Cell Calcium 38 (2005) 303–310
Intraluminal calcium as a primary regulator
of endoplasmic reticulum function
Denis Burdakov a , Ole H. Petersen b , Alexei Verkhratsky a,∗
a
Faculty of Life Sciences, The University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, UK
b MRC Group, Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Received 20 June 2005; accepted 28 June 2005
Available online 1 August 2005
Abstract
The concentration of Ca2+ inside the lumen of endoplasmic reticulum (ER) regulates a vast array of spatiotemporally distinct cellular
processes, from intracellular Ca2+ signals to intra-ER protein processing and cell death. This review summarises recent data on the mechanisms
of luminal Ca2+ -dependent regulation of Ca2+ release and uptake as well as ER regulation of cellular adaptive processes. In addition we discuss
general biophysical properties of the ER membrane, as trans-endomembrane Ca2+ fluxes are subject to basic electrical forces, determined by
factors such as the membrane potential of the ER and the ease with which Ca2+ fluxes are able to change this potential (i.e. the resistance of
the ER membrane). Although these electrical forces undoubtedly play a fundamental role in shaping [Ca2+ ]ER dynamics, at present there is
very little direct experimental information about the biophysical properties of the ER membrane. Further studies of how intraluminal [Ca2+ ]
is regulated, best carried out with direct measurements, are vital for understanding how Ca2+ orchestrates cell function. Direct monitoring of
[Ca2+ ]ER under conditions where the cytosolic [Ca2+ ] is known may also help to capture elusive biophysical information about the ER, such
as the potential difference across the ER membrane.
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Endoplasmic reticulum; Calcium; Signalling; SERCA; Neurodegeneration
1. Introduction
Integration of multiple intracellular signalling events is
largely accomplished by the endoplasmic reticulum (ER),
which extends through all parts of eukaryotic cells. The ER
membrane (endomembrane) encloses an internally continuous ER lumen, which forms a specialised pathway for communication between different cellular compartments [1–6].
The integrative function of the ER is assisted by numerous
molecular cascades, which allow it to decode incoming information and produce output signals affecting cellular function
in different temporal and spatial domains [7,8]. Ca2+ acts as
a fundamentally important signalling messenger involved in
regulation of numerous vital functions, and the ER plays a
major role in fast physiological signalling due to its ability
∗
Corresponding author. Tel.: +44 161 2755414; fax: +44 161 2755948.
E-mail address: [email protected] (A. Verkhratsky).
0143-4160/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2005.06.010
to act as a dynamic Ca2+ store, which can accumulate Ca2+
to levels 1000s of times greater than [Ca2+ ] in the cytosol.
The ER Ca2+ store participates in the generation of rapid
intracellular Ca2+ signals following chemical activation of
plasmalemmal receptors or electrical excitation of the plasma
membrane [6,9–15]. Furthermore, the ER acts as a powerful intracellular Ca2+ buffer, important for removal of the
excess Ca2+ accumulated during physiological stimulation
[16–18]. In addition, the ER functions as a key cellular transport system that traffics relevant molecules to various cellular
destinations. Finally, the ER is intimately involved in protein synthesis and posttranslational modification. The latter
is governed by multiple intra-ER resident chaperones, which
ensure correct folding of proteins [19,20].
The concentration of Ca2+ inside the ER lumen ([Ca2+ ]ER )
acts as a fundamental determinant of ER homeostasis,
controlling activation of ER Ca2+ release channels, the
endomembrane Ca2+ uptake mechanism and numerous enzy-
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D. Burdakov et al. / Cell Calcium 38 (2005) 303–310
matic cascades. In this paper, we present an overview of
how intra-ER Ca2+ coordinates cell function, and also speculate about what direct measurements of [Ca2+ ]ER may tell
us about the poorly understood biophysical properties of the
ER membrane.
2. Intraluminal Ca2+ and regulation of
endomembrane Ca2+ fluxes
Direct measurements of [Ca2+ ]ER in living cells with various techniques (e.g. ER targeted aequorin [21,22], fluorescent
Ca2+ -sensitive proteins [23,24] or synthetic fluorescent Ca2+
indicators [25–29]) convincingly demonstrated that [Ca2+ ]ER
varies within the range of 100–800 ␮M [6,15,22,26,29,30].
This very high intra-ER Ca2+ concentration provides a driving force for Ca2+ movement from the ER lumen to the
cytosol and executes direct control over the amplitude and
velocity of Ca2+ release. In addition to this direct biophysical
control, intraluminal Ca2+ allosterically regulates the availability of intracellular Ca2+ release channels and controls the
velocity of Ca2+ uptake.
2.1. [Ca2+ ]ER regulates Ca2+ release channels
Both types of Ca2+ release channels in the endomembrane, the ryanodine receptor (RyR) and the InsP3 receptor
(InsP3 R), are regulated by [Ca2+ ]ER . Several lines of evidence demonstrate that changes in [Ca2+ ]ER directly affect
the open probability of RyRs and modulate their sensitivity
to activation by both cytosolic Ca2+ and caffeine. Initial indications for direct regulation of Ca2+ release through RyRs
were obtained in skinned cardiac myocytes [31] and in isolated triades from frog and rabbit skeletal muscle [32]. In
the latter case, for example, an increase in luminal Ca2+
concentration from 0.05 to 0.7 mM resulted in a 20-fold
elevation of the release rate constant. These initial findings
were confirmed by observations on intact cardiomyocytes, in
which an elevation in sarcoplasmic reticulum Ca2+ content
increased global Ca2+ release in a highly non-linear fashion, suggesting a positive regulation of RyRs by [Ca2+ ]ER
[33,34]. This was further corroborated by direct measurements of the open probability (Po ) of single RyR channels,
which clearly demonstrated that an increase of [Ca2+ ] on the
luminal side of the receptor greatly (4–6 times) increases
the channel Po [35–39]. Such sensitivity of the RyR gating
to [Ca2+ ]ER naturally implied the existence of a Ca2+ sensor located on the luminal side of the receptor, and the first
indication for an existence of such a sensor was obtained
by trypsin digestion of the luminal part of RyRs incorporated into planar lipid membranes, which prevented luminal
Ca2+ from enhancing the Po of RyRs [36]. Further analysis revealed a rather complicated intraluminal Ca2+ -sensing
machinery associated with RyRs. The latter appeared to be a
complex of three proteins, triadin 1, junctin and calsequestrin
which, working in concert, act as a [Ca2+ ]ER sensor and regu-
late the gating of RyRs according to fluctuations of [Ca2+ ]ER
[35].
In addition, an increase in [Ca2+ ]ER affects the sensitivity
of RyRs to activation by cytosolic Ca2+ and caffeine. That
is, an increase in sarcoplasmic reticulum’s Ca2+ load significantly (several fold) elevated the frequency of Ca2+ sparks in
muscle cells [40–42]. In PC12 cells and in isolated DRG neurones, loading of the ER with Ca2+ markedly increased the
sensitivity of Ca2+ release to caffeine, decreasing the apparent EC50 for caffeine almost 3 times [43–45].
The initial suggestion that another endomembraneresident channel, the InsP3 R, may be regulated by [Ca2+ ]ER
was proposed by Irvine, who postulated the existence of a
Ca2+ sensor on the luminal part of the InsP3 R, which regulates the sensitivity of the receptor to activation by cytosolic
InsP3 [46]. This hypothesis was experimentally tested in permeabilised hepatocytes and it appeared that an increase in
[Ca2+ ]ER elevates the overall sensitivity of InsP3 -induced
Ca2+ release to InsP3 levels [47], and even promotes spontaneous Ca2+ release [48]. This [Ca2+ ]ER regulation of InsP3 induced Ca2+ release was subsequently confirmed on other
cell types (e.g. [49–51]. Yet the existence of direct regulation of InsP3 Rs by [Ca2+ ]ER remains controversial, and has
been disputed by several groups [52,53], although a recently
observed direct correlation between [Ca2+ ]ER and InsP3 induced Ca2+ release still suggests a modulatory effect of
the filling state of the store on Ca2+ release through InsP3 Rs
[54].
2.2. [Ca2+ ]ER regulates SERCA-mediated Ca2+ uptake
Ca2+ accumulation into the lumen of the ER is mediated
by specific endomembrane Ca2+ pumps, Sarco(Endo)plasmic
Reticulum Ca2+ ATPases (SERCAs) [55–57], several subtypes of which (SERCAs 1–3) are variously expressed in
eukaryotic cells. Considerable evidence indicates an important regulatory role of [Ca2+ ]ER in controlling the SERCAdependent Ca2+ uptake into the ER lumen. Already in the
early experiments on SR/ER vesicles obtained from muscle cells [58], or from HL-60 cell lines [59], it was found
that a high intra-vesicular Ca2+ level effectively inhibits Ca2+
uptake. Subsequently, it was demonstrated that clamping the
cytosolic [Ca2+ ] at relatively low levels does not significantly affect ER Ca2+ uptake as long as a sufficient quantity
of Ca2+ is present in the cytosolic compartment [22,60].
Furthermore, when [Ca2+ ]ER levels were directly correlated
with Ca2+ uptake velocity [26,29], a decrease in [Ca2+ ]ER
markedly increased the velocity of SERCA-dependent Ca2+
uptake. In these experiments, [Ca2+ ]ER was measured using a
Mag-Fura-2-based technique in two very different cell types,
mouse pancreatic acinar cells [26] and sensory neurones
isolated from dorsal root ganglia of rats [29]. The velocity of Ca2+ uptake was determined after maximal depletion
of the ER Ca2+ store by supra-maximal concentrations of
acetylcholine (pancreatic acinar cells) or caffeine (neurones),
and corrected for Ca2+ leak from the ER (determined from
D. Burdakov et al. / Cell Calcium 38 (2005) 303–310
305
Fig. 1. [Ca2+ ]ER regulates the rate of Ca2+ uptake into the ER. Upper panels shows [Ca2+ ]ER ([Ca2+ ]L/Lu ) recordings from a single DRG neurone (A) or
pancreatic acinar cell (B) treated with 20 mM caffeine or 10 ␮M acetylcholine (ACh) and 5 ␮M thapsigargin (TG) as indicated on the graph. Lower panels
show the relationship between Ca2+ uptake/Ca2+ leakage rates and [Ca2+ ]ER . In both cases [Ca2+ ]c was clamped at ∼90–100 nM by introduction of a 10 mM
BAPTA/2 mM Ca2+ buffer into the intra-pipette solution. Figures are modified from [6] (A), and [26].
thapsigargin-induced slow depletion of the store) (Fig. 1).
In both cases, agonist-induced Ca2+ depletion of the ER led
to a remarkable (5–8 times) increase in the velocity of Ca2+
uptake, which decreased in parallel with the replenishment
of the store. Most remarkably, in the neurones, clamping
the cytosolic [Ca2+ ] at ∼90 nM did not affect the relations
between [Ca2+ ]ER and the Ca2+ uptake velocity [6,29], clearly
pointing at a leading role of luminal Ca2+ in the regulation
of SERCA-dependent Ca2+ transport. In the pancreatic acinar cells, the relationship between [Ca2+ ]ER and the rate of
Ca2+ reuptake into the ER following acetylcholine-elicited
release was studied under conditions of an effective cytosolic [Ca2+ ] clamp ([Ca2+ ]c was measured to be very close to
100 nM) [26] and these experiments therefore demonstrated
directly that SERCA-mediated Ca2+ reuptake into the ER can
be regulated exclusively from the luminal side. Nevertheless,
SERCA is a Ca2+ -activated enzyme and local rises in [Ca2+ ]c
would be expected to have activating effects on the ER Ca2+
uptake. The dominant SERCA pump in the pancreatic acinar cells is SERCA2b and recently a kinetic model for this
subtype of the enzyme has been worked out [61]. The model
demonstrates clearly the very major rise in the turnover rate
of the pump when [Ca2+ ]ER is reduced from the resting level
of about 300 ␮M to the depleted level of around 30 ␮M, but
also shows the activating effect of increasing [Ca2+ ]c from
the resting level of about 100 nM towards 1 ␮M [61], which
may be responsible for an increase in [Ca2+ ]ER following
stimulation-induced cytosolic Ca2+ loads [62–64].
The molecular pathways responsible for [Ca2+ ]ER dependent regulation of SERCA pumps were recently uncovered in a series of experiments performed by Camacho and
her co-workers [65–67]. They found that co-expression of
the SERCA 2b Ca2+ pump with the ER-resident chaperones
calreticulin or ER protein 57 (ERp57; also known as ER60, GRP58) in Xenopus oocytes inhibits ER Ca2+ uptake (as
judged by a decrease in the frequency of InsP3 -dependent
Ca2+ oscillations). They further showed that ERp57 promotes
disulphide bond formation in the L4 portion of SERCA 2b,
which leads to inhibition of Ca2+ pumping activity. Finally,
they demonstrated that the complex of calreticulin-ERp57
binds to SERCA 2b in a Ca2+ -dependent manner, resulting in
greater inhibition of Ca2+ pumps at higher [Ca2+ ]ER . Lowering [Ca2+ ]ER triggered dissociation of the calreticulin-ERp57
complex from the SERCA 2b, which in turn increased the
activity of the Ca2+ pump [67].
3. Implications of intraluminal Ca2+ dynamics for
the elusive biophysics of ER membrane
In addition to being dependent on specialized Ca2+ transport pathways and the concentration gradient for Ca2+ across
the endomembrane, trans-endomembrane Ca2+ fluxes are
also influenced by the membrane potential of the ER and the
ease with which Ca2+ fluxes are able to change this potential
(i.e. the resistance of the ER membrane). Although these elec-
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D. Burdakov et al. / Cell Calcium 38 (2005) 303–310
trical factors undoubtedly play a fundamental role in shaping
[Ca2+ ]ER dynamics, at present there is very little direct experimental information about the biophysical properties of the ER
membrane. Yet, at least some information may be extracted
from the direct measurements of [Ca2+ ]ER under conditions
where the cytosolic Ca2+ concentration ([Ca2+ ]c ) is known
[6,26]. In the following sections, we discuss some potential
biophysical implications of [Ca2+ ]ER measurements. While
our discussion is largely theoretical, we hope that the arguments presented may help to establish a framework for understanding the electrophysiological determinants of [Ca2+ ]ER
dynamics.
3.1. ER membrane resistance: rapid Ca2+ release
argues for a low value
In biological membranes, transmembrane movement of
ionic charge tends to shift the membrane potential (V) towards
the equilibrium potential (E) for the charge-carrying ion(s).
If the membrane potential is easy to shift (i.e. if the resting membrane resistance is high), this process will impose
an “electrical brake” on the ionic current by reducing the
electro–chemical driving force for ionic movements (equal
to the difference between V and E), which would progressively slow down the current and substantially reduce its
maximum amplitude. Yet the reductions in [Ca2+ ]ER resulting from experimental opening of Ca2+ release channels can
be relatively large (100s of ␮M) and have rapid kinetics
[6,22,26]. This implies that Ca2+ release is unlikely to be
severely limited by the electrical brake, i.e. the membrane
potential of the ER is unlikely to be shifted markedly by the
process of Ca2+ release. Because Ca2+ release occurs through
relatively selective channels, the simplest explanation for this
phenomenon is that the ER membrane has a very low resting resistance (high resting conductance), which stabilizes
the ER membrane potential and allows fast charge compensation during Ca2+ release. The ionic nature of the baseline
ER conductance(s) that may be responsible for a low resting
resistance of the ER membrane would have important implication for Ca2+ movements in and out of the ER, because
these conductances would set the ER membrane potential
(VER ).
3.2. ER membrane potential: measurements of
[Ca2+ ]ER tell us which values are unlikely
Until now little experimental or theoretical information
emerged about what values VER may have. However, because
not all values of VER would allow Ca2+ to exit the ER, when
the release channels are open, we may obtain information
about what values VER cannot have from measurements of
channel-mediated efflux of Ca2+ from the ER at fixed and
known [Ca2+ ]c [6,26]. No net Ca2+ flow, and thus no changes
in [Ca2+ ]ER , would occur if VER is equal to the electrochemical equilibrium potential for Ca2+ (ECa ), given by the Nernst
equation:
ECa =
RT
[Ca2+ ]c
ln
zF
[Ca2+ ]ER
(1)
If VER is more inside-negative than ECa , Ca2+ will enter,
rather than leave, the ER when the Ca2+ release channels
are open. ECa thus gives the most negative value of VER
that is compatible with Ca2+ efflux from the ER. When
[Ca2+ ]c is clamped at around 90 nM by cytosolic infusion
of a BAPTA/Ca2+ mixture, a fall in [Ca2+ ]ER can occur from
a baseline of about 300 ␮M in sensory neurons and pancreatic
acinar cells (Fig. 1). Because ECa in these conditions is about
−100 mV (from Eq. (1)), this indicates that VER must be less
inside-negative than −100 mV in these cells, at least before
and during Ca2+ release. Thus, while knowledge of [Ca2+ ]ER
and [Ca2+ ]c values compatible with Ca2+ release cannot tell
us the value of VER , it at least give us an idea of what kind of
VER values would be highly unlikely; a valuable constraint
considering the almost complete lack of information about
this parameter.
3.3. ER membrane potential: can measurements of
[Ca2+ ]ER provide point estimates?
Similar theoretical arguments can be used to get an idea
of VER from measurements of the Ca2+ leak from the ER at
different [Ca2+ ]ER . When Ca2+ uptake into the ER is blocked,
the ER gradually loses Ca2+ , through a poorly understood, but
most likely passive, process of “Ca2+ leak” [26,29,68]. The
rate of this Ca2+ leak declines as [Ca2+ ]ER decreases [26,29].
The simplest explanation for this would be a fall in the electrochemical driving force for Ca2+ leakage as [Ca2+ ]ER falls.
If we assume that Ca2+ leaks through purely “passive” channels, i.e. Ohmic channels with constant expression density
and open probability, the net Ca2+ leak will become zero
when ECa is equal to VER . When [Ca2+ ]c is about 90 nM,
Ca2+ leak from the ER is zero when [Ca2+ ]ER is 160 ␮M in
sensory neurons and about 33.5 ␮M in pancreatic acinar cells
(Fig. 1), giving ECa values of about −95 and −74 mV, respectively (from Eq. (1)). If the assumption that the Ca2+ leak is a
purely Ohmic conductance is valid, these values correspond
to VER (inside-negative) of these cells.
How valid is the assumption that the Ca2+ leak current
behaves according to Ohm’s law? If it is true that VER remains
relatively constant, as suggested by the fast kinetics of Ca2+
release (see above discussion), the leak current will vary with
[Ca2+ ]ER according to a simple logarithmic relationship:
ICaLEAK ∝ VER − ECa
(2)
The experimental relationships obtained when [Ca2+ ]c is
clamped (Fig. 1) are not incompatible with curves predicted
by this equation, although it should be pointed out that
as [Ca2+ ]ER increases, it is difficult to judge whether the
Ca2+ leak continues to increase very gently (as predicted
by Eq. (2)), or whether it truly saturates. The temperature-
D. Burdakov et al. / Cell Calcium 38 (2005) 303–310
dependence of the Ca2+ leak also indicates that it is mediated by a channel [69]. Thus, although the evidence in
favour of channel-mediation is indirect [68], it is perhaps the simplest explanation compatible with the currently available data. The stable, inside-negative VER predicted by our calculations would provide a simple electrochemical explanation for the currently unexplained phenomenon of the “undepletable plateau” of [Ca2+ ]ER observed
experimentally during maximal stimulation of Ca2+ release
(e.g. [26,29]).
4. Intraluminal Ca2+ and regulation of cellular
adaptive responses
4.1. Intraluminal Ca2+ and ER chaperones
The lumen of the ER is a key site for protein synthesis, folding and trafficking. The post-translational folding of
proteins into tertiary structures is controlled by an extended
family of ER resident enzymes generally known as molecular chaperones [70–73]. Many of these chaperones, such as
calreticulin, calnexin, ORP 150 or ERp57 bear multiple Ca2+
binding sites and also act as intraluminal Ca2+ buffers. Most
importantly, Ca2+ binding to molecular chaperones regulates
307
their activity and therefore [Ca2+ ]ER directly affects posttranslational protein modification [19,20].
Molecular chaperones provide an all-important system of
protein quality control, and act as a system of defence against
any alterations in protein synthesis. Thus, over-expression of
proteins triggers an accumulation of unfolded proteins within
the ER lumen therefore creating conditions of ER stress
(manifested in forms of either unfolded protein response
or ER overload response [8,74–78], which in turn leads to
an up-regulation of ER resident chaperones and promotes
cell survival. This adaptive ER stress response is particularly
important for neuroprotection against a variety of neurodegenerative diseases associated with accumulation of misfolded and aggregation-prone proteins, such as Alzheimer’s
and Parkinson’s diseases [79]. Furthermore, there are certain
indications that chaperone malfunction may hold the responsibility for a variety of age-dependent neurological conditions
[80].
Within this context, the Ca2+ -dependence of chaperones becomes particularly important, since fluctuations in
[Ca2+ ]ER occurring during cellular activity may significantly
affect the function of intra-ER enzymatic systems. Indeed,
depletion of ER Ca2+ was shown to inhibit protein folding
in a variety of cellular systems [81]. The main impact of
changes in [Ca2+ ]ER levels is probably mediated by changes
Fig. 2. Mechanisms of [Ca2+ ]ER -dependent control of ER function. For explanation see text. SERCA – Sarco(Endo)plasmic reticulum Ca2+ ATPase, InsP3 R
– inositol 1,4,5-trisphosphate receptor, RyR – ryanodine receptor, CRT – calreticulin, CSQ – calsequestrin, NCE – sodium–calcium exchanger, PMCA –
plasmalemmal Ca2+ ATPase, PLC – phopholipase C, cADPR – cyclic ADP ribose.
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D. Burdakov et al. / Cell Calcium 38 (2005) 303–310
in functional activity of lectin-like chaperones, such as calreticulin and calnexin; these proteins also act as important
intra-ER Ca2+ buffers [82]. Calreticulin and calnexin (which
form the so-called calreticulin/calnexin cycle) are involved
in post-translational folding of the majority of glycosylated, secreted, or integral membrane proteins synthesized
within the ER. The chaperone function of both proteins is
Ca2+ -dependent because their binding to glycopropteins is
sensitive to [Ca2+ ]ER [19,83], and a decrease in [Ca2+ ]ER
below ∼50 ␮M may completely inhibit chaperone activity [83]. Furthermore, the interactions of calreticulin with
two other chaperones, the protein disulfide isomerase (PDI)
and ERp57, are also regulated by [Ca2+ ]ER [83]. The disruption of ER Ca2+ homeostasis by itself triggers the ER
stress conditions, which may eventually affect cell survival
[6,84,85].
4.2. Intraluminal Ca2+ and cell death
It is very well established that the disruption of cytosolic Ca2+ homeostasis which occurs through, for example,
excessive plasmalemmal Ca2+ influx plays a critical role in
the regulation of cell death via either apoptotic or necrotic
pathways (see, e.g. [86–89]). Recently, however, mounting
evidence suggests that alterations of ER Ca2+ homeostasis
may be similarly important in triggering apoptosis or initiating cell death [84,90]. Interestingly, either severe depletion
of ER Ca2+ or its persistent elevation may have detrimental effects on cell survival. A decrease in [Ca2+ ]ER resulting
from either inhibition of SERCA pumps by thapsigargin or
CPA, or from activation of RyRs by caffeine or ryanodine,
led to a prominent reduction in protein synthesis, disaggregation of polysomes and a remarkable increase in ER stress
markers [84,91–93], and severe decreases in [Ca2+ ]ER triggered cell death [94,95]. Cell survival was similarly affected
by increases in [Ca2+ ]ER , which can greatly facilitate apoptosis, whereas artificial lowering of ER Ca2+ content promoted
cell survival [96]. Disruptions of ER calcium homeostasis
are currently considered to be involved in various forms of
pathology, and especially in neurodegeneration. In particular, changes in ER Ca2+ handling may exacerbate neuronal
damage in hypoxic conditions [97,98], may underlie central nervous system remodelling during ageing [99–102], and
could be critical for initiation of cell death in Alzheimer disease [90,103–105].
5. Conclusions
[Ca2+ ]ER influences and coordinates a truly staggering
array of spatiotemporally diverse intracellular processes
(Fig. 2). Much of the global physiological and pathological
state of a cell may thus be potentially encoded in this single
parameter. Determining how [Ca2+ ]ER dynamics is generated and kept inside physiological boundaries is thus critical
to understanding cell function, and methods of direct mea-
surements of [Ca2+ ]ER are likely to be essential to achieve
this aim.
Acknowledgements
The authors thank BBSRC, Royal Society, MRC, INTAS
and The Wellcome Trust for support.
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