antiport in acid vesicles
from Dictyostelium . Proc. Natl. Acad. Sci. U.S.A. 89, 8025–8029
105 Cunningham, K. W. (2011) Acidic calcium stores of Saccharomyces cerevisiae . Cell
Calcium 50, 129–138
106 Moniakis, J., Coukell, M. B. and Forer, A. (1995) Molecular cloning of an intracellular
P-type ATPase from Dictyostelium that is up-regulated in calcium-adapted cells. J. Biol.
Chem. 270, 28276–28281
107 Ramos, I. B., Miranda, K., Pace, D. A., Verbist, K. C., Lin, F. Y., Zhang, Y., Oldfield, E.,
Machado, E. A., De Souza, W. and Docampo, R. (2010) Calcium- and
polyphosphate-containing acidic granules of sea urchin eggs are similar to
acidocalcisomes, but are not the targets for NAADP. Biochem. J. 429, 485–495
108 Fasolato, C., Zottini, M., Clementi, E., Zacchetti, D., Meldolesi, J. and Pozzan, T. (1991)
Intracellular Ca2 + pools in PC12 cells. Three intracellular pools are distinguished by
their turnover and mechanisms of Ca2 + accumulation, storage, and release. J. Biol.
Chem. 266, 20159–20167
109 Srinivas, S. P., Ong, A., Goon, L. and Bonanno, J. A. (2002) Lysosomal Ca2 + stores in
bovine corneal endothelium. Invest. Ophthalmol. Vis. Sci. 43, 2341–2350
110 Styrt, B. and Klempner, M. S. (1988) Lysosomotropic amines modulate neutrophil
calcium homeostasis. J. Cell. Physiol. 135, 309–316
111 Yagodin, S., Pivovarova, N. B., Andrews, S. B. and Sattelle, D. B. (1999) Functional
characterization of thapsigargin and agonist-insensitive acidic Ca2 + stores in
Drosophila melanogaster S2 cell lines. Cell Calcium 25, 429–438
112 Dickinson, G. D., Churchill, G. C., Brailoiu, E. and Patel, S. (2010) Deviant nicotinic acid
adenine dinucleotide phosphate (NAADP)-mediated Ca2 + signaling upon lysosome
proliferation. J. Biol. Chem. 285, 13321–13325
113 Vasudevan, S. R., Lewis, A. M., Chan, J. W., Machin, C. L., Sinha, D., Galione, A. and
Churchill, G. C. (2010) The calcium-mobilizing messenger nicotinic acid adenine
dinucleotide phosphate participates in sperm activation by mediating the acrosome
reaction. J. Biol. Chem. 285, 18262–18269
114 Yamasaki, M., Masgrau, R., Morgan, A. J., Churchill, G. C., Patel, S., Ashcroft, S. J. and
Galione, A. (2004) Organelle selection determines agonist-specific Ca2 + signals in
pancreatic acinar and beta cells. J. Biol. Chem. 279, 7234–7240
115 Brailoiu, G. C., Brailoiu, E., Parkesh, R., Galione, A., Churchill, G. C., Patel, S. and Dun,
N. J. (2009) NAADP-mediated channel ‘chatter’ in neurons of the rat medulla oblongata.
Biochem. J. 419, 91–97
c The Authors Journal compilation c 2011 Biochemical Society
116 Gerasimenko, J. V., Sherwood, M., Tepikin, A. V., Petersen, O. H. and Gerasimenko, O. V.
(2006) NAADP, cADPR and IP3 all release Ca2 + from the endoplasmic reticulum and an
acidic store in the secretory granule area. J. Cell Sci. 119, 226–238
117 Kinnear, N. P., Boittin, F. X., Thomas, J. M., Galione, A. and Evans, A. M. (2004)
Lysosome-sarcoplasmic reticulum junctions. A trigger zone for calcium signaling by
nicotinic acid adenine dinucleotide phosphate and endothelin-1. J. Biol. Chem. 279,
54319–54326
118 Rah, S. Y., Mushtaq, M., Nam, T. S., Kim, S. H. and Kim, U. H. (2010) Generation of
cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate by CD38 for Ca2 +
signaling in interleukin-8-treated lymphokine-activated killer cells. J. Biol. Chem. 285,
21877–21887
119 Russell, J. T. (1984) δpH, H + diffusion potentials, and Mg2 + ATPase in neurosecretory
vesicles isolated from bovine neurohypophyses. J. Biol. Chem. 259, 9496–9507
120 Moseley, R. H. and Van Dyke, R. W. (1995) Organic cation transport by rat liver
lysosomes. Am. J. Physiol. 268, G480–G486
121 Malecki, J., Wiedlocha, A., Wesche, J. and Olsnes, S. (2002) Vesicle transmembrane
potential is required for translocation to the cytosol of externally added FGF-1. EMBO J.
21, 4480–4490
122 Camacho, M., Machado, J. D., Alvarez, J. and Borges, R. (2008) Intravesicular calcium
release mediates the motion and exocytosis of secretory organelles: a study with adrenal
chromaffin cells. J. Biol. Chem. 283, 223832–2389
123 Brailoiu, E., Churamani, D., Cai, X., Schrlau, M. G., Brailoiu, G. C., Gao, X., Hooper, R.,
Boulware, M. J., Dun, N. J., Marchant, J. S. and Patel, S. (2009) Essential requirement
for two-pore channel 1 in NAADP-mediated calcium signaling. J. Cell Biol. 186,
201–209
124 Brailoiu, E., Hooper, R., Cai, X., Brailoiu, G. C., Keebler, M. V., Dun, N. J., Marchant,
J. S. and Patel, S. (2010) An ancestral deuterostome family of two-pore channels
mediates nicotinic acid adenine dinucleotide phosphate-dependent calcium release from
acidic organelles. J. Biol. Chem. 285, 2897–2901
125 Brailoiu, E., Rahman, T., Churamani, D., Prole, D. L., Brailoiu, G. C., Hooper, R., Taylor,
C. W. and Patel, S. (2010) An NAADP-gated two-pore channel targeted to the plasma
membrane uncouples triggering from amplifying Ca2 + signals. J. Biol. Chem. 285,
38511–38516
126 Brailoiu, G. C., Gurzu, B., Gao, X., Parkesh, R., Aley, P. K., Trifa, D. I., Galione, A., Dun,
N. J., Madesh, M., Patel, S. et al. (2010) Acidic NAADP-sensitive calcium stores in the
endothelium: agonist-specific recruitment and role in regulating blood pressure. J. Biol.
Chem. 285, 37133–37137
127 Calcraft, P. J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., Tang, J., Rietdorf,
K., Teboul, L., Chuang, K. T. et al. (2009) NAADP mobilizes calcium from acidic
organelles through two-pore channels. Nature 459, 596–600
128 Ruas, M., Rietdorf, K., Arredouani, A., Davis, L. C., Lloyd-Evans, E., Koegel, H., Funnell,
T. M., Morgan, A. J., Ward, J. A., Watanabe, K. et al. (2010) Purified TPC isoforms form
NAADP receptors with distinct roles for Ca2 + signaling and endolysosomal trafficking.
Curr. Biol. 20, 703–709
129 Vercesi, A. E., Moreno, S. N. and Docampo, R. (1994) Ca2 + /H + exchange in acidic
vacuoles of Trypanosoma brucei . Biochem. J. 304, 227–233
130 Goncalves, P. P., Meireles, S. M., Neves, P. and Vale, M. G. (2000) Methods for analysis
of Ca2 + /H + antiport activity in synaptic vesicles isolated from sheep brain cortex. Brain
Res. Brain Res. Protoc. 5, 102–108
131 Milani, G., Schereiber, A. Z. and Vercesi, A. E. (2001) Ca2 + transport into an
intracellular acidic compartment of Candida parapsilosis . FEBS Lett. 500, 80–84
132 Hilden, S. A. and Madias, N. E. (1989) H + /Ca2 + exchange in rabbit renal cortical
endosomes. J. Membr. Biol. 112, 131–138
133 Blaustein, M. P. and Lederer, W. J. (1999) Sodium/calcium exchange: its physiological
implications. Physiol. Rev. 79, 763–854
134 LeFurgey, A., Spencer, A. J., Jacobs, W. R., Ingram, P. and Mandel, L. J. (1991)
Elemental microanalysis of organelles in proximal tubules. I. Alterations in transport and
metabolism. J. Am. Soc. Nephrol. 1, 1305–1320
135 Krieger-Brauer, H. I. and Gratzl, M. (1983) Effects of monovalent and divalent cations on
Ca2 + fluxes across chromaffin secretory membrane vesicles. J. Neurochem. 41,
1269–1276
136 Mahapatra, N. R., Mahata, M., Hazra, P. P., McDonough, P. M., O’Connor, D. T. and
Mahata, S. K. (2004) A dynamic pool of calcium in catecholamine storage vesicles:
exploration in living cells by a novel vesicle-targeted chromogranin A/aequorin chimeric
photoprotein. J. Biol. Chem. 279, 51107–51121
137 Lytton, J. (2007) Na + /Ca2 + exchangers: three mammalian gene families control Ca2 +
transport. Biochem. J. 406, 365–382
138 Roux, M. M., Townley, I. K., Raisch, M., Reade, A., Bradham, C., Humphreys, G.,
Gunaratne, H. J., Killian, C. E., Moy, G., Su, Y. H. et al. (2006) A functional genomic and
proteomic perspective of sea urchin calcium signaling and egg activation. Dev. Biol.
300, 416–433
Endolysosomal Ca2 + signalling in health and disease
139 Altimimi, H. F. and Schnetkamp, P. P. (2007) Na + /Ca2 + -K + exchangers (NCKX):
functional properties and physiological roles. Channels 1, 62–69
140 Pan, C.-Y., Tsai, L. L., Jiang, J. H., Chen, L. W. and Kao, L. S. (2008) The co-presence of
Na + /Ca2 + -K + exchanger and Na + /Ca2 + exchanger in bovine adrenal chromaffin
cells. J. Neurochem. 107, 658–667
141 Lamason, R. L., Mohideen, M. A., Mest, J. R., Wong, A. C., Norton, H. L., Aros, M. C.,
Jurynec, M. J., Mao, X., Humphreville, V. R., Humbert, J. E. et al. (2005) SLC24A5, a
putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310,
1782–1786
142 Genazzani, A. A. and Galione, A. (1996) Nicotinic acid-adenine dinucleotide phosphate
mobilizes Ca2 + from a thapsigargin-insensitive pool. Biochem. J. 315, 721–725
143 Rooney, E. K., Gross, J. D. and Satre, M. (1994) Characterisation of an intracellular
Ca2 + pump in Dictyostelium . Cell Calcium 16, 509–522
144 Zong, X., Schieder, M., Cuny, H., Fenske, S., Gruner, C., Rotzer, K., Griesbeck, O.,
Harz, H., Biel, M. and Wahl-Schott, C. (2009) The two-pore channel TPCN2 mediates
NAADP-dependent Ca2 + -release from lysosomal stores. Pflügers Arch. 458, 891–899
145 Johnson, J. D., Kuang, S., Misler, S. and Polonsky, K. S. (2004) Ryanodine receptors in
human pancreatic β cells: localization and effects on insulin secretion. FASEB J. 18,
878–880
146 Mitchell, K. J., Lai, F. A. and Rutter, G. A. (2003) Ryanodine receptor type I and nicotinic
acid adenine dinucleotide phosphate receptors mediate Ca2 + release from
insulin-containing vesicles in living pancreatic β cells (MIN6). J. Biol. Chem. 278,
11057–11064
147 Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R.
and Rutter, G. A. (2001) Dense core secretory vesicles revealed as a dynamic Ca2 + store
in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.
J. Cell Biol. 155, 41–51
148 Duman, J. G., Chen, L., Palmer, A. E. and Hille, B. (2006) Contributions of intracellular
compartments to calcium dynamics: implicating an acidic store. Traffic 7, 859–872
149 Haller, T., Volkl, H., Deetjen, P. and Dietl, P. (1996) The lysosomal Ca2 + pool in MDCK
cells can be released by Ins(1,4,5)P3 - dependent hormones or thapsigargin but does not
activate store-operated Ca2 + entry. Biochem. J. 319, 909–912
150 Mandal, P. K., Mandal, A. and Ahearn, G. A. (2006) 65 Zn2 + Transport by lobster
hepatopancreatic lysosomal membrane vesicles. J. Exp. Zool. A Comp. Exp. Biol. 305,
203–214
151 Lytton, J., Westlin, M., Burk, S. E., Shull, G. E. and MacLennan, D. H. (1992) Functional
comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of
calcium pumps. J. Biol. Chem. 267, 14483–14489
152 Goncalves, P. P., Meireles, S. M., Neves, P. and Vale, M. G. (2000) Distinction between
Ca2 + pump and Ca2 + /H + antiport activities in synaptic vesicles of sheep brain cortex.
Neurochem. Int. 37, 387–396
153 Pinton, P., Pozzan, T. and Rizzuto, R. (1998) The Golgi apparatus is an inositol
1,4,5-trisphosphate-sensitive Ca2 + store, with functional properties distinct from those
of the endoplasmic reticulum. EMBO J. 17, 5298–5308
154 Nunez, M. T., Gaete, V. and Escobar, A. (1990) Endocytic vesicles contain a
calmodulin-activated Ca2 + pump that mediates the inhibition of acidification by
calcium. Biochim. Biophys. Acta 1028, 21–24
155 Thevenod, F. and Schulz, I. (1988) H + -dependent calcium uptake into an IP3-sensitive
calcium pool from rat parotid gland. Am. J. Physiol. 255, G429–G440
156 Thevenod, F., Dehlinger-Kremer, M., Kemmer, T. P., Christian, A. L., Potter, B. V. and
Schulz, I. (1989) Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and
-insensitive (IisCaP) nonmitochondrial Ca2 + pools in rat pancreatic acinar cells.
J. Membr. Biol. 109, 173–186
157 Ezaki, J., Himeno, M. and Kato, K. (1992) Purification and characterization of
(Ca2 + -Mg2 + )-ATPase in rat liver lysosomal membranes. J. Biochem. 112, 33–39
158 Hicks, B. W. and Parsons, S. M. (1992) Characterization of the P-type and V-type
ATPases of cholinergic synaptic vesicles and coupling of nucleotide hydrolysis to
acetylcholine transport. J. Neurochem. 58, 1211–1220
159 Salceda, R. and Sanchez-Chavez, G. (2000) Calcium uptake, release and ryanodine
binding in melanosomes from retinal pigment epithelium. Cell Calcium 27, 223–229
160 Krause, K. H. and Lew, P. D. (1987) Subcellular distribution of Ca2 + pumping sites in
human neutrophils. J. Clin. Invest. 80, 107–116
161 Lopez, J. J., Camello-Almaraz, C., Pariente, J. A., Salido, G. M. and Rosado, J. A. (2005)
Ca2 + accumulation into acidic organelles mediated by Ca2 + - and vacuolar
H + -ATPases in human platelets. Biochem. J. 390, 243–252
162 Lopez, J. J., Redondo, P. C., Salido, G. M., Pariente, J. A. and Rosado, J. A. (2006) Two
distinct Ca2 + compartments show differential sensitivity to thrombin, ADP and
vasopressin in human platelets. Cell. Signaling 18, 373–381
163 Jardin, I., Lopez, J. J., Pariente, J. A., Salido, G. M. and Rosado, J. A. (2008)
Intracellular calcium release from human platelets: different messengers for multiple
stores. Trends Cardiovasc. Med. 18, 57–61
371
164 Papp, B., Enyedi, A., Paszty, K., Kovacs, T., Sarkadi, B., Gardos, G., Magnier, C.,
Wuytack, F. and Enouf, J. (1992) Simultaneous presence of two distinct
endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization
by radio-immunoblotting and inhibition by 2,5-di-(t -butyl)-1,4-benzohydroquinone.
Biochem. J. 288, 297–302
165 Wuytack, F., Papp, B., Verboomen, H., Raeymaekers, L., Dode, L., Bobe, R., Enouf, J.,
Bokkala, S., Authi, K. S. and Casteels, R. (1994) A sarco/endoplasmic reticulum
Ca2 + -ATPase 3-type Ca2 + pump is expressed in platelets, in lymphoid cells, and in
mast cells. J. Biol. Chem. 269, 1410–1416
166 Zbidi, H., Jardin, I., Woodard, G. E., Lopez, J. J., Berna, A., Salido, G. M. and Rosado,
J. A. (2011) STIM1 and STIM2 are located in the acidic Ca2 + stores and associates with
Orai1 upon depletion of the acidic stores in human platelets.
J. Biol. Chem. 286, 12257–12270
167 Prasad, V., Okunade, G. W., Miller, M. L. and Shull, G. E. (2004) Phenotypes of SERCA
and PMCA knockout mice. Biochem. Biophys. Res. Commun. 322, 1192–1203
168 Aulestia, F. J., Redondo, P. C., Rodriguez-Garcia, A., Rosado, J. A., Salido, G. M.,
Alonso, M. T. and Garcia-Sancho, J. (2011) Two distinct calcium pools in the
endoplasmic reticulum of HEK-293T cells. Biochem. J. 435, 227–235
169 Toyoshima, C. and Inesi, G. (2004) Structural basis of ion pumping by Ca2 + -ATPase of
the sarcoplasmic reticulum. Annu. Rev. Biochem. 73, 269–292
170 Miller, A. J., Vogg, G. and Sanders, D. (1990) Cytosolic calcium homeostasis in fungi:
roles of plasma membrane transport and intracellular sequestration of calcium. Proc.
Natl. Acad. Sci. U.S.A. 87, 9348–9352
171 Steen, M., Kirchberger, T. and Guse, A. H. (2007) NAADP mobilizes calcium from the
endoplasmic reticular Ca2 + store in T-lymphocytes. J. Biol. Chem. 282, 18864–18871
172 Gerasimenko, J. V., Tepikin, A. V., Petersen, O. H. and Gerasimenko, O. V. (1998)
Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr.
Biol. 8, 1335–1338
173 Sherwood, M. W., Prior, I. A., Voronina, S. G., Barrow, S. L., Woodsmith, J. D.,
Gerasimenko, O. V., Petersen, O. H. and Tepikin, A. V. (2007) Activation of trypsinogen
in large endocytic vacuoles of pancreatic acinar cells. Proc. Natl. Acad. Sci. U.S.A. 104,
5674–5679
174 Lundqvist-Gustafsson, H., Gustafsson, M. and Dahlgren, C. (2000) Dynamic Ca2 +
changes in neutrophil phagosomes. A source for intracellular Ca2 + during
phagolysosome formation? Cell Calcium 27, 353–362
175 Menteyne, A., Burdakov, A., Charpentier, G., Petersen, O. H. and Cancela, J. M. (2006)
Generation of specific Ca2 + signals from Ca2 + stores and endocytosis by differential
coupling to messengers. Curr. Biol. 16, 1931–1937
176 Berchtold, M. W., Brinkmeier, H. and Muntener, M. (2000) Calcium ion in skeletal
muscle: its crucial role for muscle function, plasticity, and disease. Physiol. Rev. 80,
1215–1265
177 Gelebart, P., Opas, M. and Michalak, M. (2005) Calreticulin, a Ca2 + -binding chaperone
of the endoplasmic reticulum. Int. J. Biochem. Cell Biol. 37, 260–266
178 Michalak, M., Robert Parker, J. M. and Opas, M. (2002) Ca2 + signaling and calcium
binding chaperones of the endoplasmic reticulum. Cell Calcium 32, 269–278
179 Gyorke, S. and Terentyev, D. (2008) Modulation of ryanodine receptor by luminal calcium
and accessory proteins in health and cardiac disease. Cardiovasc. Res. 77, 245–255
180 Mogami, H., Gardner, J., Gerasimenko, O. V., Camello, P., Petersen, O. H. and Tepikin, A.
V. (1999) Calcium binding capacity of the cytosol and endoplasmic reticulum of mouse
pancreatic acinar cells. J. Physiol. 518, 463–467
181 Docampo, R., de Souza, W., Miranda, K., Rohloff, P. and Moreno, S. N. (2005)
Acidocalcisomes: conserved from bacteria to man. Nat. Rev. Microbiol. 3, 251–261
182 Ruiz, F. A., Lea, C. R., Oldfield, E. and Docampo, R. (2004) Human platelet dense
granules contain polyphosphate and are similar to acidocalcisomes of bacteria and
unicellular eukaryotes. J. Biol. Chem. 279, 44250–44257
183 Kendall, M. D. and Warley, A. (1986) Elemental content of mast cell granules measured
by X-ray microanalysis of rat thymic tissue sections. J. Cell Sci. 83, 77–87
184 Hoogduijn, M. J., Smit, N. P., van der Laarse, A., van Nieuwpoort, A. F., Wood, J. M. and
Thody, A. J. (2003) Melanin has a role in Ca2 + homeostasis in human melanocytes.
Pigment Cell Res. 16, 127–132
185 Nguyen, T., Chin, W. C. and Verdugo, P. (1998) Role of Ca2 + /K + ion exchange in
intracellular storage and release of Ca2 + . Nature 395, 908–912
186 Clapper, D. L., Walseth, T. F., Dargie, P. J. and Lee, H. C. (1987) Pyridine nucleotide
metabolites stimulate calcium release from sea urchin egg microsomes desensitized to
inositol trisphosphate. J. Biol. Chem. 262, 9561–9568
187 Whitaker, M. J. and Irvine, R. F. (1984) lnositol 1,4,5-trisphosphate microinjection
activates sea urchin eggs. Nature 312, 636–639
188 Clapper, D. L. and Lee, H. C. (1985) Inositol trisphosphate induces calcium release from
nonmitochondrial stores in sea urchin egg homogenates. J. Biol. Chem. 260,
13947–13954
189 Epel, D., Patton, C., Wallace, R. W. and Cheung, W. Y. (1981) Calmodulin activates NAD
kinase of sea urchin eggs: an early event of fertilization. Cell 23, 543–549
c The Authors Journal compilation c 2011 Biochemical Society
372
A. J. Morgan and others
190 Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N. and Clapper, D. L. (1989) Structural
determination of a cyclic metabolite of NAD with intracellular calcium-mobilizing
activity. J. Biol. Chem. 264, 1608–1615
191 Rusinko, N. and Lee, H. C. (1989) Widespread occurrence in animal tissues of an
enzyme catalyzing the conversion of NAD into a cyclic metabolite with intracellular
calcium-mobilizing activity. J. Biol. Chem. 264, 11725–11731
192 Lee, H. C. and Aarhus, R. (1995) A derivative of NADP mobilizes calcium stores
insensitive to inositol trisphosphate and cyclic ADP-ribose. J. Biol. Chem. 270,
2152–2157
193 Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiba, K. (1989)
Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding
protein P400. Nature 342, 32–38
194 Lai, F. A., Erickson, H. P., Rousseau, E., Liu, Q. Y. and Meissner, G. (1988) Purification
and reconstitution of the calcium release channel from skeletal muscle. Nature 331,
315–319
195 Bezprozvanny, I., Watras, J. and Ehrlich, B. E. (1991) Bell-shaped calcium-response
curves of Ins(1,4,5)P3 - and calcium-gated channels from endoplasmic reticulum of
cerebellum. Nature 351, 751–754
196 Roderick, H. L., Berridge, M. J. and Bootman, M. D. (2003) Calcium-induced calcium
release. Curr. Biol. 13, R425
197 Galione, A., Lee, H. C. and Busa, W. B. (1991) Ca2 + -induced Ca2 + release in sea
urchin egg homogenates: modulation by cyclic ADP-ribose. Science 253, 1143–1146
198 Genazzani, A. A., Mezna, M., Dickey, D. M., Michelangeli, F., Walseth, T. F. and Galione,
A. (1997) Pharmacological properties of the Ca2 + -release mechanism sensitive to
NAADP in the sea urchin egg. Br. J. Pharmacol. 121, 1489–1495
199 Churchill, G C. and Galione, A. (2001) NAADP induces Ca2 + oscillations via a two-pool
mechanism by priming IP3- and cADPR-sensitive Ca2 + stores. EMBO J. 20,
2666–2671
200 Lee, H. C. and Aarhus, R. (2000) Functional visualization of the separate but interacting
calcium stores sensitive to NAADP and cyclic ADP-ribose. J. Cell Sci. 113, 4413–4420
201 Galione, A. (2006) NAADP, a new intracellular messenger that mobilizes Ca2 + from
acidic stores. Biochem. Soc. Trans. 34, 922–926
202 Lee, H. C. (2005) NAADP-mediated calcium signaling. J. Biol. Chem. 280,
33693–33696
203 Reference deleted
204 Cancela, J. M., Churchill, G. C. and Galione, A. (1999) Coordination of agonist-induced
Ca2 + signalling patterns by NAADP in pancreatic acinar cells. Nature 398, 74–76
205 Chini, E. N. and Dousa, T. P. (1996) Nicotinate-adenine dinucleotide phosphate-induced
Ca2 + -release does not behave as a Ca2 + -induced Ca2 + -release system. Biochem. J.
316, 709–711
206 Bak, J., White, P., Timar, G., Missiaen, L., Genazzani, A. A. and Galione, A. (1999)
Nicotinic acid adenine dinucleotide phosphate triggers Ca2 + release from brain
microsomes. Curr. Biol. 9, 751–754
207 Churchill, G. C. and Galione, A. (2000) Spatial control of Ca2 + signaling by nicotinic
acid adenine dinucleotide phosphate diffusion and gradients. J. Biol. Chem. 275,
38687–38692
208 Patel, S., Churchill, G. C. and Galione, A. (2001) Coordination of Ca2 + signalling by
NAADP. Trends Biochem. Sci. 26, 482–489
209 Rizzuto, R., Marchi, S., Bonora, M., Aguiari, P., Bononi, A., De Stefani, D., Giorgi, C.,
Leo, S., Rimessi, A., Siviero, R. et al. (2009) Ca2 + transfer from the ER to mitochondria:
when, how and why. Biochim. Biophys. Acta 1787, 1342–1351
210 de Brito, O. M. and Scorrano, L. (2008) Mitofusin 2 tethers endoplasmic reticulum to
mitochondria. Nature 456, 605–610
211 Kinnear, N. P., Wyatt, C. N., Clark, J. H., Calcraft, P. J., Fleischer, S., Jeyakumar,
L. H., Nixon, G. F. and Evans, A. M. (2008) Lysosomes co-localize with ryanodine
receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat
pulmonary arterial smooth muscle. Cell Calcium 44, 190–201
212 Davis, L. C., Morgan, A. J., Ruas, M., Wong, J. L., Graeff, R. M., Poustka, A. J., Lee, H.
C., Wessel, G. M., Parrington, J. and Galione, A. (2008) Ca2 + signaling occurs via
second messenger release from intraorganelle synthesis sites. Curr. Biol. 18,
1612–1618
213 Morgan, A. J. and Galione, A. (2007) Fertilization and nicotinic acid adenine
dinucleotide phosphate induce pH changes in acidic Ca2 + stores in sea urchin eggs.
J. Biol. Chem. 282, 37730–37737
214 Cosker, F., Cheviron, N., Yamasaki, M., Menteyne, A., Lund, F. E., Moutin, M. J.,
Galione, A. and Cancela, J. M. (2010) The ecto-enzyme CD38 is a nicotinic acid adenine
dinucleotide phosphate (NAADP) synthase that couples receptor activation to Ca2 +
mobilization from lysosomes in pancreatic acinar cells. J. Biol. Chem. 285,
38251–38259
215 Sun-Wada, G. H., Wada, Y. and Futai, M. (2003) Lysosome and lysosome-related
organelles responsible for specialized functions in higher organisms, with special
emphasis on vacuolar-type proton ATPase. Cell. Struct. Funct. 28, 455–463
c The Authors Journal compilation c 2011 Biochemical Society
216 Dammermann, W. and Guse, A. H. (2005) Functional ryanodine receptor expression is
required for NAADP-mediated local Ca2 + signaling in T-lymphocytes. J. Biol. Chem.
280, 21394–21399
217 Mojzisova, A., Krizanova, O., Zacikova, L., Kominkova, V. and Ondrias, K. (2001) Effect
of nicotinic acid adenine dinucleotide phosphate on ryanodine calcium release channel
in heart. Pflügers Arch. 441, 674–677
218 Hohenegger, M., Suko, J., Gscheidlinger, R., Drobny, H. and Zidar, A. (2002) Nicotinic
acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.
Biochem. J. 367, 423–431
219 Copello, J. A., Qi, Y., Jeyakumar, L. H., Ogunbunmi, E. and Fleischer, S. (2001) Lack of
effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine
receptors. Cell Calcium 30, 269–284
220 Pitt, S. J., Funnell, T. M., Sitsapesan, M., Venturi, E., Rietdorf, K., Ruas, M., Ganesan, A.,
Gosain, R., Churchill, G. C. et al. (2010) TPC2 is a novel NAADP-sensitive Ca2 + release
channel, operating as a dual sensor of luminal pH and Ca2 + . J. Biol. Chem. 285,
35039–35046
221 Ogunbayo, O. A., Zhu, Y., Rossi, D., Sorrentino, V., Ma, J., Zhu, M. X. and Evans, A. M.
(2011) Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas
NAADP activates two-pore domain channels. J. Biol. Chem. 286, 9136–9140
222 Bach, G. (2001) Mucolipidosis type IV. Mol. Genet. Metab. 73, 197–203
223 Sun, M., Goldin, E., Stahl, S., Falardeau, J. L., Kennedy, J. C., Acierno, Jr, J. S., Bove,
C., Kaneski, C. R., Nagle, J., Bromley, M. C. et al. (2000) Mucolipidosis type IV is
caused by mutations in a gene encoding a novel transient receptor potential channel.
Hum. Mol. Genet. 9, 2471–2478
224 Cheng, X., Shen, D., Samie, M. and Xu, H. (2010) Mucolipins: intracellular TRPML1–3
channels. FEBS Lett. 584, 2013–2021
225 Pryor, P. R., Reimann, F., Gribble, F. M. and Luzio, J. P. (2006) Mucolipin-1 is a
lysosomal membrane protein required for intracellular lactosylceramide traffic. Traffic 7,
1388–1398
226 Vergarajauregui, S. and Puertollano, R. (2006) Two di-leucine motifs regulate trafficking
of mucolipin-1 to lysosomes. Traffic 7, 337–353
227 LaPlante, J. M., Falardeau, J., Sun, M., Kanazirska, M., Brown, E. M., Slaugenhaupt,
S. A. and Vassilev, P. M. (2002) Identification and characterization of the single channel
function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting
the lysosomal pathway. FEBS Lett. 532, 183–187
228 Cantiello, H. F., Montalbetti, N., Goldmann, W. H., Raychowdhury, M. K.,
Gonzalez-Perrett, S., Timpanaro, G. A. and Chasan, B. (2005) Cation channel activity of
mucolipin-1: the effect of calcium. Pflügers Arch. 451, 304–312
229 Dong, X. P., Cheng, X., Mills, E., Delling, M., Wang, F., Kurz, T. and Xu, H. (2008) The
type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release
channel. Nature 455, 992–996
230 Zhang, F. and Li, P. L. (2007) Reconstitution and characterization of a nicotinic acid
adenine dinucleotide phosphate (NAADP)-sensitive Ca2 + release channel from liver
lysosomes of rats. J. Biol. Chem. 282, 25259–25269
231 Zhang, F., Jin, S., Yi, F. and Li, P. L. (2009) TRP-ML1 functions as a lysosomal
NAADP-sensitive Ca2 + release channel in coronary arterial myocytes. J. Cell. Mol.
Med. 13, 3174–3185
232 Yamaguchi, S., Jha, A., Li, Q., Soyombo, A. A., Dickinson, G. D., Churamani, D.,
Brailoiu, E., Patel, S. and Muallem, S. (2011) TRPML1 and two-pore channels are
functionally independent organellar ion channels. J. Biol. Chem. 286, 22934–22942
233 Dong, X.-P., Shen, D., Wang, X., Dawson, T., Li, X., Zhang, Q., Cheng, X., Zhang, Y.,
Weisman, L. S., Delling, M. and Xu, H. (2010) PI(3,5)P2 controls membrane trafficking
by direct activation of mucolipin Ca2 + release channels in the endolysosome. Nat.
Commun. 1, 38
234 Sumoza-Toledo, A. and Penner, R. (2011) TRPM2: a multifunctional ion channel for
calcium signaling. J. Physiol. 589, 1515–1525
235 Perraud, A. L., Fleig, A., Dunn, C. A., Bagley, L. A., Launay, P., Schmitz, C., Stokes, A. J.,
Zhu, Q., Bessman, M. J., Penner, R. et al. (2001) ADP-ribose gating of the
calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature 411,
595–599
236 Lange, I., Yamamoto, S., Partida-Sanchez, S., Mori, Y., Fleig, A. and Penner, R. (2009)
TRPM2 functions as a lysosomal Ca2 + -release channel in β cells. Sci. Signal. 2, ra23
237 Toth, B. and Csanady, L. (2010) Identification of direct and indirect effectors of the
transient receptor potential melastatin 2 (TRPM2) cation channel. J. Biol. Chem. 285,
30091–30102
238 Qureshi, O. S., Paramasivam, A., Yu, J. C. and Murrell-Lagnado, R. D. (2007)
Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis.
J. Cell Sci. 120, 3838–3849
239 Zhu, M. X., Ma, J., Parrington, J., Galione, A. and Evans, A. M. (2010) TPCs:
endolysosomal channels for Ca2 + mobilization from acidic organelles triggered by
NAADP. FEBS Lett. 584, 1966–1974
Endolysosomal Ca2 + signalling in health and disease
240 Patel, S., Marchant, J. S. and Brailoiu, E. (2010) Two-pore channels: regulation by
NAADP and customized roles in triggering calcium signals. Cell Calcium 47, 480–490
241 Ishibashi, K., Suzuki, M. and Imai, M. (2000) Molecular cloning of a novel form
(two-repeat) protein related to voltage-gated sodium and calcium channels. Biochem.
Biophys. Res. Commun. 270, 370–376
242 Hille, B. (2001) Ion Channels of Excitable Membranes. Sinauer, New York
243 Durell, S. R. and Guy, H. R. (2001) A putative prokaryote voltage-gated Ca2 + channel
with only one 6TM motif per subunit. Biochem. Biophys. Res. Commun. 281, 741–746
244 Furuichi, T., Cunningham, K. W. and Muto, S. (2001) A putative two pore channel
AtTPC1 mediates Ca2 + flux in Arabidopsis leaf cells. Plant Cell Physiol. 42, 900–905
245 Kadota, Y., Furuichi, T., Ogasawara, Y., Goh, T., Higashi, K., Muto, S. and Kuchitsu, K.
(2004) Identification of putative voltage-dependent Ca2 + -permeable channels involved
in cryptogein-induced Ca2 + transients and defense responses in tobacco BY-2 cells.
Biochem. Biophys. Res. Commun. 317, 823–830
246 Peiter, E., Maathuis, F. J., Mills, L. N., Knight, H., Pelloux, J., Hetherington, A. M. and
Sanders, D. (2005) The vacuolar Ca2 + -activated channel TPC1 regulates germination
and stomatal movement. Nature 434, 404–408
247 Beyhl, D., Hörtensteiner, S., Martinoia, E., Farmer, E. E., Fromm, J., Marten, I. and
Hedrich, R. (2009) The fou2 mutation in the major vacuolar cation channel TPC1 confers
tolerance to inhibitory luminal calcium. Plant J. 58, 715–723
248 Hedrich, R. and Marten, I. (2011) TPC1–SV channels gain shape. Mol. Plant 4, 428–441
249 Navazio, L., Bewell, M. A., Siddiqua, A., Dickinson, G. D., Galione, A. and Sanders, D.
(2000) Calcium release from the endoplasmic reticulum of higher plants elicited by the
NADP metabolite nicotinic acid adenine dinucleotide phosphate. Proc. Natl. Acad. Sci.
U.S.A. 97, 8693–8698
250 Hooper, R., Churamani, D., Brailoiu, E., Taylor, C. W. and Patel, S. (2011) Membrane
topology of NAADP-sensitive two-pore channels and their regulation by N-linked
glycosylation. J. Biol. Chem. 286, 9141–9149
251 Cai, X. and Patel, S. (2010) Degeneration of an intracellular ion channel in the primate
lineage by relaxation of selective constraints. Mol. Biol. Evol. 27, 2352–2359
252 Schieder, M., Rotzer, K., Bruggemann, A., Biel, M. and Wahl-Schott, C. (2010) Planar
patch clamp approach to characterize ionic currents from intactlysosomes. Sci. Signal.
3, pl3
253 Naylor, E., Arredouani, A., Vasudevan, S. R., Lewis, A. M., Parkesh, R., Mizote, A.,
Rosen, D., Thomas, J. M., Izumi, M., Ganesan, A. et al. (2009) Identification of a
chemical probe for NAADP by virtual screening. Nat. Chem. Biol. 5, 220–226
254 Aley, P. K., Noh, H. J., Gao, X., Tica, A. A., Brailoiu, E. and Churchill, G. C. (2010) A
functional role for nicotinic acid adenine dinucleotide phosphate in oxytocin-mediated
contraction of uterine smooth muscle from rat. J. Pharmacol. Exp. Ther. 333,
726–735
255 Yamasaki, M., Thomas, J. M., Churchill, G. C., Garnham, C., Lewis, A. M., Cancela,
J. M., Patel, S. and Galione, A. (2005) Role of NAADP and cADPR in the induction and
maintenance of agonist-evoked Ca2 + spiking in mouse pancreatic acinar cells. Curr.
Biol. 15, 874–878
256 Kim, B. J., Park, K. H., Yim, C. Y., Takasawa, S., Okamoto, H., Im, M. J. and Kim, U. H.
(2008) Generation of nicotinic acid adenine dinucleotide phosphate and cyclic
ADP-ribose by glucagon-like peptide-1 evokes Ca2 + signal that is essential for insulin
secretion in mouse pancreatic islets. Diabetes 57, 868–878
257 Kim, S. Y., Cho, B. H. and Kim, U. H. (2010) CD38-mediated Ca2 + signaling
contributes to angiotensin II-induced activation of hepatic stellate cells: attenuation of
hepatic fibrosis by CD38 ablation. J. Biol. Chem. 285, 576–582
258 Cancela, J. M., Van Coppenolle, F., Galione, A., Tepikin, A. V. and Petersen, O. H. (2002)
Transformation of local Ca2 + spikes to global Ca2 + transients: the combinatorial roles
of multiple Ca2 + releasing messengers. EMBO J. 21, 909–919
259 Petersen, O. H., Gerasimenko, O. V., Tepikin, A. V. and Gerasimenko, J. V. (2011)
Aberrant Ca2 + signalling through acidic calcium stores in pancreatic acinar cells. Cell
Calcium 50, 193–199
260 Moccia, F., Lim, D., Kyozuka, K. and Santella, L. (2004) NAADP triggers the fertilization
potential in starfish oocytes. Cell Calcium 36, 515–524
261 Churchill, G. C., O’Neill, J. S., Masgrau, R., Patel, S., Thomas, J. M., Genazzani, A. A.
and Galione, A. (2003) Sperm deliver a new second messenger: NAADP. Curr. Biol. 13,
125–128
262 Moccia, F., Billington, R. A. and Santella, L. (2006) Pharmacological characterization of
NAADP-induced Ca2 + signals in starfish oocytes. Biochem. Biophys. Res. Commun.
348, 329–336
263 Pryor, P. R., Mullock, B. M., Bright, N. A., Gray, S. R. and Luzio, J. P. (2000) The role of
intraorganellar Ca2 + in late endosome-lysosome heterotypic fusion and in the
reformation of lysosomes from hybrid organelles. J. Cell Biol. 149, 1053–1062
264 Piper, R. C. and Luzio, J. P. (2004) CUPpling calcium to lysosomal biogenesis. Trends
Cell Biol. 14, 471–473
373
265 Luzio, J. P., Pryor, P. R. and Bright, N. A. (2007) Lysosomes: fusion and function. Nat.
Rev. Mol. Cell Biol. 8, 622–632
266 Tugba Durlu-Kandilci, N., Ruas, M., Chuang, K. T., Brading, A., Parrington, J. and
Galione, A. (2010) TPC2 proteins mediate nicotinic acid adenine dinucleotide phosphate
(NAADP)- and agonist-evoked contractions of smooth muscle. J. Biol. Chem. 285,
24925–24932
267 Esposito, B., Gambara, G., Lewis, A. M., Palombi, F., D’Alessio, A., Taylor, L. X.,
Genazzani, A. A., Ziparo, E., Galione, A., Churchill, G. C. and Filippini, A. (2011) NAADP
links histamine H1 receptors to secretion of von Willebrand factor in human endothelial
cells. Blood 117, 4968–4977
268 Aley, P. K., Mikolajczyk, A. M., Munz, B., Churchill, G. C., Galione, A. and Berger, F.
(2010) Nicotinic acid adenine dinucleotide phosphate regulates skeletal muscle
differentiation via action at two-pore channels. Proc. Natl. Acad. Sci. U.S.A. 107,
19927–19932
269 Brailoiu, E., Churamani, D., Pandey, V., Brailoiu, G. C., Tuluc, F., Patel, S. and Dun, N. J.
(2006) Messenger-specific role for nicotinic acid adenine dinucleotide phosphate in
neuronal differentiation. J. Biol. Chem. 281, 15923–15928
270 Sulem, P., Gudbjartsson, D. F., Stacey, S. N., Helgason, A., Rafnar, T., Jakobsdottir, M.,
Steinberg, S., Gudjonsson, S. A., Palsson, A., Thorleifsson, G. et al. (2008) Two newly
identified genetic determinants of pigmentation in Europeans. Nat. Genet. 40, 835–837
271 Pryor, P. R. and Raines, S. A. (2010) Manipulation of the host by pathogens to survive
the lysosome. Biochem. Soc. Trans. 38, 1417–1419
272 Meikle, P. J., Hopwood, J. J., Clague, A. E. and Carey, W. F. (1999) Prevalence of
lysosomal storage disorders. JAMA, J. Am. Med. Assoc. 281, 249–254
273 Verity, C., Winstone, A. M., Stellitano, L., Will, R. and Nicoll, A. (2010) The
epidemiology of progressive intellectual and neurological deterioration in childhood.
Arch. Dis. Child. 95, 361–364
274 Ioannou, Y. A. (2004) Defects in transmembrane proteins. In Lysosomal Disorders of the
Brain (Platt, F. M. and Walkley, S. U., eds), pp. 206–208, Oxford University Press, Oxford
275 Vanier, M. T. and Millat, G. (2003) Niemann-Pick disease type C. Clin. Genet. 64,
269–281
276 Ko, D. C., Milenkovic, L., Beier, S. M., Manuel, H., Buchanan, J. and Scott, M. P. (2005)
Cell-autonomous death of cerebellar purkinje neurons with autophagy in Niemann-Pick
type C disease. PLoS Genet. 1, 81–95
277 Patterson, M. C., Vanier, M. T., Suzuki, K., Morris, J. A., Carstea, E., Neufeld, E. B.,
Blanchette-Mackie, J. E. and Penchev, P. G. (2001) Niemann-Pick disease type C: a lipid
trafficking disorder. In The Metabolic and Molecular Bases of Inherited Disease (Scriver,
C. R., Beadet, A. L., Valle, D. and Sly, W. S., eds), pp. 3611–3633, McGraw Hill, New
York
278 Love, S., Bridges, L. R. and Case, C. P. (1995) Neurofibrillary tangles in Niemann-Pick
disease type C. Brain 118, 119–129
279 Lloyd-Evans, E. and Platt, F. M. (2010) Lipids on trial: the search for the offending
metabolite in Niemann-Pick type C disease. Traffic 11, 419–428
280 Ikonen, E. and Holtta-Vuori, M. (2004) Cellular pathology of Niemann-Pick type C
disease. Semin. Cell Dev. Biol. 15, 445–454
281 Storch, J. and Xu, Z. (2009) Niemann-Pick C2 (NPC2) and intracellular cholesterol
trafficking. Biochim. Biophys. Acta 1791, 671–678
282 Karten, B., Peake, K. B. and Vance, J. E. (2009) Mechanisms and consequences of
impaired lipid trafficking in Niemann-Pick type C1-deficient mammalian cells. Biochim.
Biophys. Acta 1791, 659–670
283 Ioannou, Y. A. (2000) The structure and function of the Niemann-Pick C1 protein. Mol.
Genet. Metab. 71, 175–181
284 Davies, J. P., Chen, F. W. and Ioannou, Y. A. (2000) Transmembrane molecular pump
activity of Niemann-Pick C1 protein. Science 290, 2295–2298
285 Ioannou, Y. A. (2001) Multidrug permeases and subcellular cholesterol transport. Nat.
Rev. Mol. Cell Biol. 2, 657–668
286 Bach, G., Zeevi, D. A., Frumkin, A. and Kogot-Levin, A. (2010) Mucolipidosis type IV
and the mucolipins. Biochem. Soc. Trans. 38, 1432–1435
287 Bargal, R., Avidan, N., Ben-Asher, E., Olender, Z., Zeigler, M., Frumkin, A.,
Raas-Rothschild, A., Glusman, G., Lancet, D. and Bach, G. (2000) Identification of the
gene causing mucolipidosis type IV. Nat. Genet. 26, 118–123
288 Puertollano, R. and Kiselyov, K. (2009) TRPMLs: in sickness and in health. Am. J.
Physiol. Renal Physiol. 296, F1245–F1254
289 Altarescu, G., Sun, M., Moore, D. F., Smith, J. A., Wiggs, E. A., Solomon, B. I., Patronas,
N. J., Frei, K. P., Gupta, S., Kaneski, C. R. et al. (2002) The neurogenetics of
mucolipidosis type IV. Neurology 59, 306–313
290 Sardiello, M., Palmieri, M., di Ronza, A., Medina, D. L., Valenza, M., Gennarino, V. A.,
Di Malta, C., Donaudy, F., Embrione, V., Polishchuk, R. S. et al. (2009) A gene network
regulating lysosomal biogenesis and function. Science 325, 473–477
291 Bargal, R. and Bach, G. (1988) Phospholipids accumulation in mucolipidosis IV
cultured fibroblasts. J. Inherit. Metab. Dis. 11, 144–150
c The Authors Journal compilation c 2011 Biochemical Society
374
A. J. Morgan and others
292 Goldin, E., Blanchette-Mackie, E. J., Dwyer, N. K., Pentchev, P. G. and Brady, R. O.
(1995) Cultured skin fibroblasts derived from patients with mucolipidosis 4 are
auto-fluorescent. Pediatr. Res. 37, 687–692
293 Bach, G., Cohen, M. M. and Kohn, G. (1975) Abnormal ganglioside accumulation in
cultured fibroblasts from patients with mucolipidosis IV. Biochem. Biophys. Res.
Commun. 66, 1483–1490
294 Bach, G., Ziegler, M., Kohn, G. and Cohen, M. M. (1977) Mucopolysaccharide
accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV.
Am. J. Hum. Genet. 29, 610–618
295 Chen, C. S., Bach, G. and Pagano, R. E. (1998) Abnormal transport along the lysosomal
pathway in mucolipidosis, type IV disease. Proc. Natl. Acad. Sci. U.S.A. 95,
6373–6378
296 Fares, H. and Greenwald, I. (2001) Regulation of endocytosis by CUP-5, the
Caenorhabditis elegans mucolipin-1 homolog. Nat. Genet. 28, 64–68
297 Nakae, I., Fujino, T., Kobayashi, T., Sasaki, A., Kikko, Y., Fukuyama, M., Gengyo-Ando,
K., Mitani, S., Kontani, K. and Katada, T. (2010) The arf-like GTPase Arl8 mediates
delivery of endocytosed macromolecules to lysosomes in Caenorhabditis elegans . Mol.
Biol. Cell 21, 2434–2442
298 Bach, G. (2005) Mucolipin 1: endocytosis and cation channel: a review. Pflügers Arch.
451, 313–317
299 Bach, G., Chen, C. S. and Pagano, R. E. (1999) Elevated lysosomal pH in Mucolipidosis
type IV cells. Clin. Chim. Acta 280, 173–179
300 Soyombo, A. A., Tjon-Kon-Sang, S., Rbaibi, Y., Bashllari, E., Bisceglia, J., Muallem, S.
and Kiselyov, K. (2006) TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid
hydrolytic activity. J. Biol. Chem. 281, 7294–7301
301 Kogot-Levin, A., Zeigler, M., Ornoy, A. and Bach, G. (2009) Mucolipidosis type IV: the
effect of increased lysosomal pH on the abnormal lysosomal storage. Pediatr. Res. 65,
686–690
302 LaPlante, J. M., Ye, C. P., Quinn, S. J., Goldin, E., Brown, E. M., Slaugenhaupt, S. A. and
Vassilev, P. M. (2004) Functional links between mucolipin-1 and Ca2 + -dependent
membrane trafficking in mucolipidosis IV. Biochem. Biophys. Res. Commun. 322,
1384–1391
303 Raychowdhury, M. K., Gonzalez-Perrett, S., Montalbetti, N., Timpanaro, G. A., Chasan,
B., Goldmann, W. H., Stahl, S., Cooney, A., Goldin, E. and Cantiello, H. F. (2004)
Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the
mucolipin-1 cation channel. Hum. Mol. Genet. 13, 617–627
304 Vergarajauregui, S., Martina, J. A. and Puertollano, R. (2009) Identification of the
penta-EF-hand protein ALG-2 as a Ca2 + -dependent interactor of mucolipin-1. J. Biol.
Chem. 284, 36357–36366
305 Lelouvier, B. and Puertollano, R. (2011) Mucolipin-3 regulates luminal calcium,
acidification, and membrane fusion in the endosomal pathway. J. Biol. Chem. 286,
9826–9832
Received 1 June 2011/5 July 2011; accepted 18 July 2011
Published on the Internet 13 October 2011, doi:10.1042/BJ20110949
c The Authors Journal compilation c 2011 Biochemical Society
306 Pelled, D., Lloyd-Evans, E., Riebeling, C., Jeyakumar, M., Platt, F. M. and Futerman,
A. H. (2003) Inhibition of calcium uptake via the sarco/endoplasmic reticulum
Ca2 + -ATPase in a mouse model of Sandhoff disease and prevention by treatment with
N -butyldeoxynojirimycin. J. Biol. Chem. 278, 29496–29501
307 Hackam, D. J., Rotstein, O. D., Zhang, W. J., Demaurex, N., Woodside, M., Tsai, O. and
Grinstein, S. (1997) Regulation of phagosomal acidification. Differential targeting of
Na + /H + exchangers, Na + /K + -ATPases, and vacuolar-type H + -ATPases. J. Biol.
Chem. 272, 29810–29820
308 Thevenod, F. (2002) Ion channels in secretory granules of the pancreas and their role in
exocytosis and release of secretory proteins. Am. J. Physiol. Cell Physiol. 283,
C651–C672
309 Machen, T. E., Leigh, M. J., Taylor, C., Kimura, T., Asano, S. and Moore, H. P. (2003) pH
of TGN and recycling endosomes of H + /K + -ATPase-transfected HEK-293 cells:
implications for pH regulation in the secretory pathway. Am. J. Physiol. Cell Physiol.
285, C205–C214
310 Seksek, O., Biwersi, J. and Verkman, A. S. (1995) Direct measurement of trans-Golgi pH
in living cells and regulation by second messengers. J. Biol. Chem. 270, 4967–4970
311 Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren,
A. P., Bindokas, V. et al. (2006) CFTR regulates phagosome acidification in macrophages
and alters bactericidal activity. Nat. Cell Biol. 8, 933–944
312 Van Dyke, R. W., Root, K. V. and Hsi, R. A. (1996) cAMP and protein kinase A stimulate
acidification of rat liver endosomes in the absence of chloride. Biochem. Biophys. Res.
Commun. 222, 312–316
313 Deriy, L. V., Gomez, E. A., Jacobson, D. A., Wang, X., Hopson, J. A., Liu, X. Y., Zhang,
G., Bindokas, V. P., Philipson, L. H. and Nelson, D. J. (2009) The granular chloride
channel ClC-3 is permissive for insulin secretion. Cell. Metab. 10, 316–323
314 Li, D. Q., Jing, X., Salehi, A., Collins, S. C., Hoppa, M. B., Rosengren, A. H., Zhang, E.,
Lundquist, I., Olofsson, C. S., Morgelin, M. et al. (2009) Suppression of sulfonylureaand glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride
transport protein ClC-3. Cell. Metab. 10, 309–315
315 Mitchell, J., Wang, X., Zhang, G., Gentzsch, M., Nelson, D. J. and Shears, S. B. (2008)
An expanded biological repertoire for Ins(3,4,5,6)P4 through its modulation of ClC-3
function. Curr. Biol. 18, 1600–1605
316 Hara-Chikuma, M., Yang, B., Sonawane, N. D., Sasaki, S., Uchida, S. and Verkman, A. S.
(2005) ClC-3 chloride channels facilitate endosomal acidification and chloride
accumulation. J. Biol. Chem. 280, 1241–1247
317 Llopis, J., McCaffery, J. M., Miyawaki, A., Farquhar, M. G. and Tsien, R. Y. (1998)
Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green
fluorescent proteins. Proc. Natl. Acad. Sci. U.S.A. 95, 6803–6808
318 Jentsch, T. J., Maritzen, T., Keating, D. J., Zdebik, A. A. and Thévenod, F. (2010) ClC-3 a
granular anion transporter involved in insulin secretion? Cell. Metab. 12, 307–308
319 Renström, E. (2010) Response to Jentsch et al. Cell. Metab. 12, 309
Biochem. J. (2011) 439, 349–374 (Printed in Great Britain)
doi:10.1042/BJ20110949
SUPPLEMENTARY ONLINE DATA
Molecular mechanisms of endolysosomal Ca2 + signalling in health
and disease
Anthony J. MORGAN*1 , Frances M. PLATT*, Emyr LLOYD-EVANS† and Antony GALIONE*1
*Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K., and †School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, U.K.
Table S1
Acid vesicle elemental composition determined by microprobe analysis
√ √√√
Data expressed in nmol/mg dry weight (equivalent to mmol/kg), mM (§) or as relative abundance ( to
) when not quantified. nmol/mg dry weight ≈mM [8].
Organelle
Source
Na
K
Mg
Cl
Ca
P
S
Zn
Reference
Acidocalcisome
Trypansoma cruzi
Leishmania major
Sea urchin egg (Paracentrotus lividus )
Sea urchin egg (Arbacia lixula )
Sea urchin egg (Arbacia punctulata )
Porcine platelet
Pancreatic β cell
Vascular smooth muscle
Duodenal absorptive cells
Renal proximal tubules
Drosophila S2 cells
Murine Balb/c macrophages
Murine C57BL/6 macrophages
Sea urchin egg (P. lividus )
Sea urchin egg (A. lixula )
161
148
37
237
646
515
√
√
171
39
32
97
√√
1390
1216
34
34
√√√
10
−3
64
159
√√
148
74
√√√
2
68
370
536
√
77
50*
169
73
436
202
5
479
31
3
14
√√√
290
√√√
333
170
34
412
263
0.8
36.8
36
21
222
56
10.6
151.5
192
503
7.7
4.6
9.5
0.2
4.6
10.3
201
482
54.4
14.0
19.8
29.5
389
122
20.5
18.5
19.7
60
[1]
[1]
[2]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]§
[10]§
[2]
[2]
Cortical granule
Dense (‘clear’) granule
Alpha granule
Insulin granule
Lysosome
Phagosome
Yolk granule
√
0.8
0.13
*Determined by flame photometry.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
A. J. Morgan and others
Table S2
Acidic store free [ion] and membrane potential
CHO, Chinese hamster ovary; eGFP, enhanced green fluorescent protein; YFP, yellow fluorescent protein.
Parameter
Organelle
Cell type
Resting value*
Method
Reference(s)
Ca2 +
Trans -Golgi
HeLa cell
HeLa cell
3T3 Swiss fibroblast
Phagosome
Human neutrophils
0.58 μM
Secretory vesicle
MIN-6 pancreatic β cell
Bovine adrenal chromaffin cell
PC12
Porcine Platelet
Bovine adrenal chromaffin cell
Mouse macrophage
40–51 μM
50–100 μM‡
1.4 μM
12 μM
5.6 μM
400–600 μM
Human fibroblast
550 μM
6.7
6.2
6.4
6.2
6.3
5.7
6.9 (3 min† ) 5.7 (20 min† )
5.9
5.6
5.9
6.4
5.0
5–6
[12]
[23]
[24]
[25]
[26]
[25]
[13]
[14]
[25]
[26]
[24]
[27]
[28]
Golgi
Endosome
Phagosome
Secretory vesicle
Lysosome
Lysosome
Secretory Vesicle
Lysosome
Golgi
Endosome
HeLa cell
Human fibroblast
HEK-293 cell
HeLa cells
Human foreskin keratinocyte
Hippocampal neuron
3T3 Swiss fibroblast
Pancreatic acinar cell
HeLa cells
Human foreskin keratinocyte
HEK-293 cell
Mouse macrophage
Mouse pancreatic β cell and
MIN-6 cell
MIN-6 pancreatic β cell
Bovine adrenal chromaffin cell
PC12
Sea urchin egg
Mouse macrophage
Human fibroblast
MDCK II Epithelial
Natural killer cell
Vero kidney epithelium
Rat liver
RAW264.7 macrophage
Bovine adrenal chromaffin cell
RAW264.7 macrophage
Rat liver
Bovine adrenal chromaffin cell
RAW264.7 macrophage
Vero kidney epithelium
J744 cell and CHO cell
Golgi–aequorin
Golgi–D1cpv
Endocytosed Oregon Green
BAPTA-5N
Endocytosed Oregon Green
BAPTA-5N
Phagocytosed fura-2 zymosan
particle
VAMP–aequorin
VAMP–aequorin
Chromogranin–aequorin
Null-point
Null-point
Endocytosed fura-2 dextran/Oregon
Green BAPTA dextran
Endocytosed Rhod dextran (low
affinity)
Golgi–D1cpv (YFP fluorescence)
Fluorescein (via liposomes)
TGN38–pHLuorin
TGN–pHLuorin
TGN38–pHLuorin
Synapto–pHLuorin
Endocytosed FITC–dextran
Endocytosed FITC–dextran
pHLuorin
Cellubrevin–pHLuorin
Synaptobrevin–pHLuorin
Carboxyfluorescein beads
Lysosensor Yellow/Blue DND-160
[11]
[12]
[13]
Pancreatic acinar cell
300 μM
130 μM
29 μM (3 min† )
3 μM (20 min† )
37 μM
VAMP–pHLuorin
VAMP–EGFP
Neuropeptide Y–ClopHensor
Lysosensor Yellow/Blue DND-160
Endocytosed FITC–dextran
Endocytosed FITC–dextran
Endocytosed Oregon Green–dextran
Granzyme B–eGFP
Null-point
Null-point
Null-point
Null-point
Null-point
Atomic absorption spectrometry
Null-point
Null-point
Endocytosed BAC–dextran
Endocytosed BAC–dextran
[16]
[18]
[29]
[30]
[21]
[22]
[31]
[32]
[33]
[34]
[35]
[20]
[36]
[37]
[20]
[36]
[38]
[39]
Recycling endosome
J744 cell and CHO cell
Endocytosed BAC–dextran
[38]
Recycling endosome
Late endosome
Murine hepatocyte
J744 & CHO cells
6.3
5.5
5.2
5.5
4.0
4.7
4.5
<5.0
107 mM
67 mM
∼ 20 mM
22.8 mM (30–42 mM total)
50-62 mM
50 mM (total)
18.8 mM (17–36 mM total)
20.6 mM
49 mM
17 mM (53 mM, 45 min later)
28 mM (73 mM, 45 min
later)
18–24 mM (40–46 mM, 15 min
later)
16 mM (46 mM, 15 min later)
28 mM (58 mM, 45 min later)
[40]
[38]
Murine hepatocyte
∼ 20 mM (58 mM 45 min later)
Secretory vesicle
Secretory vesicle
Phagosome
PC12
INS-1 pancreatic β cell
RAW264.7 cell
110 mM
1–100 μM
+ 27 mV
Endocytosed BAC–dextran
Endocytosed
BAC-α 2 –macroglobulin
Endocytosed
BAC-α 2 –macroglobulin
Neuropeptide Y–ClopHensor
VAMP2–eCALWY
DiBAC4 & DACCA (FRET pair)
Endosome [MVB
(multivesicular body)]
Recycling endosome
Lysosome
Rat liver
Rat liver
+ 6–42 mV**
[14 C]methylamine or 36 Cl −
[42]
Rat liver
Rat kidney cortex
+ 1–69 mV**
+ 33 mV
+ 20–40 mV
+ 19 mV
[14 C]methylamine or 36 Cl −
S[14 C]CN −
diS–C3 -(5)
DiBAC4 /Rhodamine or
DiSBAC2 /DACCA (FRET pairs)
[42]
[43]
[37]
[44]
Endosome
Lysosome
pHL
Trans -Golgi
Endosome
Recycling endosome
Phagosome
Secretory vesicle
Lysosome
K+
Na +
Cl −
Zn2 +
Resting membrane
potential (ψ)§
RAW264.7 cell
c The Authors Journal compilation c 2011 Biochemical Society
[14]
[15]
[16,17]
[18]
[19]
[4]
[20]
[21]
[22]
[40]
[29]
[41]
[35]
Endolysosomal Ca2 + signalling in health and disease
Table S2
Continued
Parameter
Organelle
Cell type
Resting value*
Method
Reference(s)
Secretory vesicle
Bovine adrenal
Bovine pituitary
Barley
Green alga
+ 100 mV
+ 10 mV¶
+ 9– + 23 mV
+ 2 mV
S[14 C]CN −
S[14 C]CN −
Electrophysiology
Electrophysiology
[45]
[46]
[47]
[48]
Vacuole
*Free concentration of ion or pH value.
†Time after endocytosis.
‡Probably a 3–4-fold underestimate because the actual pHL was not factored in, as conceded by the authors [18].
§Lumen-positive ψ.
¶In presence of KCl, but is possibly an overestimate since the extra-granular pH was 6.1.
For consistency, the sign has been reversed from the cytosol-negative values originally reported.
**Depending on the extra-vesicular KCl concentration.
REFERENCES
1 Docampo, R., de Souza, W., Miranda, K., Rohloff, P. and Moreno, S. N. (2005)
Acidocalcisomes: conserved from bacteria to man. Nat. Rev. Microbiol. 3, 251–261
2 Gillot, I., Ciapa, B., Payan, P. and Sardet, C. (1991) The calcium content of cortical
granules and the loss of calcium from sea urchin eggs at fertilization. Dev. Biol. 146,
396–405
3 Ramos, I. B., Miranda, K., Pace, D. A., Verbist, K. C., Lin, F. Y., Zhang, Y., Oldfield, E.,
Machado, E. A., De Souza, W. and Docampo, R. (2010) Calcium- and
polyphosphate-containing acidic granules of sea urchin eggs are similar to
acidocalcisomes, but are not the targets for NAADP. Biochem. J. 429, 485–495
4 Grinstein, S., Furuya, W., Vander Meulen, J. and Hancock, R. G. (1983) The total and free
concentrations of Ca2 + and Mg2 + inside platelet secretory granules. Measurements
employing a novel double null point technique. J. Biol. Chem. 258, 14774–14777
5 Nakagaki, I., Sasaki, S., Hori, S. and Kondo, H. (2000) Ca2 + and electrolyte mobilization
following agonist application to the pancreatic beta cell line HIT. Pflügers Arch. 440,
828–834
6 James-Kracke, M. R., Sloane, B. F., Shuman, H. and Somlyo, A. P. (1979) Lysosomal
composition in cultured vascular smooth muscle cells: electron probe analysis. Proc.
Natl. Acad. Sci. U.S.A. 76, 6461–6465
7 Davis, W. L., Jones, R. G. and Hagler, H. K. (1979) Calcium-containing lysosomes in the
normal chick duodenum: a histochemical and analytical electron microscopic study.
Tissue Cell 11, 127–138
8 LeFurgey, A., Spencer, A. J., Jacobs, W. R., Ingram, P. and Mandel, L. J. (1991) Elemental
microanalysis of organelles in proximal tubules. I. Alterations in transport and
metabolism. J. Am. Soc. Nephrol. 1, 1305–1320
9 Yagodin, S., Pivovarova, N. B., Andrews, S. B. and Sattelle, D. B. (1999) Functional
characterization of thapsigargin and agonist-insensitive acidic Ca2 + stores in Drosophila
melanogaster S2 cell lines. Cell Calcium 25, 429–438
10 Wagner, D., Maser, J., Moric, I., Vogt, S., Kern, W. V. and Bermudez, L. E. (2006)
Elemental analysis of the Mycobacterium avium phagosome in Balb/c mouse
macrophages. Biochem. Biophys. Res. Commun. 344, 1346–1351
11 Pinton, P., Pozzan, T. and Rizzuto, R. (1998) The Golgi apparatus is an inositol
1,4,5-trisphosphate-sensitive Ca2 + store, with functional properties distinct from those
of the endoplasmic reticulum. EMBO J. 17, 5298–5308
12 Lissandron, V., Podini, P., Pizzo, P. and Pozzan, T. (2010) Unique characteristics of Ca2 +
homeostasis of the trans-Golgi compartment. Proc. Natl. Acad. Sci. U.S.A. 107,
9198–9203
13 Gerasimenko, J. V., Tepikin, A. V., Petersen, O. H. and Gerasimenko, O. V. (1998) Calcium
uptake via endocytosis with rapid release from acidifying endosomes. Curr. Biol. 8,
1335–1338
14 Sherwood, M. W., Prior, I. A., Voronina, S. G., Barrow, S. L., Woodsmith, J. D.,
Gerasimenko, O. V., Petersen, O. H. and Tepikin, A. V. (2007) Activation of trypsinogen in
large endocytic vacuoles of pancreatic acinar cells. Proc. Natl. Acad. Sci. U.S.A. 104,
5674–5679
15 Lundqvist-Gustafsson, H., Gustafsson, M. and Dahlgren, C. (2000) Dynamic Ca2 +
changes in neutrophil phagosomes A source for intracellular Ca2 + during
phagolysosome formation? Cell Calcium 27, 353–362
16 Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R.
and Rutter, G. A. (2001) Dense core secretory vesicles revealed as a dynamic Ca2 + store
in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera.
J. Cell Biol. 155, 41–51
17 Mitchell, K. J., Lai, F. A. and Rutter, G. A. (2003) Ryanodine receptor type I and nicotinic
acid adenine dinucleotide phosphate receptors mediate Ca2 + release from
insulin-containing vesicles in living pancreatic β cells (MIN6). J. Biol. Chem. 278,
11057–11064
18 Santodomingo, J., Vay, L., Camacho, M., Hernandez-Sanmiguel, E., Fonteriz, R. I.,
Lobaton, C. D., Montero, M., Moreno, A. and Alvarez, J. (2008) Calcium dynamics in
bovine adrenal medulla chromaffin cell secretory granules. Eur. J. Neurosci. 28,
1265–1274
19 Mahapatra, N. R., Mahata, M., Hazra, P. P., McDonough, P. M., O’Connor, D. T. and
Mahata, S. K. (2004) A dynamic pool of calcium in catecholamine storage vesicles:
exploration in living cells by a novel vesicle-targeted chromogranin A/aequorin chimeric
photoprotein. J. Biol. Chem. 79, 51107–51121
20 Haigh, J. R., Parris, R. and Phillips, J. H. (1989) Free concentrations of sodium,
potassium and calcium in chromaffin granules. Biochem. J. 259,
485–491
21 Christensen, K. A., Myers, J. T. and Swanson, J. A. (2002) pH-dependent regulation of
lysosomal calcium in macrophages. J. Cell Sci. 115, 599–607
22 Lloyd-Evans, E., Morgan, A. J., He, X., Smith, D. A., Elliot-Smith, E., Sillence, D. J.,
Churchill, G. C., Schuchman, E. H., Galione, A. and Platt, F. M. (2008) Niemann-Pick
disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal
calcium. Nat. Med. 14, 1247–1255
23 Seksek, O., Biwersi, J. and Verkman, A. S. (1995) Direct measurement of trans-Golgi pH
in living cells and regulation by second messengers. J. Biol. Chem. 270,
4967–4970
24 Machen, T. E., Leigh, M. J., Taylor, C., Kimura, T., Asano, S. and Moore, H. P. (2003) pH
of TGN and recycling endosomes of H + /K + -ATPase-transfected HEK-293 cells:
implications for pH regulation in the secretory pathway. Am. J. Physiol. Cell Physiol.
285, C205–C214
25 Miesenbock, G., De Angelis, D. A. and Rothman, J. E. (1998) Visualizing secretion and
synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394,
192–195
26 Disbrow, G. L., Hanover, J. A. and Schlegel, R. (2005) Endoplasmic reticulum-localized
human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH.
J. Virol. 79, 5839–5846
27 Yates, R. M., Hermetter, A. and Russell, D. G. (2005) The kinetics of phagosome
maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic
activity. Traffic 6, 413–420
28 Stiernet, P., Guiot, Y., Gilon, P. and Henquin, J. C. (2006) Glucose acutely decreases pH of
secretory granules in mouse pancreatic islets. Mechanisms and influence on insulin
secretion. J. Biol. Chem. 281, 22142–22151
29 Arosio, D., Ricci, F., Marchetti, L., Gualdani, R., Albertazzi, L. and Beltram, F. (2010)
Simultaneous intracellular chloride and pH measurements using a GFP-based sensor.
Nat. Methods 7, 516–518
30 Haley, S. A. and Wessel, G. M. (2004) Regulated proteolysis by cortical granule serine
protease-1 at fertilization. Mol. Biol. Cell 15, 2084–2092
31 Haggie, P. M. and Verkman, A. S. (2009) Unimpaired lysosomal acidification in
respiratory epithelial cells in cystic fibrosis. J. Biol Chem. 284,
7681–7686
c The Authors Journal compilation c 2011 Biochemical Society
A. J. Morgan and others
32 Bird, C. H., Rizzitelli, A., Harper, I., Prescott, M. and Bird, P. I. (2010) Use of granzyme
B-based fluorescent protein reporters to monitor granzyme distribution and granule
integrity in live cells. Biol. Chem. 391, 999–1004
33 Schapiro, F. B. and Grinstein, S. (2000) Determinants of the pH of the Golgi complex.
J. Biol. Chem. 275, 21025–21032
34 Van Dyke, R. W. (1995) Na + /H + exchange modulates acidification of early rat liver
endocytic vesicles. Am. J. Physiol. 269, C943–C954
35 Steinberg, B. E., Touret, N., Vargas-Caballero, M. and Grinstein, S. (2007) In situ
measurement of the electrical potential across the phagosomal membrane using FRET
and its contribution to the proton-motive force. Proc. Natl. Acad. Sci. U.S.A. 104,
9523–9528
36 Steinberg, B. E., Huynh, K. K., Brodovitch, A., Jabs, S., Stauber, T., Jentsch, T. J. and
Grinstein, S. (2010) A cation counterflux supports lysosomal acidification. J. Cell Biol.
189, 1171–1186
37 Ohkuma, S., Moriyama, Y. and Takano, T. (1983) Electrogenic nature of lysosomal proton
pump as revealed with a cyanine dye. J. Biochem. 94, 1935–1943
38 Sonawane, N. D. and Verkman, A. S. (2003) Determinants of [Cl − ] in recycling and late
endosomes and Golgi complex measured using fluorescent ligands. J. Cell Biol. 160,
1129–1138
39 Sonawane, N. D., Thiagarajah, J. R. and Verkman, A. S. (2002) Chloride concentration in
endosomes measured using a ratioable fluorescent Cl − indicator: evidence for chloride
accumulation during acidification. J. Biol. Chem. 277, 5506–5513
Received 1 June 2011/5 July 2011; accepted 18 July 2011
Published on the Internet 13 October 2011, doi:10.1042/BJ20110949
c The Authors Journal compilation c 2011 Biochemical Society
40 Hara-Chikuma, M., Yang, B., Sonawane, N. D., Sasaki, S., Uchida, S. and Verkman, A. S.
(2005) ClC-3 chloride channels facilitate endosomal acidification and chloride
accumulation. J. Biol. Chem. 280, 1241–1247
41 Vinkenborg, J. L., Nicolson, T. J., Bellomo, E. A., Koay, M. S., Rutter, G. A. and Merkx, M.
(2009) Genetically encoded FRET sensors to monitor intracellular Zn2 + homeostasis.
Nat. Methods 6, 737–740
42 Van Dyke, R. W. and Belcher, J. D. (1994) Acidification of three types of liver endocytic
vesicles: similarities and differences. Am. J. Physiol. 266, C81–C94
43 Harikumar, P. and Reeves, J. P. (1983) The lysosomal proton pump is electrogenic.
J. Biol. Chem. 258, 10403–10410
44 Koivusalo, M., Steinberg, B. E., Mason, D. and Grinstein, S. (2011) In situ measurement
of the electrical potential across the lysosomal membrane using FRET. Traffic 12, 972–982
45 Rottenberg, H. (1979) The measurement of membrane potential and pH in cells,
organelles, and vesicles. Methods Enzymol. 55, 547–569
46 Scherman, D., Nordmann, J. and Henry, J. P. (1982) Existence of an adenosine
5’-triphosphate-dependent proton translocase in bovine neurosecretory granule
membrane. Biochemistry 21, 687–694
47 Walker, D. J., Leigh, R. A. and Miller, A. J. (1996) Potassium homeostasis in vacuolate
plant cells. Proc. Natl. Acad. Sci. U.S.A. 93, 10510–10514
48 Bethmann, B., Thaler, M., Simonis, W. and Schonknecht, G. (1995) Electrochemical
potential gradients of H + , K + , Ca2 + , and Cl − across the tonoplast of the green alga
Eremosphaera viridis . Plant Physiol. 109, 1317–1326