Physiol Rev 86: 369 – 408, 2006;
doi:10.1152/physrev.00004.2005.
Microdomains of Intracellular Ca2⫹: Molecular Determinants
and Functional Consequences
ROSARIO RIZZUTO AND TULLIO POZZAN
Department of Experimental and Diagnostic Medicine and Interdisciplinary Center for the Study of Inflammation,
University of Ferrara, Ferrara; and Department of Biomedical Sciences, National Research Council Institute
of Neuroscience, and Venetian Institute of Molecular Medicine, University of Padua, Padua, Italy
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I. Introduction
II. Plasma Membrane Calcium Channels
A. Generation of subplasma membrane Ca2⫹ microdomains
B. Functional role of subplasma membrane microdomains
III. The Endoplasmic and the Sarcoplasmic Reticulum
A. Organelle morphology
B. Membrane and luminal continuity
C. Molecular heterogeneity
D. Generation of Ca2⫹ microdomains by RyRs
E. Elementary and global signals in IP3-dependent systems
F. Turning puffs into global signals: role of other second messengers
IV. Mitochondria
A. Stores, sinks, or inactive elements in Ca2⫹ homeostasis?
B. Mitochondrial heterogeneity in Ca2⫹ handling
C. Functional role of mitochondrial Ca2⫹ uptake
V. Other Intracellular Calcium Stores
A. Endosomes
B. Golgi apparatus
C. Secretory granules
D. Lysosomes
E. IP3R and RyR expression
F. Channels other than the IP3Rs and the RyRs and their subcellular localization
V. Concluding Remarks
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Rizzuto, Rosario, and Tullio Pozzan. Microdomains of Intracellular Ca2⫹: Molecular Determinants and Functional
Consequences. Physiol Rev 86: 369 – 408, 2006; doi:10.1152/physrev.00004.2005.—Calcium ions are ubiquitous and
versatile signaling molecules, capable of decoding a variety of extracellular stimuli (hormones, neurotransmitters,
growth factors, etc.) into markedly different intracellular actions, ranging from contraction to secretion, from
proliferation to cell death. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca2⫹]
rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be
initiated. In this review, we discuss the structural and functional bases that generate the subcellular heterogeneity
in cellular Ca2⫹ levels at rest and under stimulation. This complex choreography requires the concerted action of
many different players; the central role is, of course, that of the calcium ion, with the main supporting characters
being all the entities responsible for moving Ca2⫹ between different compartments, while the cellular architecture
provides a determining framework within which all the players have their exits and their entrances. In particular, we
concentrate on the molecular mechanisms that lead to the generation of cytoplasmic Ca2⫹ microdomains, focusing
on their different subcellular location, mechanism of generation, and functional role.
I. INTRODUCTION
A very steep gradient of Ca2⫹, over four orders of
magnitude, exists across the plasma membrane of all
eukaryotic cells: cytosolic free calcium is maintained
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roughly at 100 nM while the extracellular milieu generally
has a [Ca2⫹] of over 1 mM. This gradient is maintained
due to the action of plasma membrane Ca2⫹-ATPases
(PMCA) and Na⫹/Ca2⫹ exchangers (NCX); whereas the
former are ubiquitous, the latter are predominantly ex-
0031-9333/06 $18.00 Copyright © 2006 the American Physiological Society
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alterations of the resting [Ca2⫹]c. Indeed, loading cells
with Ca2⫹ indicators implies the addition of an exogenous
Ca2⫹ buffer to the cytoplasm, with a variation of several
tens to hundreds of micromolar (6); it is obvious that a
different Ca2⫹ buffering capacity does influence the amplitude of the [Ca2⫹]c rises that are observed upon activation, but this can be taken into consideration and accounted for (181, 301).
With regard to the inward “Ca2⫹ leak,” its exact
nature is still undetermined. Controlled, Ca2⫹ influx paths
most likely exist to ensure this key aspect of cell physiology (2, 3, 406).
In addition to the largely mysterious leak channels
mentioned above, cells express in their plasma membrane
a variety of well-characterized Ca2⫹ channels. The gating
properties of these channels can depend on membrane
potential [in the so-called voltage-operated Ca2⫹ channels
(VOC); Ref. 46], or ligand binding [in the case of receptoroperated Ca2⫹ channels (ROC); Ref. 213]. Furthermore, a
heterogeneous group of channels [most of which belong
to the so-called transient receptor potential family (TRPs)]
are activated by a variety of factors, including mechanical
stretch, osmolarity, temperature, second messengers, G
proteins, protein-protein interactions, etc. (226, 227; see
also Ref. 249). The molecular nature of an additional
group of channels, in this case activated by arachidonic
acid, is still undetermined (337). An exhaustive description of all these channels is well beyond the scope of the
present text, and the interested reader is referred to recent reviews that provide detailed analyses of this topic
(46, 226, 227). The different molecular nature of these
channels, their subcellular distribution, and their regulation are the key components of the generation of subplasma membrane Ca2⫹ microdomains, the focus of this
review, and this latter aspect of the problem is addressed
in detail in the next section.
A similar conceptual framework, i.e., a Ca2⫹ pump
and leak equilibrium, regulates the steady-state level of
[Ca2⫹] within intracellular Ca2⫹ stores, i.e., the endo/
sarcoplasmic reticulum, the mitochondria, and a heterogeneous group of vesicular organelles including the Golgi
apparatus, endosomes/lysosomes, and secretory vesicles.
Functionally speaking, the sidedness of the intracellular
Ca2⫹ store membrane is identical to that of the plasma
membrane, i.e., the active Ca2⫹ transport accumulates
Ca2⫹ within the organelle lumen (that thus corresponds to
the extracellular milieu) and the channels return it to the
cytoplasm. Ca2⫹ uptake within the stores depends on
mechanisms molecularly and functionally completely different from those of the plasma membrane (see below)
and, as in the case of the plasma membrane, the leak
mechanism is still highly mysterious, although evidence
has been recently provided that the protein import complex of the ER, the so-called translocon (193), or, under
some conditions, the inositol 1,4,5-trisphosphate (IP3) re-
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pressed in excitable tissues. Mammalian PMCAs are encoded by 4 genes, and ⬃30 isoform variants can be generated via alternative RNA splicing of the primary gene
transcripts; the expression of different PMCA isoforms
and splice variants is regulated in a developmental as well
as a tissue- and cell type-specific manner (45, 352, 353).
Three distinct genes are known to exist for the NCXs, and
several splice variants are known for NCX1 and NCX3
(277, 278, 297). A closely related family of exchangers, the
Na⫹/Ca2⫹-K⫹ exchange (NCKX) gene family, has been
recently discovered (138). This family of proteins, first
discovered in the outer segments of vertebrate rod photoreceptors, now comprises four members (NCKX1 to 4);
a partial sequence of a fifth human NCKX gene has also
appeared in the databases. In situ NCKX1 and heterologously expressed NCKX2 operate at a 4Na⫹:1Ca2⫹ ⫹1K⫹
stoichiometry.
The energy to extrude Ca2⫹ against its electrochemical gradient has different origins: ATP hydrolysis in the
case of PMCAs, with one Ca2⫹ being extruded per ATP
consumed, and the electrochemical Na⫹ gradient in the
case of NC(K)Xs, with the extrusion of one Ca2⫹ at the
expense of three Na⫹ (and 4 Na⫹ in the case of NCKX)
(45, 73, 277, 278, 294, 297, 352, 353).
A general concept, obvious to specialists but often
not appreciated enough by many cell biologists, is that the
long-term steady-state free Ca2⫹ concentration in the cytoplasm ([Ca2⫹]c) depends exclusively on the equilibrium
between the rates of the efflux mechanisms and the rate
of inward Ca2⫹ “leak” across the plasma membrane. Indeed, any modulation of the activity of organelles that
accumulate or release Ca2⫹, or even changes in the level
of Ca2⫹ buffers within the cytoplasm, only affect [Ca2⫹]c
in a transient manner and have no direct role in the
long-term maintenance of this parameter. In other words,
a cell could theoretically maintain its steady-state [Ca2⫹]c
even without any Ca2⫹ storage organelles or cytoplasmic
Ca2⫹ buffers. Thus inhibition of the sarco/endoplasmic
reticulum Ca2⫹-ATPase (SERCA pumps; see below)
causes the release of Ca2⫹ accumulated in the stores and
a transient, reversible, increase in [Ca2⫹]c, but has no
persistent effect on steady-state Ca2⫹, unless, as is the
case in many cell types, it modifies the so-called capacitative Ca2⫹ entry (CCE; see below). Similarly, inhibition
of mitochondrial Ca2⫹ uptake can only indirectly lead to
long-term modifications of steady-state Ca2⫹ level in the
cytosol, e.g., if this inhibition affects the cellular ATP
levels or the plasma membrane potential. Along the same
line of reasoning, the Ca2⫹ buffering capacity of the cell
cytoplasm can vary by over an order of magnitude among
different cells, yet this results in no obvious change of the
resting [Ca2⫹]c (15, 20, 65, 95, 404). More than a mere
biochemical curiosity, this concept is of primary importance: if it were not true, the classical methods used to
monitor [Ca2⫹]c with fluorescent dyes would lead to gross
MICRODOMAINS OF INTRACELLULAR CALCIUM
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brane potential depolarization or Ca2⫹ mobilization from
intracellular stores (188, 189). Since then, localized increases of cytoplasmic Ca2⫹ have been repeatedly measured by direct imaging with fluorescent indicators or
inferred from indirect approaches. The existence, amplitude, and functional role of Ca2⫹ microdomains have
been the subject of intense investigation over the last
decades, in particular, but not only, concerning their role
in neurotransmitter release.
Overall, the review focuses on the [Ca2⫹] microheterogeneity detected in the cell domains participating in
the generation and/or functional translation of cytosolic
Ca2⫹ signals: the plasma membrane, sarco/endoplasmic
reticulum, Golgi apparatus and secretory vesicles, and the
mitochondria. We here discuss the heterogeneity in the
molecular repertoire of Ca2⫹ handling of these cell domains, as well as the large differences in their resting and
stimulated [Ca2⫹].
Section II of this review is dedicated to a brief summary of the vast literature dealing with the generation of
subplasma membrane microdomains, with particular emphasis on the microscopic local events occurring a few
nanometers away from the mouth of plasma membrane
Ca2⫹ channels in neuronal cells (see, for example, Ref.
398). The generation of subplasma membrane Ca2⫹ microdomains is, however, not a peculiarity of neuronal
cells, and their existence and possible function have been
described in many nonexcitable cells. In the latter cell
types, such microdomains can again remain restricted to
the few nanometers of cytoplasm around the inner mouth
of the channels or they can be represented by more large
heterogeneities (tens of ␮m2 from their site of generation). The second part of the first chapter is dedicated to
discuss the functional role of the Ca2⫹ microdomains
generated below the plasma membrane, from the activation of neurotransmitter release in synaptic terminals to
the modulation of specific plasma membrane enzymes,
e.g., Ca2⫹-sensitive adenylate cyclases, NO synthase, and,
last but not least, Ca2⫹-dependent gene activation. This
latter event, though occurring far away from the plasma
membrane, yet in some cases depends on very local Ca2⫹
increases occurring at the mouth of plasma membrane
Ca2⫹ channels.
Section III is dedicated to reviewing the structural and
functional bases that permit the generation of microdomains in cytoplasmic Ca2⫹ due to mobilization of Ca2⫹
from the sarco/endoplasmic reticulum. The nature and
main structural features of the molecular machinery that
allow the uptake, storage, and release of Ca2⫹ in the
sarco/endoplasmic reticulum (e.g., Ca2⫹ pumps, Ca2⫹ release channels, and Ca2⫹ buffering proteins) and their
heterogeneous distribution within the endomembrane
system that forms the basis for the subcellular Ca2⫹ heterogeneities due to Ca2⫹ mobilization is discussed. The
mechanism of generation of the microdomains named
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ceptor themselves (253), may contribute to the Ca2⫹ leak
from the ER. As to the regulated release of Ca2⫹ from
intracellular stores, it also depends on a group of channels whose molecular properties have been the subject in
the last years of intense investigation. Their distribution
within cells and their physiological regulation will be
discussed in some detail in the sections below.
The complexity of the Ca2⫹ handling machinery
briefly summarized above is at the basis of the heterogeneity of Ca2⫹ distribution within cells, “the Ca2⫹ microdomains,” not only at rest, but also, and most important,
during stimulation. The definition of Ca2⫹ microdomain is
far from being univocous, and it has been used with
different meanings (in particular as far as its spatial dimensions) by investigators in different fields. Thus neuroscientists, in general, use the term Ca2⫹ microdomains
primarily referring to the very local elevations of Ca2⫹ in
the vicinity (nm) of the mouth of presynaptic Ca2⫹ channels that play a pivotal role in regulating neurotransmitter
release (9). Cell biologists, on the other hand, refer to
Ca2⫹ microdomains in more general terms, i.e., any increase in cytoplasmic Ca2⫹ that remains localized in one
part of the cell, from the very local hot spots close to Ca2⫹
channels, to larger subcellular Ca2⫹ gradients such as
those formed between the complex arborization of astrocyte processes and the nuclear region (24, 268), in the
apical pole of pancreatic acinar cells (8, 271, 370), during
cytotoxic T lymphocyte attack of target cells (288) or
oriented locomotion (119) (for recent reviews see, for
example, Refs. 22, 32, 55, 291). In this contribution, the
term Ca2⫹ microdomain is used in the most general way,
i.e., all the increases in cellular Ca2⫹ that do not involve
the generality of the cell cytoplasm, but remain localized
to part of the cell.
In the 1970s and 1980s (and often even to date), the
existence of Ca2⫹ microdomains was invoked to explain
results otherwise uninterpretable, but without a solid,
direct experimental support. The existence of Ca2⫹ microdomains is intrinsic to the physicochemical characteristics of the cell interior. In fact, the high Ca2⫹ buffering
capacity of the cytoplasm and hence the very low Ca2⫹
diffusion rate, 10 –50 ␮m2/s (4), the existence of highly
organized subcellular structures endowed with the capacity to take up and release Ca2⫹, the clustering of Ca2⫹
channels (both of the plasma membrane and of organelles) in discrete membrane domains, all appear ideally suited to generate transient (or even prolonged) local
elevations of Ca2⫹. However, discrete, transient, heterogeneities in Ca2⫹ levels within the cytoplasm of living
cells were eventually experimentally measured only in the
late 1980s when fluorescent Ca2⫹ indicators became
widely available. One of the first direct measurements of
a localized Ca2⫹ increase was reported in 1988: a subplasma membrane Ca2⫹ increase (in the cell body and
growth cones) activated in sympathetic neurons by mem-
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II. PLASMA MEMBRANE CALCIUM CHANNELS
A. Generation of Subplasma Membrane
Ca2ⴙ Microdomains
Given the key importance of Ca2⫹ influx for the
maintenance of cytoplasmic and organelle steady-state
Ca2⫹ levels, as well as for the induction of Ca2⫹ increases
during cell activation, it comes as no surprise that most
cells express more than one type of Ca2⫹ channel. In
addition, particularly in cells with a complex morphology,
these channels can be differently distributed along the
plasma membrane (see also below). Moreover, the repertoire of Ca2⫹ channels of any given cell can vary not only
depending on its specific function and developmental
stage, but also during its life span. For example, new
channels can be inserted into the plasma membrane upon
activation of specific receptors (see, for example, Refs.
340, 386); it has also been proposed that the mechanisms
of Ca2⫹ influx that are activated by store depletion may
depend exclusively on the insertion of channels into the
plasma membrane (397). The Ca2⫹ selectivity of these
channels is highly variable, ranging from very high (over 3
orders of magnitude in favor of Ca2⫹ compared with Na⫹,
in the case of VOCs) to rather poor (in the case of some
ROCs and some members of the TRP family). A common
characteristic of the most Ca2⫹-selective channels is their
capability of transporting efficiently monovalent cations
when all divalent cations are removed from the medium,
suggesting the existence in the Ca2⫹-selective channels of
a common mechanism that ensures their ionic specificity.
Whatever the nature and gating properties of a channel, it is easily envisaged that upon its opening a high
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concentration of Ca2⫹ will be generated at its mouth, on
the inner side of the plasma membrane (9). In terms of
peak amplitude, it has been calculated that, at physiological extracellular Ca2⫹ concentration and at 0 mV of membrane potential, the Ca2⫹ concentration should be in the
order of 100 ␮M within 100 nm of the mouth of an open
Ca2⫹ channel (244). Even higher local concentrations will
be transiently reached in voltage-clamped cells during the
so-called tail current (i.e., during the repolarization
phase). These values have been calculated for bovine
adrenal medullary cells, a cell type where the nature of
the channels, and the concentration and binding kinetics
of the soluble Ca2⫹ buffers within the cytoplasm, are
known in great detail (243); for most other cell types,
these parameters are much less defined. In general terms,
the peak amplitude and spatial diffusion of the Ca2⫹
microdomain formed at the mouth of a Ca2⫹ channel and
its immediate neighborhood will depend on the conductance of the channel itself, its Ca2⫹ selectivity, the concentration of Ca2⫹ in the extracellular medium, the membrane potential, and the nature and amount of the intracellular Ca2⫹ buffers.
Over the last two decades, several experimental approaches have been adopted in the attempt to measure,
directly or indirectly, the free Ca2⫹ concentration close to
a Ca2⫹ channel’s mouth. Back in 1980, Llinas et al. (192)
injected a low-affinity mutant of the Ca2⫹-sensitive photoprotein aequorin into squid giant axons and calculated
that hot spots in the range of 200 –300 ␮M are reached in
the synapse during action potential discharge. Neher and
co-workers (139), comparing the rate of exocytosis induced by uncaging Ca2⫹ from a high-affinity buffer and by
physiological stimulation, calculated values in the order
of 100 –200 ␮M in the vicinity of the channels in synaptic
terminals of retinal bipolar cells. Later studies in the large
synapses of the calices of Held led to the conclusion that
under physiological conditions fast vesicle release can be
accounted for by increases in Ca2⫹ concentration to
lower values, on the order of 20 ␮M (326).
The question of the [Ca2⫹] necessary to trigger fast
vesicle exocytosis is not a mere problem of accuracy
measurement but has major functional implications. Indeed, if a [Ca2⫹] of 10 –20 ␮M is sufficient to trigger
vesicle release, it can be concluded that channels other
than those in close proximity to the vesicles are involved;
if [Ca2⫹] in the 200 –300 ␮M range is instead necessary to
trigger vesicle fusion, the consequence is that only the
channels closely apposed to the secretory site contribute
to the neurotransmission event. A conservative conclusion on this point would be that, depending on the synapse type, either type of arrangements is possible.
Subplasma membrane [Ca2⫹] significantly higher
than those measured in the bulk cytosol have been determined by several groups by localizing Ca2⫹-sensitive
probes on the inner surface of the plasma membrane,
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Ca2⫹ sparks and puffs and their importance as building
blocks for the spreading of the Ca2⫹ signal to the whole
cytoplasm (to ensure the physiological response of the
cells) is dealt with in some detail, in particular in relation
to their role in the physiopathology of the Ca2⫹ signal in
cardiac myocytes.
Section IV is dedicated to mitochondria, organelles
that are primarily the targets of cellular Ca2⫹ microdomains, rather than the generators of subcellular Ca2⫹
heterogeneity. The importance of cytoplasmic Ca2⫹ microdomains in shaping the capacity of mitochondria to
accumulate Ca2⫹, the functional consequences of mitochondrial Ca2⫹ accumulation and their derangements under pathological conditions, particularly during programmed
cell death, are reviewed in detail.
Finally, section V is dedicated to the mechanisms of
Ca2⫹ handling by other cellular organelles (e.g., the Golgi
apparatus, secretory vesicles, lysosomes) and to the potential role played by such noncanonical Ca2⫹ stores as
further potential generators of Ca2⫹ microdomains during
stimulation.
MICRODOMAINS OF INTRACELLULAR CALCIUM
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both at rest and during opening of Ca2⫹ channels in cells
other than neurons. Thus, by using a SNAP-25 aequorin
chimera, Marsault et al. (202) concluded that on the inner
surface of the plasma membrane of the smooth muscle
cell line A7r5 at rest, the mean Ca2⫹ concentration is
⬃1–2 ␮M (possibly reflecting the existence of hot spots in
the vicinity of flickering channels, rather than an homogeneous high level underneath the plasma membrane),
while during Ca2⫹ influx due to CCE activation, values as
high as 50 ␮M can be reached (see Fig. 1). Similarly,
Nakahashi et al. (241) concluded that an aequorin adenylate cyclase chimera bound to the inner surface of the
plasma membrane is capable of detecting, upon activation
of CCE, values in the order of 10 –20 ␮M. Higher levels of
Ca2⫹ in the subplasma membrane region (compared with
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the bulk cytoplasm) have been calculated also using
green fluorescent protein (GFP)-based Ca2⫹ probes (cameleons): in particular, the same Ca2⫹ probe either selectively localized within caveolae, homogeneously distributed along the inner surface of the plasma membrane, or
in the cytoplasm measured quite different values both at
rest and upon activation of CCE (151). In particular, the
[Ca2⫹] in the cytoplasmic rim around caveolae at rest was
⬃320 nM, significantly higher than the mean value below
the plasma membrane (260 nM) and ⬃10-fold higher than
that monitored in the bulk cytoplasm (22 nM). Upon
activation of CCE, the [Ca2⫹] peak was much larger in the
caveolae region (but the latter values were not quantified). More recently, using the same probe, Isshiki et al.
(150) demonstrated that in resting conditions there is a
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2⫹
FIG. 1. [Ca ] changes close to plasma membrane
Ca2⫹ channels. Top: schematic view of the Ca2⫹ microdomains generated around an open channel. The
pseudocolor scale (on the right) indicates the approximate values of [Ca2⫹], and the bar indicates the approximate distance. A and B: Ca2⫹ changes in the bulk
cytosol and in the cytoplasmic rim underneath the
plasma membrane as revealed by recombinant aequorin
localized in the cytosol or bound to the inner surface of
the plasma membrane. Bottom, A: A7r5 cells (a smooth
muscle cell line) expressing cytosolic aequorin. B: same
cells expressing a fusion protein between SNAP25 and
aequorin that binds selectively to the inner leaflet of the
plasma membrane. Please note that while Ca2⫹ mobilization from intracellular stores results in a very similar
Ca2⫹ rise with either probe, the Ca2⫹ influx elicited by
Ca2⫹ addition (to induce capacitative Ca2⫹ entry)
causes a dramatically higher increase in [Ca2⫹] when
measured with the aequorin bound to the plasma membrane. For other details, see Marsault et al. (202). [A and
B from Marsault et al. (202).]
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ROSARIO RIZZUTO AND TULLIO POZZAN
rather inhomogeneous high Ca2⫹ level along the subplasma membrane cytoplasmic rim. This relatively elevated [Ca2⫹] could be abolished by removing Ca2⫹ from
the medium and restored upon Ca2⫹ readdition (Fig. 2).
Along the same line, also Nagai et al. (240), using new
cameleons either bound to the inner leaflet of the plasma
membrane or free in the cytoplasm, found a higher level
of subplasma membrane [Ca2⫹] compared with that in the
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2⫹
FIG. 2. Changes of [Ca ] on the
inner side of plasma membrane rafts and
their importance in the activation of specific cellular functions. Top: schematic
view of the plasma membrane organization including membrane proteins and
lipid rafts. Phospholipids forming rafts
are depicted in greenish blue and cholesterol (enriched in rafts) as the red symbols. A: region of the cytoplasm close to
the CCE channel. B: bulk cytosol. C: a
membrane-anchored effector system, e.g.,
NO synthase. In this scheme the channel
responsible for CCE is hypothesized to be
located in the lipid raft. Bottom, A: endothelial cells expressing a raft targeted cameleon; measurement of [Ca2⫹] in the region
depicted as A in the top panel. B: endothelial cells expressing a cytosolic cameleon;
measurement of [Ca2⫹] in the region depicted as B in the top panel. Note the large
change in fluorescence ratio (a measure of
the change in [Ca2⫹]) upon changing the
[Ca2⫹] of the medium only in the case of
the cameleon bound to the inner side of the
rafts. C: [Ca2⫹] increases elicited by activation of CCE in HeLA cells expressing an
aequorin-NO synthase chimera; a cytosolic
aequorin and the aequorin-NO synthase
chimera, whose membrane-anchoring domain has been deleted. Note the much
larger increase in [Ca2⫹] upon activation of
CCE when using the aequorin-NO synthase
construct. [A, B, and D from Isshiki et al.
(150); C from Lin et al. (185).]
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there is no doubt that Ca2⫹ waves triggered by stimulation
with hormones or neurotransmitters (cholecystokinin,
bombesin, acetylcholine) always originate from the apical
pole. However, the underlying mechanism of this polarization is still debated. According to one view, based on
patch-clamp experiments with two separate stimulatory
and recording pipettes, signaling complexes at the apical
pole are more sensitive to agonist stimulation, implying a
higher density and/or more efficient coupling in this portion of the cell (183). According to a radically different
view, receptors are not clustered at the apical pole, but
rather at the basal pole (318, 393). Nevertheless, the stimulation of receptors located in the basal membrane causes
a cytosolic Ca2⫹ concentration ([Ca2⫹]c) rise that initiates
in the apical pole (7, 370), because the latter region is
endowed with a higher density of IP3 receptors (this
conclusion is undisputed), and IP3 produced at the basal
pole can rapidly diffuse throughout the cell. In support of
this view is the observation that intracellular application
of IP3 and/or cyclic ADP ribose (cADPR) (Fig. 3) through
the patch pipette always causes Ca2⫹ spikes in the apical
pole (370). Whatever the mechanism, the functional significance of this polarization is striking. Specific functions
can be triggered at the apical pole (e.g., stimulation of
exocytosis, but also activation of Ca2⫹-regulated Cl⫺
channels) and at the basal pole (e.g., regulation of nuclear
transcription, but also activation of Ca2⫹ regulation of K⫹
channels). In general, it can be concluded that the spatial
clustering of receptors and Ca2⫹ channels, and the ensuing spatial patterns of Ca2⫹ diffusion within the cell, as
well as regulatory effects within membrane microdomains, contribute to increase the flexibility of Ca2⫹-mediated signals.
B. Functional Role of Subplasma
Membrane Microdomains
The generation of local hot spots at the mouth of
Ca2⫹ channels (or in general just below the plasma membrane) critically controls a number of different cellular
functions. By far the most intensely investigated is the
secretion of neurotransmitters at the presynaptic membrane. This latter topic has been extensively and repeatedly reviewed in the last years. Accordingly, here we will
limit ourselves to summarize the fundamental facts and
concepts, referring the reader to other contributions for a
more thorough analysis of this problem (23, 31, 61, 85,
162, 207, 208, 246, 247, 326, 345, 355). Below we will deal
more extensively with other Ca2⫹-dependent events, similarly modulated by local [Ca2⫹] at the subplasma membrane level, that have received relatively less attention in
the recent past.
As discussed above, although the Ca2⫹ levels reached
at the mouth of the open channels and the spatial diffu-
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bulk cytoplasm, both at rest (⬃80 vs. 30 nM, respectively)
and upon stimulation (well above 1 ␮M below the plasma
membrane compared with ⬃700 nM in the cytoplasm).
Less dramatic, but still measurable differences between
the cytoplasmic rim at the inner surface of the plasma
membrane and the bulk cytosol have been monitored
with other probes, e.g., fluorescent Ca2⫹ dyes endowed
with a lipid anchor or with other GFP probes bound to the
inner surface of the plasma membrane. Thus, using the
GFP-based Ca2⫹ indicator ratiometric pericam, it has
been reported that the peak value of [Ca2⫹] in the bulk
cytosol or on the inner leaflet of the plasma membrane in
pancreatic ␤-cells depolarized by high K⫹ reached 1.38
and 1.82 ␮M, respectively (284).
Although Ca2⫹ microdomains below the plasma
membrane are primarily due to opening of plasma membrane Ca2⫹ channels, it should be mentioned that they
can also depend on IP3-induced Ca2⫹ release from intracellular stores evoked in the immediate vicinity of the
plasma membrane (see, for example, Ref. 26). Close contacts between IP3-sensitive Ca2⫹ stores and plasma membrane have indeed been found in several cell types (for a
recent review, see Ref. 22). An emerging theme is the
elucidation of the cytoskeletal scaffolds that allow the
formation of signaling clusters on the inner side of the
plasma membrane. In the Drosophila photoreceptor cells,
INAD, a protein consisting of tandem arrays of five PDZ
domains, was shown to assemble in the rhabdomere a
complex including TRP and TRPL channels, protein kinase C (PKC), phospholipase C (PLC), rhodopsin, calmodulin, and myosin III, that is necessary for the G protein-mediated signaling cascade triggered by light exposure (182). Neurons express different isoforms of Homer,
a scaffolding protein with an EV1 domain that binds to a
consensus sequence present in various signaling proteins
[e.g., mGluRs, IP3Rs, ryanodine receptors (RyRs), TRPs,
and the Shank postsynaptic proteins, part of the NMDAassociated PSD-95 complex] and a coiled-coil dimerization domain (376). These intermolecular bridges may allow the formation of stable receptor complexes at the
level of the synapse that favor the cross-talk of glutamatedependent signaling pathways. This structural arrangement is not limited to excitable cells, although in other
cell types the molecular information is still scant. It is
likely, however, that similar, when not the same, scaffolding proteins play a role. Indeed, the Homer proteins are
ubiquitously expressed, and in nonexcitable cells, Homer
was shown to regulate the physical association between
TRPC1 and the IP3R, that in turn controls the gating of
these plasma membrane channels (400).
Polarized epithelial cells, such as those forming exocrine glands, provide another interesting example of
membrane clustering of receptors and transducing proteins and allow some insight into the functional significance of these molecular arrangements. In these cells,
375
376
ROSARIO RIZZUTO AND TULLIO POZZAN
2⫹
sion of Ca2⫹ within the cell may vary quite extensively
depending on the channel type, synapse considered, and
the buffering characteristics of the cytoplasm, there is a
general consensus that under physiological conditions
small synaptic vesicles require increases of [Ca2⫹]c at
least 5–10 times higher than those reached in the bulk
cytoplasm to efficiently and rapidly fuse with the plasma
membrane. Synaptic vesicles are indeed known to fuse
nonrandomly with the presynaptic membrane, but at specific sites, the so-called active zones. As discussed above,
the latter are enriched with Ca2⫹ channels, and thus the
vesicles containing the neurotransmitters are strategically
located to take full advantage of the Ca2⫹ microdomains
generated at, or close to, the mouth of the channels
themselves. It should be stressed that the microdomains
generated at the active zones are responsible for the
fusion of small synaptic vesicles, while other slower exocytotic events, such as those occurring in endocrine tissues probably depend more on bulk, or less localized,
cytosolic Ca2⫹ increases (52). The release of dense-core
vesicles from synaptic terminals is also presumably dependent on bulk increases of [Ca2⫹]c (53).
Physiol Rev • VOL
Exocytosis is by no mean the only physiological
event that depends on the amplitude of the hot spots
occurring close to the mouth of plasma membrane Ca2⫹
channels. The modulation of the Ca2⫹ channels themselves by Ca2⫹ is another key event occurring at that site.
Indeed, many Ca2⫹ channels are inactivated by the incoming cation, and most experimental evidence supports the
notion that the site of this inactivation resides on the
inner side of the membrane and thus depends on the
amplitude of the Ca2⫹ level reached at the mouth of the
channel itself (5, 92, 173, 344). Thus, not only the substitution of Ca2⫹ with Ba2⫹ prevents inactivation, but inclusion in the patch pipette of a fast Ca2⫹ buffer such as
BAPTA or a slow one, such as EGTA, has dramatically
different effects on the inactivation of the Ca2⫹ current,
particularly in the case of some VOCs (184), i.e., BAPTA
largely prevents inactivation, whereas EGTA does not.
Ca2⫹ inhibition is not a unique characteristic of Ca2⫹
VOCs and other Ca2⫹-permeable channels (e.g., Ca2⫹ release-activated Ca2⫹ channel, CRAC) are potently inactivated by Ca2⫹ itself (191, 261, 407). Last, but not least, it
should be mentioned that local [Ca2⫹] elevations may
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3. Localized Ca2⫹ changes in pancreatic acinar cells. Effects of cADPR (A), inositol 1,4,5-trisphosphate (IP3) (B), and cADPR ⫹ IP3 (C)
on Ca -activated Cl current and localized increases of [Ca2⫹] at the apical pole in pancreatic acinar cells. [From Cancela et al. (43).]
FIG.
MICRODOMAINS OF INTRACELLULAR CALCIUM
Physiol Rev • VOL
other model systems and for other types of Ca2⫹ channels, namely, the P/Q type (358) and the NMDA receptors
(364). In particular, using either HEK cells transfected
with P/Q-type channels or cerebellar granule neurons, it
was found that the synaptic protein syntaxin 1A is upregulated in response to P/Q-type channel activation. This
signaling appears to require communication between the
channel and internal Ca2⫹ stores because it is blocked by
inhibitors of the SERCAs (155). Thus the P/Q channel
regulation of syntaxin 1A may be a mechanism by which
Ca2⫹ channels regulate the expression of proteins that
participate in synaptic vesicle release. Along the same
line, Greenberg’s group (64, 364) showed that stimulation
of neurons in culture with ephrin B triggers clustering of
NMDA and ephrin receptors and association of the receptor complex with the Src family of tyrosine kinases. In
turn, the phosphorylation of the NMDA receptor increases the Ca2⫹ flux through the channels themselves
and potentiates their activation of CREB-dependent gene
expression. Unlike the situation of L-type Ca2⫹ channels,
however, in the latter cases it is still unclear whether
activation of gene expression depends on microdomains
of Ca2⫹ around the channels or on bulk nuclear and
cytoplasmic Ca2⫹ rises. Last, but not least, major functional differences in terms of conveying the Ca2⫹ signal
from the channels to the nucleus have been demonstrated
between the activation of synaptic versus nonsynaptic
NMDA receptors (137). Activation of synaptic NMDA receptors resulted in a robust activation of CREB-dependent transcription, whereas activation of extrasynaptic
NMDA receptors not only caused an inhibition of CREB
activity, but also triggered cell death. The conclusion is
that, given the different subunit composition of the NMDA
receptors (NR2B extrasynaptic and NR2A synaptic), local
events around the NR2A NMDA receptor subunit at synapses may underlie synapse-specific signaling to the nucleus.
Similarly striking appears the efficacy of Ca2⫹ entering through CCE channels in eliciting activation-inhibition
of different isoforms of Ca2⫹-dependent adenylate cyclases (86). When recombinantly expressed, Ca2⫹-sensitive
adenylate cyclases give rise to the anticipated changes in
cAMP levels in response to CCE. However, CCE appeared
far more efficacious at regulating their activity than other
means of elevating bulk [Ca2⫹]c to similar levels, i.e., 1)
release from intracellular stores mediated by IP3, 2) Ca2⫹
ionophores, or 3) arachidonic acid-mediated Ca2⫹ influx
(81). It has thus been concluded that a very close apposition exists between Ca2⫹-sensitive adenylate cyclases
and CCE channels, and more important, that the local
microdomains of high [Ca2⫹] close to the channels are the
key events in the control of cAMP synthesis by these
enzymes (81, 225). Of interest, some of the most Ca2⫹sensitive adenylate cyclases (AC5, AC6, and AC8) are
concentrated in caveolae, from which the Ca2⫹-insensi-
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have the opposite effect in some channels, i.e., a facilitating role on Ca2⫹ channel gating (182).
A striking effect of the hot spots generated at the
mouth of L-type Ca2⫹ channels, totally unrelated to secretion or Ca2⫹ channel modulation, is that reported in References 67, 71, and 163. These authors investigated the
transmission of the Ca2⫹ signal evoked by opening of
plasma membrane L-type Ca2⫹ channels to the nucleus
using as models primary cultures of hippocampal or cortical neurons. This consists in the activation of CREB
phosphorylation and downstream activation of gene transcription. The authors showed that in their model systems
the phosphorylation of CREB is dependent on the nature
of the Ca2⫹ channel (it depends critically on the activation
of L-type channels and in particular on their COOH-terminal domain, while the role of other Ca2⫹ VOCs or
NMDA receptors was negligible) and not on the amplitude
of the bulk increases in [Ca2⫹]c. Most surprisingly for an
event that occurs at a distance of several microns from
the plasma membrane, CREB phosphorylation in the nucleus is critically dependent on the peak [Ca2⫹] reached
close to the mouth of the channel itself. Indeed, the slow
Ca2⫹ buffer EGTA was totally inefficient at blocking the
activation of nuclear CREB phosphorylation, while the
fast Ca2⫹ buffer BAPTA completely prevented it, despite
the fact that the two Ca2⫹ chelators were equally effective
at inhibiting the increases in bulk [Ca2⫹]c (67). Dolmetsch
et al. (71) provided an elegant molecular explanation for
this effect. In particular, they showed that the COOH
terminus of L-type channels interacts with calmodulin in
a Ca2⫹-dependent fashion that leads to the activation of
the mitogen-activated protein kinase (MAPK) pathway;
this in turn causes the phosphorylation of the CREB
kinases Rsk1 and -2 and eventually to long-term CREB
phosphorylation. All these events are thus critically dependent on the elevation of Ca2⫹ in the immediate vicinity
of L-type channels and the isoleucine-glutamine (“IQ”)
motif in the COOH terminus of the L-type Ca2⫹ VOCs (that
binds Ca2⫹-calmodulin) is critical for conveying the Ca2⫹
signal to the nucleus from the mouth of the channels.
Interestingly, while the phosphorylation of CREB depends on the microdomains of Ca2⫹ around the L-type
Ca2⫹ channels, CREB-dependent gene transcription appears to depend also on the increases in nuclear [Ca2⫹],
which occur after relatively intense stimuli. The dual
regulation of signaling pathways by the [Ca2⫹] near the
channels and in the nucleus may permit neurons to precisely tailor transcriptional activation to specific types of
electrical or chemical stimuli and at the same time ensures that only robust stimuli that generate substantial
nuclear [Ca2⫹] elevations are converted into long-term
changes in gene expression (10, 135, 136; for a review, see
Ref. 70).
Evidence not yet as clear linking local Ca2⫹ events to
activation of gene transcription has been reported in
377
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ROSARIO RIZZUTO AND TULLIO POZZAN
III. THE ENDOPLASMIC AND THE
SARCOPLASMIC RETICULUM
The use of the term sarco/endoplasmic reticulum
(SR/ER) was introduced (44) to suggest that the two
tubular networks are the same cytological entity, with the
SR being the muscle-specialized version of the ER of all
other cell types (44). In reality, the similarities between
ER and SR are not so obvious. Morphologically, the SR of
striated muscles (cardiac and skeletal) comprises a network of tubules (longitudinal SR) and cisternae (TC, terminal cisternae) with a very specific anatomical distribution with respect to the plasma membrane invaginations,
the transverse tubules (t tubules), and the myofilaments
(98, 289, 346). On the contrary, since the classical studies
Physiol Rev • VOL
of Palade in the 1950s (258 –260), it was clear that the ER
of all other cells does not have an obvious ordinate morphology, but fills up the cytoplasm with an intricate network of tubules and cisternae that are continuous with
the nuclear membrane. Not only are the morphological
features of the ER and SR dramatically different, but their
protein composition is also quantitatively and qualitatively quite diverse. In the longitudinal SR, the SERCAs
represent up to 90% of the total membrane proteins while
the region of the SR juxtaposed to the t tubules, the
junctional SR, is highly enriched in a Ca2⫹ release channel
(also known as RyR) (44, 291). The RyRs are not present
in any other location along the SR membrane. The
SERCAs, on the contrary, represent up to 1% of the total
membrane proteins of the ER and membrane regions so
highly enriched in Ca2⫹ release channels such as the
junctional SR have never been found in the ER. In addition, a large part of the ER is covered with ribosomes (the
so-called rough ER) while no ribosomes are found on the
surface of the SR. At the same time, however, the SR
expresses several proteins typical of the ER, e.g., the
immunoglobulin heavy-chain binding protein, BiP, calnexin, calreticulin and protein disulfide isomerase (PDI)
(211). Finally, it must be stressed that a classical ER
network does exist also in striated muscle, close to the
nuclei, and endowed with all the typical morphological
features of the ER of other cells, e.g., covered by ribosomes and continuous with the nuclear membrane. Thus
a conservative answer to the question of whether the SR
and the ER are the same organelle is that the SR is a
differentiation of an ER-derived reticular network, that
has progressively evolved into a unique highly specialized
morphofunctional structure, characteristic of the striated
muscle fibers.
Does the structural and functional heterogeneity of
the SR, as far as Ca2⫹ handling is concerned, have an
equivalent in the ER of other cell types? Tentatively one
could try to answer the question using several criteria: 1)
morphology, 2) membrane and luminal continuity, and 3)
protein composition. These criteria are addressed separately below.
A. Organelle Morphology
Three morphologically distinct regions can be identified in the ER: the smooth, the rough ER, and the nuclear
membrane. The first two regions differ by the presence of
bound ribosomes (258 –260), while the nuclear membrane
is significantly different from the rough ER in as much as
the ribosomes are bound only to the outer surface, it
contains the nuclear pores, and the inner surface has a
unique protein composition and function (121, 363, 382).
To the best of our knowledge, however, to this morphologically distinct regions there is no correspondent qual-
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tive adenylate cyclase (AC7) is excluded (225, 342). We
have mentioned above that the rises of Ca2⫹ underneath
caveolae upon CCE activation are larger than those occurring in the cytosol or in other parts of the subplasma
membrane cytoplasmic rim (150, 151).
Another physiologically important example is that
provided by endothelial nitric oxide synthase (eNOS),
another enzyme normally localized at the cytoplasmic
surfaces of caveolae, via an acylation process (87, 88,
332). Mislocalization of eNOS outside the caveolar membrane markedly reduces the sensitivity of the enzyme to
Ca2⫹ influx as a trigger for NO production. Furthermore,
Isshiki et al. (150) showed that the increase in subplasma
membrane Ca2⫹ elicited simply by shifting cells from
Ca2⫹-free to Ca2⫹-containing medium results in robust
activation of eNOS, under conditions where there is no
appreciable increase in bulk [Ca2⫹]c, but significant increases in the subplasma membrane Ca2⫹ levels (150)
(and see Fig. 2).
The role of subplasma membrane Ca2⫹ microdomains is also of key importance in modulating the Ca2⫹
uptake capacity of a mitochondrial subpopulation (304,
306); this issue will be addressed in detail below. Suffice
it to say here that mitochondria appear to be both the
target of these microdomains (thus allowing a very rapid
and efficient uptake of Ca2⫹ by the organelles upon Ca2⫹
influx) as well as the key player for maintaining the Ca2⫹
flux through some channels, by buffering Ca2⫹ locally and
thus preventing their Ca2⫹-dependent inactivation.
Finally, a particularly striking, and apparently counterintuitive, role of subplasma membrane Ca2⫹ microdomains is that occurring in smooth muscles. In these cells,
a local mobilization of Ca2⫹ from strategically located ER
cisternae can activate Ca2⫹-sensitive plasma membrane
K⫹ channels leading to cellular hyperpolarization and
muscle relaxation, an effect that is the opposite of that
elicited by a global Ca2⫹ elevation, i.e., myofilament contraction (274).
MICRODOMAINS OF INTRACELLULAR CALCIUM
itative functional differentiation in terms of Ca2⫹ handling: they all possess Ca2⫹ channels and pumps, and
Ca2⫹ can be taken up and released from all three ER
regions (212, 291). Quantitatively, though, the density and
distribution of the proteins involved in Ca2⫹ handling are
far from being homogeneous, an important aspect that is
discussed in detail below.
379
different levels of Ca2⫹ in their lumen has been suggested
not only by experiments using recombinant aequorin
(230) but also by electron microscopic (EM) determination of Ca2⫹ content (275). However, it should be pointed
out that it is still unclear whether these functional differences occur within the ER sensu strictu or in other tubovesicular compartments endowed with Ca2⫹ uptake
and release mechanisms (see below).
B. Membrane and Luminal Continuity
C. Molecular Heterogeneity
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By far the most convincing approach to demonstrate
heterogeneity in the ER is to determine its protein composition and, using classical cell biological methods (subcellular fractionation and immunocytochemical localization), to establish whether indeed there are functionally
distinct subdomains.
1. The SERCAs
A key component that could lead to heterogeneity in
Ca2⫹ handling is obviously the mechanism of Ca2⫹ accumulation. The SERCA family includes the products of
three genes, named SERCA1 (ATP2A1), SERCA2 (ATP2A2),
and SERCA3 (ATP2A3), each giving rise to alternatively
spliced mRNA and protein isoforms. Until recently, the
situation appeared relatively simple. It has been known
for some years that SERCA1 and SERCA2 have two 3⬘-end
splice variants encoding isoforms differing in their COOH
termini, mainly expressed in skeletal muscles of adult
(SERCA1a) and neonatal fibers (SERCA1b), in cardiac
muscle (SERCA2a), and in all other cell types (SERCA2b)
(127, 194). A third SERCA2 variant has been recently
identified and named SERCA2c (109). The third gene,
SERCA3, was found in most nonmuscle cells (SERCA3a,
according to the new nomenclature). More recently, the
SERCA3 genes have been shown to possess a higher
degree of complexity. Mice, rats, and humans express a
variety of species-specific SERCA3 isoforms, in addition
to the species-unspecific SERCA3a. Indeed, SERCA3b and
-3c mRNAs and proteins were first described in mice and
humans (287); these mRNAs originated from the partial or
complete insertion of a new exon 21, respectively. Rats
were then found to be devoid of SERCA3b and SERCA3c
mRNAs, which are replaced by a so-called SERCA3b/c
isoform (203). Next, it was found that the insertion of an
additional exon 22 in human SERCA3b and SERCA3c
mRNAs gave rise to the SERCA3d and SERCA3e isoforms
(204). Finally, functional differences in the human SERCA3a,
-3b, and -3c isoforms were also pointed out (69). An
additional SERCA3f (h3f) mRNA has been very recently
identified, derived from new alternative splicing of the
human SERCA3 gene, and its distribution pattern in human cell lines of different origins and normal human
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There is strong, functional, and morphological evidence that both membrane and lumen are continuous
throughout the ER network. Direct evidence for membrane and luminal continuity has been provided by experiments of fluorescence photobleaching in cells expressing
an ER-located GFP. Prolonged light-induced photobleaching of GFP in a single small region of the ER results in
diffuse reduction (up to complete disappearance) of fluorescence from the whole reticular network, including
the nuclear envelope; on the other hand, short bleaching
periods are followed by a rapid restoration of fluorescence due to the diffusion of GFP from neighboring ER
tubules (77, 187, 230). Functional continuity within the ER
has also been demonstrated in terms of Ca2⫹ handling
properties. Petersen and co-workers (223) have demonstrated the concept of Ca2⫹ tunneling, in which ER in the
apical region of acinar cells of the pancreas is rapidly
refilled with Ca2⫹ originating from a pipette attached to
the basolateral membrane (i.e., on the opposite site with
respect to the apical pole), without measurable increases
in bulk cytosolic Ca2⫹ concentration. More recently, direct monitoring of Ca2⫹ diffusion within the ER lumen has
been carried out in pancreatic acinar cells by Park et al.
(262), confirming the conclusion that, at least in pancreatic acinar cells, the ER lumen is continuous and Ca2⫹ is
in rapid equilibrium within the network, from the cisternae located at the basolateral side up to the ER intermingled with the zymogen granules in the apical pole. A
similar conclusion was reached by Verkhratsky (384) for
different neurons. However, continuity in the ER lumen is
not always the rule. In cultured hippocampal neurons, for
example, vesicular ER compartments endowed with the
capacity of accumulating and releasing Ca2⫹, but clearly
luminally discontinuous from the rest of reticular ER,
have been described in dendrites (13). Functional evidence of noncontinuity, at least in terms of equilibration
of Ca2⫹ between different ER compartments, has been
provided in several model systems. For example, in some
smooth muscle cells it has been demonstrated that IP3
and caffeine (that activates RyRs) can release Ca2⫹ independently of each other, and the two Ca2⫹ pools apparently do not equilibrate (27, 122). Similar results have
been obtained in chromaffin cells (48, 254; but see also
Ref. 149), and the existence of subregions of the ER with
380
ROSARIO RIZZUTO AND TULLIO POZZAN
tissues does not completely overlap with that of the other
SERCAs (29).
Subcellular fractionation experiments of microsomal
fractions have often revealed partial dissociation in the
distribution of SERCAs and other ER marker proteins
involved in Ca2⫹ handling (see, for example, Refs. 251,
316). They have been, to the best of our knowledge,
mainly probed with nonisoform specific antibodies and
accordingly an even further subcellular heterogeneity in
SERCA distribution could have been underscored.
2. Ca2⫹ release channels
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The second most important molecular component of
the Ca2⫹ handling machinery of the ER is represented by
the Ca2⫹ release channels. They belong to two families,
the so-called IP3R and RyR. The heterogeneous distribution of these channels within intracellular membranes is
potentially one of the key factors in determining a spatial
heterogeneity in the Ca2⫹ signals and thus in the formation of Ca2⫹ microdomains; accordingly, this aspect will
be dealt with here in some detail.
A) RYRS. RyRs are expressed in three different isoforms, the product of three independent genes (347, 357);
a fourth, apparently fish-specific RyR (96, 234) will not be
dealt with here. The gene products assemble in homotetramers, giving rise to a multi-subunit protein channel of
very high molecular mass (⬎2,000 kDa). The isoforms
show different degrees of tissue specificity: RyR1 is expressed essentially in skeletal muscle, although recent
reports show its expression and activity in other cell
types, such as cerebellum (168) and B lymphocytes (120,
329). RyR2 is expressed in heart (257) and, comparatively
less abundantly, in brain (103). RyR3 is ubiquitously expressed at very low levels (including those tissues, such
as cardiac and skeletal muscle, in which one of the other
two isoforms is highly expressed) (349).
As to the subcellular distribution and the correlation
with function, the simplest case is that of muscle cells, in
which the abundant RyR repertoire represents the prevailing mechanism for rapidly generating the [Ca2⫹]c rise
driving muscle contraction. In both skeletal and cardiac
cells, RyRs are present in specialized portions of the SR,
the TC facing the t tubule (97). In this domain, the bulky
cytosolic protrusion of the RyR represents the “foot” (the
electron-dense material interposed between the two
membranes) and is part of the machinery that translates
membrane depolarization into SR Ca2⫹ release. This process requires either the transfer of a conformational
change from voltage-gated plasma membrane Ca2⫹ channels to RyR1 (in skeletal muscle) or the triggering of
Ca2⫹-induced Ca2⫹ release (CICR) by Ca2⫹ entry in the
case of RyR2 (in heart), as described below.
The brain represents a complex case, in which RyRs
(mostly RyR2) are expressed at moderate levels in spe-
cific subsets of Ca2⫹ stores. Both glial cells and a variety
of neuronal subpopulations (cerebral cortex, hippocampus, and cerebellum) have been shown to express RyRs
(for a review, see Ref. 33), and both isoform specificity
and expression levels appear to be developmentally regulated (233, 317). The wide expression of RyRs, and their
fine regulation, appear to correlate with an important role
of specific Ca2⫹ stores in modulating a variety of neuronal
functions, ranging from morphogenesis (145) to synaptic
plasticity (78), to cite but two interesting examples. In the
first case, axonal guidance was correlated with RyR-mediated Ca2⫹ release; in the latter, in hippocampal dendritic spines, Ca2⫹ entry through NMDA receptors was
shown to trigger a much larger CICR from RyR-dependent
Ca2⫹ stores (in other reports, however, this effect was not
observed, as store depletion did not reduce synaptically
evoked Ca2⫹ signals; Ref. 164). The situation of RyR
functions in glial cells is enigmatic. As mentioned above
its expression is well documented (for a review, see Ref.
33), although most groups failed to reveal clear RyRdependent Ca2⫹ release (see, for example, Ref. 267).
Finally, a challenging case is that of RyR3, given its
very low levels of expression in essentially all cell types
(117). What is its function, given that in most cases other
Ca2⫹ release channels (RyR1 and -2 or IP3Rs) are much
more abundant? An intriguing possibility was put forward
in the most unexpected case, i.e., skeletal muscle, in
which the amount of RyR3 is exceedingly small compared
with the main isoform, RyR1 (348). In some nonmammalian vertebrates, such as frogs, RyR1 and RyR3 are expressed in similar amounts, and thus their localization
and functional properties could be independently assessed. EM results showed that, although both isoforms
are restricted to the TC, RyR3 are located in a more
peripheral position (perijunctional feet) (84). As to function, CICR activity of RyR1 was shown be suppressed in
situ, thus leaving RyR3 as the prevailing system for extending the Ca2⫹ signal by CICR in this cell model (236,
237). In these concepts (inhibition of RyR1 CICR in situ
vs. high efficiency of RyR3) the apparent paradox of low
level RyR3 expression in adult mammalian muscle could
find an explanation. More in general, this weakly expressed RyR isoform could prove a relevant potentiating
mechanism of Ca2⫹ signals. Coherent with this possibility, the RyR3 knockouts show an overt phenotype, which
includes behavioral abnormalities (12, 105) but also reduced spark activity (59) and sensitivity to caffeine-induced muscle contracture (320).
Although by far the best-characterized mechanisms
of activation of RyRs are on the one hand the proteinprotein interaction between RyR1 and Ca2⫹ VOCs in skeletal muscle and on the other CICR in heart and most other
cells (affecting primarily RyR2 and -3), over the last few
years much interest has attracted the demonstration that
cADPR can also modulate the opening probability of the
MICRODOMAINS OF INTRACELLULAR CALCIUM
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Ca2⫹ signals are initiated (235, 331, 370). This reflects a
selective enrichment of the receptors in a subcompartment of the ER, given that the bulk of ER is localized in
the basolateral region of the cell. A similar situation is
true for other polarized epithelial cells, such as salivary
glands (178, 179, 245) and parotid acinar cells. No such
obvious polarization has been described for other cell
types, whether they express primarily one or all subtypes
of IP3Rs (for a review, see Ref. 385).
The distribution of IP3Rs on the nuclear membrane is
still debated. Evidence has been provided that in some
cells IP3R2 is particularly abundant on the nuclear envelope. The localization of IP3Rs on the nuclear membrane
is not surprising, given that the nuclear envelope is continuous with the ER and the molecular composition of the
outer surface of the nuclear envelope is known to be
indistinguishable (as far as other protein markers is concerned) from the rest of the rough ER. Indeed, in isolated
nuclei, IP3Rs have been clearly demonstrated on the outer
surface also by electrophysiological, direct measurement
of IP3-activated currents (197, 198). More debated is the
expression of IP3Rs on the inner surface of the nuclear
membrane, and conflicting results have been reported.
Several functional data have been published in the last
years supporting the possibility that IP3Rs are located
also on the inner surface of the nuclear membrane (see,
for example, Refs. 112, 113, 141, 273). Although these
functional experiments are apparently convincing, to the
best of our knowledge, however, no immunocytochemical
evidence at the EM level for the presence of IP3Rs on the
inner surface of the nuclear envelope (rather the opposite) has yet been obtained. It also needs to be stressed
that the nuclear envelope often branches into the nucleus,
and thus it may occur that the apparent IP3-induced release of Ca2⫹ from the inner nuclear envelope depends on
these invaginations, rather than on IP3Rs located on the
inner surface of the membrane. Indeed, Echevarria et al.
(76) have recently provided convincing evidence for the
existence of such a nucleoplasmic reticulum, continuous
with the nuclear envelope and endowed with IP3Rs. More
convincing in favor of the localization of the IP3R on the
inner surface of the nuclear membrane appears the situation of starfish oocytes, but the situation in these cells
may be unique (323).
In several cell types, the IP3Rs appear to be often
concentrated close to the plasma membrane (e.g., in neurons, in some smooth muscle, or in atrial cardiomyocytes), and it has been also suggested that the IP3Rs may
be endowed with the capacity to interact directly with
some plasma membrane proteins, e.g., TRP channels (25,
400) or G protein (402) regulating their opening under a
variety of conditions. Finally, the IP3Rs in the postsynaptic densities appear to be part of a complex scaffolding
machinery composed of several proteins, including metabo-
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RyRs (for a recent review see Ref. 176). Although it is
undisputed that cADPR can cause the release of Ca2⫹
through the RyRs, it remains undetermined whether this
molecule acts as a long-term modulator of Ca2⫹ release or
whether its concentration can rapidly vary within cells
upon receptor activation. It is worth mentioning here that
another soluble messenger, NAADP, may also activate the
RyRs, although the identification of the Ca2⫹ release
channels sensitive to NAADP is still a matter of controversy, particularly in mammalian cells (see sect. V for a
more detailed discussion of this point).
B) IP3RS. IP3Rs were first identified and cloned in the
mouse in 1989 (104). Currently, three genes are known,
with several alternatively spliced variants. The full-length
sequences for three distinct IP3R genes from human and
rodents are now available; the primary sequences of IP3Rs
from other species (e.g., Xenopus, Caenorhabditis elegans, and Drosophila) have also been determined (for
recent reviews, see Refs. 270, 368, 385). The type 1 isoform of the IP3R is spliced at three regions, with five
distinct variants resulting from splicing at the S2 site. The
type 2 isoform has also been proposed to undergo alternative splicing (for reviews, see Refs. 269, 270, 368, 385).
Whereas RyRs can form only homotetramers, IP3R
channels can form homo- or heterotetramers. Characteristics such as structure-function relationship, activation
by IP3, modulation by Ca2⫹, ATP, phosphorylation, or
accessory proteins have all been thoroughly investigated,
and several recent reviews have been published on this
topic in the past few years (see, for example, Refs. 269,
270, 368, 385); here we limit ourselves to the discussion of
IP3R distribution in the ER membrane as a potential
source of heterogeneity in Ca2⫹ signaling. In cerebellar
Purkinje neurons, the cell type with by far the highest
expression of IP3Rs, IP3R1 is the most abundant (⬎90% of
the total); these channels are found throughout the whole
cell (cell body, dendrites up to the spines), apart from the
axon, but within ER membranes the distribution is far
from homogeneous (214, 319, 324, 365). The density on
smooth ER cisternae is much higher than on rough membranes, and the density of the receptors on the outer
surface of the nuclear membrane is comparatively rather
low. No immunohistochemical evidence for the expression of IP3Rs on the inner surface of the nuclear membrane has ever been reported in these cells, nor significant
labeling of the plasma membrane has been observed.
The situation in other cell types, that most often
express more than one subtype of IP3Rs, is far more
complex. In the central nervous system, for example,
IP3R1 predominates in neurons [when present, IP3R3 is
primarily at the level of the nuclear envelope (330)], and
IP3R2 is exclusively expressed in astrocytes (333, 339). In
the exocrine pancreas, a tissue where all three types of
IP3Rs are expressed, the channels are concentrated in the
apical pole, the trigger zone where all receptor-activated
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ROSARIO RIZZUTO AND TULLIO POZZAN
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although initial studies in the 1980s provided strong evidence in favor of a role for IP3 in Ca2⫹ release from SR
(383, 390), there is recent data that convincingly argue
against this possibility (290). Unfortunately, this problem
has been largely neglected in the last years.
3. Luminal Ca2⫹ binding proteins
Finally, heterogeneity in ER Ca2⫹ handling may depend on heterogeneous distribution of luminal Ca2⫹ binding proteins (215). Such heterogeneity is clear in the SR of
striated muscle where the high-capacity, low-affinity Ca2⫹
binding protein calsequestrin (CS) is exclusively located
in the TC (18, 42). Two isoforms of CS are known, one
typical of skeletal and the other of cardiac muscle. This
protein is expressed, in addition to striated muscle, in
some smooth muscle cell types (250, 298, 388, 389) and in
avian cerebellar Purkinje neurons (365, 387, 391). In these
cells too, CS has an inhomogeneous distribution within
the ER, presumably due to the unique sorting characteristics of this protein (107, 108). Other ubiquitous Ca2⫹
binding proteins (calreticulin, BiP, PDI, etc), on the other
hand, appear to have a rather diffuse and homogeneous
distribution (for a review, see Ref. 211), but this problem
has not been analyzed in great detail.
D. Generation of Ca2ⴙ Microdomains by RyRs
In cardiac and skeletal muscle, a highly structured
morphological architecture allows the generation of Ca2⫹
microdomains at the surface of the SR; these microdomains are a key component of the trigger for firing the
Ca2⫹ signals necessary for cell activation (97, 356). Indeed, in both cell types, the physiological, stimulatory
signal leading to contraction is conveyed by an action
potential: a plasma membrane depolarization travels via
the opening of voltage-dependent Na⫹ channels and
reaches the cell interior through invaginations (the t tubules) in which VOCs are located; this causes the influx of
Ca2⫹ that is insufficient to induce the physiological response (the sliding of the acto-myosinic contractile apparatus), but represents the trigger for the release of Ca2⫹
(particularly in the heart) from the large intracellular
Ca2⫹ reservoir (the SR), through the RyRs (302). In skeletal muscle, direct coupling between the two molecules
(VOCs and RyRs) is believed to cause the opening of
RyRs. In the latter tissue, thus the influx of Ca2⫹ through
VOCs plays a facilitatory, but not necessary role. Indeed,
the interaction between the two channels is thought to be
the necessary event to cause activation of the RyR isoform of skeletal muscle (RyR1) (295), and then Ca2⫹
release through the RyR1 is amplified by CICR. In heart,
no direct physical interaction occurs between the two
types of channels, and thus a high [Ca2⫹] in the proximity
of the RyR2 represents the essential activatory signal
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tropic receptors for glutamate, homer, etc. (377, 400; for
recent reviews, see Refs. 270, 385).
Further heterogeneity can be provided by different
functional modifications of the IP3Rs in selected compartments of the cells. Thus, in neurons, phosphorylated
IP3Rs appear to be differently distributed between dendrites and cell bodies (279).
Still highly debated is the question of whether and to
what extent IP3Rs, and in particular IP3R3, reach the
plasma membrane. IP3R1 is endowed with strong ER
retention signals (263), but whether this is the case or not
for the other isoforms is not known. Good functional
evidence for the existence of IP3-gated Ca2⫹ channels has
been reported for the plasma membrane of olfactory neurons (41, 80, 190) and lymphocytes (167, 367).
To complicate matters even further, the distribution
of the IP3Rs can vary during cell differentiation and upon
stimulation. A major reorganization of the whole ER and
of the IP3R distribution occurs during oocyte maturation
(see, for example, Refs. 93, 354). Similarly, a spectacular
redistribution of IP3Rs occurs in MDCK cells during the
formation of the polarized epithelium (58), and a similar
result had been observed in keratinocytes (101). In these
cases, however, it is yet unclear whether the dramatic
rearrangement involves complete or partial redistribution
of the ER as a whole or whether it reflects a selective
mobility of the IP3Rs themselves within the network.
Several proteins interact with IP3Rs. A few have a
modulatory role, while others may be essential in determining its subcellular localization. Among the latter, a key
role is probably played by actin, ankyrin, homer, and
protein 4.1 (for recent reviews, see Refs. 270, 385). A
unique regulation of IP3R1, dependent on pH, Ca2⫹ concentration, and redox state within the ER lumen is mediated by the resident ER protein of the thioredoxin family,
ERp44 (142).
A last, but not secondary, question concerns the subtle microscopic heterogeneity of IP3R distribution, i.e.,
their aggregation in microscopic clusters. Indeed, ion
channels and receptors in the cell membranes and internal membranes are often distributed in discrete clusters.
With the use of mathematical modeling, it has been concluded that channel clustering can enhance the cell’s
Ca2⫹ signaling capability (334, 335). Mapping of functional channels (in the ER of Xenopus oocytes) indicates
indeed that IP3Rs tend to aggregate into microscopic (⬍1
␮m) as well as macroscopic (⬃10 ␮m) clusters. IP3R
clustering may contribute to the spatiotemporal complexity of the [Ca2⫹]c signals (197, 198; see also below).
While the presence of IP3Rs in the SR of smooth
muscle, from different tissues, and in cardiac myocytes,
including the Purkinje fibers (125), is undisputed, the
expression, localization, and functional role, if any, of
IP3Rs in the SR of skeletal muscle is largely undetermined. The literature on this topic is highly contradictory:
MICRODOMAINS OF INTRACELLULAR CALCIUM
1. The Ca2⫹ sparks
Rapid Ca2⫹ imaging of both cardiac and skeletal
muscle has revealed the occurrence of transient, local
increases in Ca2⫹ concentration, denominated Ca2⫹
sparks (50) that were attributed to the opening of single
RyR channels or, more likely, a cluster of RyRs (186, 375).
Typically, these localized events are 2 ␮m wide and last
for ⬍0.05 s (10 ms to peak and 20 ms half-decay) (see, for
example, Fig. 4). They represent elementary excitation
events, are self-limited, and in a nonstimulated heart
cell occur with a frequency of ⬃100 Hz. Two aspects
are relevant for the understanding of their basic properties.
The first regards the number of RyRs involved and
their activation mechanism. Is a spark, by definition,
the smallest [Ca2⫹]c rise detected by fluorescence microscopy with Ca2⫹ indicators, the opening of a single
or multiple channels? This issue is still subject to debate, but the data currently available seem to argue
against the possibility that a spark represents the activity of a single channel. Indeed, when taking into
account the amplitude of the [Ca2⫹]c rise, the area
involved and the buffering capacity of the cytosol, the
ion flux generating a spark was estimated to be 2– 4 pA
(50). Electrophysiological estimates of the current
through a single open RyR channel are ⬃0.35 pA (and
the expectation is that in physiological conditions in
situ the current is even markedly smaller) (210). Thus it
appears more likely that a spark is due to the synchronous
opening of a cluster of RyRs. How does this occur? At
least in part, the anatomical architecture provides the
mechanism for the synchronous activation. Indeed, the
crucial site for muscle excitability is the junction between
the t tubules (containing the VOCs) and the TC of the SR
(where clusters of RyRs are located). In this restricted
cytosolic domain upon opening of the plasma membrane
2⫹
FIG. 4. Ca
sparks. A: typical Ca2⫹ sparks, taken by high-speed confocal microscopy, in cardiac myocytes loaded with fluo 3. On the left,
control cells; on the right, cells from hypertrophic heart. Signal-averaged Ca2⫹ sparks are shown as line-scan images (top) and as surface plot
(bottom). B: sulfo-rhodamine B was used to identify the extracellular space in the TT (red), whereas Ca2⫹ sparks were imaged simultaneously
(green). These images demonstrate that Ca2⫹ release takes place where the TT and TC are located. Cells are from control (SD), salt-resistant animals
(SR/Jr), and salt-sensitive, hypertensive animals (SS/Jr). [From Gomez et al. (123), copyright 1997 AAAS.]
Physiol Rev • VOL
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(238). Therefore, in heart Ca2⫹ influx through VOCs is
necessary to trigger SR Ca2⫹ release through the RyR2 via
the process of CICR.
In this context, the Ca2⫹ sensitivity of the RyRs, as
well as the evaluation of the local Ca2⫹ concentration to
which they are exposed, represents a key factor in determining the excitability of the muscle fiber and the efficiency and duration of contraction. For this reason, a
great effort has been placed on identifying the “fundamental” Ca2⫹ signaling events and clarifying the mechanisms
that allow their extension and lead to the contraction of
the whole fiber. As will be discuss later, it is now clear
that alterations in this ordered series of events leads to
dysfunctions observed in a number of pathological conditions (ranging, in heart, from cardiac arrhythmias to
heart failure). For reasons of brevity, we will focus on
heart cells, in which both the regulatory mechanisms and
the pathological consequences have been studied in great
detail, to provide an outline of the molecular basis of
elementary events, and the transition of these events into
global rises in the cell. However, despite the known differences in the molecular identity of RyR isoforms and in
the mechanism of channel activation, it should be remembered that the concept of spatially and temporally restricted “elementary” Ca2⫹ signals elicited by RyR opening (“sparks”) holds true also in skeletal muscle, and the
regulatory mechanisms controlling their frequency and
the extension into global signals are largely the same.
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ROSARIO RIZZUTO AND TULLIO POZZAN
Physiol Rev • VOL
2. Generating a global Ca2⫹ signal
How, and when, do these elementary events turn into
global signals, capable of causing the activation of the
contractile apparatus, and thus the functional response of
muscle cells? The simplest, and most physiological, mechanism is that of the action potential. In this case, the rapid
propagation of a depolarization wave across the plasma
membrane leads to the virtually synchronous opening of
VOCs and thus the summation of Ca2⫹ sparks into a
global rise (99). In this case, the expected increase in
subplasmalemmal [Ca2⫹] (due to the concerted activity of
multiple VOCs) leads to a dramatic enhancement of the
spark rate (up to 106 higher than in resting conditions),
which is further amplified by the greater efficiency of
CICR. This leads to a large [Ca2⫹]c rise and the prompt
activation of myocyte contraction.
In pathological conditions, such as cardiac arrhythmias, Ca2⫹ sparks can generate Ca2⫹ waves gradually
diffusing through the cell. Specifically, conditions of
“higher excitability” of SR Ca2⫹ release, such as that
conferred by a higher luminal Ca2⫹ concentration (129,
341), may prevent the self-limitation of the elementary
event and cause a feed-forward mechanism that causes
the opening of neighboring RyR clusters, and thus the
slow propagation of the Ca2⫹ rise to the whole cell (49).
The asynchronicity of the Ca2⫹ rise (and consequently of
the refractory period) may lead to the establishment of
looping signals and thus generate alternative pace-making
regions in the heart, leading to cardiac arrhythmias. Apart
from the higher intrinsic excitability of RyRs, also the
cross-talk of Ca2⫹ release channels in signaling microdomains has been shown to increase cardiac excitability
(195). Type 2 IP3Rs, known to be expressed in heart (125),
colocalize with RyRs in the junctional area, i.e., in the cell
domain critical for triggering the [Ca2⫹]c rise. Thus stimulation by endothelin-1, angiotensin II, or ␣-adrenergic
agonists (all known to be arrhythmogenic) lowers the
threshold for SR Ca2⫹ release (195). This through the
activation of depolarizing currents, such as ITI, the Ca2⫹activated transient depolarizing current (159) causes delayed after-depolarizations (DADs) that may trigger premature action potentials and thus the generation of an
arrhythmia.
3. The regulation of RyR activity in vivo
In this context, it is easy to understand why significant emphasis has thus been placed on identifying the
regulatory mechanisms operating in the RyR microenvironment and controlling Ca2⫹ release through the
channels (for a review, see Ref. 91). At least in vitro, it
has been shown that a number of factors play an active
role; they include Ca2⫹ itself, both on the cytosolic side
(i.e., the positive feedback at the basis of CICR and the
inhibitory effect described at millimolar Ca2⫹ concen-
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Ca2⫹ channels, the RyR clusters are exposed to a microdomain of high [Ca2⫹] that triggers RyR opening and
releases SR Ca2⫹ (97, 356). In turn, the opening of the
first RyRs of the cluster will further increase the local
[Ca2⫹] and thus recruit a larger subset of RyRs, if not
the whole cluster, thus generating a measurable Ca2⫹
spark.
The second essential property of the spark is its
spatial limitation. In principle, this is a contradiction,
given the inherent self-propagability of the Ca2⫹ signal
(i.e., the fact that Ca2⫹ itself is the main activator of RyRs,
and that the process of CICR is necessary for generating
the Ca2⫹ spark, as described above). The simplest explanation for the spatial restriction rests most likely in the
organization of the microdomain, which allows a closely
packed group of RyRs to act synchronously (“sticky cluster”). Indeed, in normal conditions the [Ca2⫹]c rise in the
microdomain elicited by the opening of one, or few,
plasma membrane or RyR channels rapidly recruits most
of the cluster, giving rise to the visible Ca2⫹ spark. In
conditions of normal SR excitability, cytosolic Ca2⫹ buffers prevent the involvement of neighboring clusters (but
see below the effect of SR Ca2⫹ overload). As to the
activated cluster, local depletion of junctional SR leads to
a rapid drop of Ca2⫹ flux through the tightly coupled
group of RyRs, and thus promptly terminates the Ca2⫹
spark (343). This spatial limitation could be overcome
either by the simultaneous opening of plasma membrane
Ca2⫹ channels (such as the opening of VOCs triggered by
the plasma membrane depolarization of the action potential) or by a higher open probability of RyRs, as we will
discuss later.
Alternative possibilities for the spatial restriction of
sparks cannot be excluded. These may include the heavily
debated issue of Ca2⫹ inactivation, suggested by the evidence of the bell-shaped sensitivity of RyRs to Ca2⫹ (79),
but questioned by the demonstration that Ca2⫹ inhibition
occurs at ⬃1 mM [Ca2⫹] for RyR1 (209) and 5–10 mM
[Ca2⫹] for RyR2 and RyR3 (54, 60). Thus, if any physiological role has to be postulated for this process, it most
likely refers to the termination phase of action potentials
and the induction of a refractory phase. Another proposed
mechanism is “Ca2⫹ adaptation” of RyRs, i.e., the slow
(time constant ⬃1 s) decay of channel opening (Po) observed in flash photolysis experiments (130), that could
be relieved by a second Ca2⫹ pulse and thus appeared
different from classical Ca2⫹ inactivation. Occurrence
and significance of RyR adaptation is still debated (for a
review, see Ref. 91). It should be noted that, at physiological Mg2⫹ concentration, the time constant is 10-fold
lower (378), and thus the role of adaptation in rapid
events, such as the control of RyR excitability in sparks,
appears more plausible.
MICRODOMAINS OF INTRACELLULAR CALCIUM
Physiol Rev • VOL
(ARVD2) or increasing (VTSIP) it (373). Thus it has been
proposed that ARVD2 mutations, with different mechanisms, render RyR2 leaky, while the VTSIP mutations
reduce the probability of channel opening. In the case of
phospholamban, the clinical phenotype of human mutations adds complexity to the picture. In this case, null
mutations (and the consequent hyperactivation of SERCA)
lead to dilated cardiomyopathy (325), in complete contrast to the mouse knockout in which enhancement of
cardiac activity was reported (196). A sensible explanation for the discrepancy is that while in mouse (which
exhibits a high frequency of heart rate) phospholamban is
mostly inhibited, in humans this is not the case, and
chronic inotropic stimulation leads to development of
heart hypertrophy (131).
E. Elementary and Global Signals
in IP3-Dependent Systems
The occurrence of elementary signals, due to the
opening of a spatially restricted group of channels (264)
and denominated “Ca2⫹ puffs” (396), is also shared by the
IP3-dependent cell systems. In Xenopus oocytes, imaging
experiments at high spatial and temporal resolution demonstrated the appearance, at low agonist concentrations,
of local increases that start abruptly, peak in ⬃50 ms, and
decay in 2–300 ms (265). These localized [Ca2⫹]c increases represent the opening of a tightly packed cluster
of IP3Rs (336). As in the case of RyR-dependent systems,
Ca2⫹ puffs denote the “Ca2⫹ excitability” of the cell, but
the induction of a physiological response requires the
coalescence of these elementary events into a larger rise,
which may be limited to a portion of a polarized cell, or
diffuse to the whole cell body in a truly global Ca2⫹ signal.
Moreover, the “pacemaker” activity of Ca2⫹ puffs participates in controlling the frequency of repetitive Ca2⫹ spiking, the phenomenon known as “Ca2⫹ oscillations” that
regulates downstream events such as gene transcription
(72) or metabolism (133). The initiation and propagation
of a global Ca2⫹ signal relies on the cumulative recruitment of Ca2⫹ puffs as the slow rise in [Ca2⫹] sensitizes
neighboring IP3Rs, thus increasing the probability of puff
occurrence and their merging into propagating Ca2⫹
waves (200). Interestingly, in a series of studies carried
out in HeLa cells, it could be demonstrated that the “pacemaker” activity during the latency period (i.e., before the
initiation of a global Ca2⫹ rise) originates from a limited
number of puff sites, which repetitively fire during consecutive stimulations (369). Whether this represents a
structural organization of IP3R clusters, or the convergence of modulatory inputs, remains to be ascertained
and may vary in different cell types.
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trations) and on the luminal side (sensitivity of RyRs to
cytosolic agonists increases at high levels of SR Ca2⫹
loading), Mg2⫹ and ATP (although it is unlikely that
major fluctuations occur in their cytosolic levels),
cADP ribose (that has been proposed to play an important regulatory role not only in nonexcitable cells, e.g.,
sea urchin eggs, as discussed elsewhere in this review,
but also in heart), oxidation of critical residues, and
phosphorylation/dephosphorylation events. In addition,
several clear-cut examples of regulation by proteinprotein interaction have been reported; these include
calmodulin (with a reported inhibitory effect on RyR2
and a bell-shaped effect on RyR1, activatory and inhibitory at low and high Ca2⫹, respectively), FKBP12.6,
and calsequestrin. The relative importance of these potential regulatory mechanisms in vivo is difficult to ascertain and remains inferred by either the interpretation of
the in vitro studies or by analysis of informative clinical
phenotypes.
On the first aspect, a large body of work correlates
the state of filling of the SR store with both the excitability
of RyRs (129) and the control of Ca2⫹-activated inward
currents (56), such as ITI, two mechanisms that may
concur in the generation of arrhythmias. SR Ca2⫹ overload may also increase the contractility of the myocardium, as directly demonstrated in a mouse model in
which SR Ca2⫹ accumulation was increased by overexpressing a SERCA and ablating the SERCA inhibitor phospholamban (403).
Given that the relative importance of these potential
regulatory mechanisms in vivo is difficult to ascertain, the
information deduced from informative clinical phenotypes appears particularly useful. Hyperactivation of protein kinase A (PKA), reported to occur in the failing heart,
hyperphosphorylates the RyR channels, and hence their
binding to FKBP12.6, with consequent alteration of the
open probability of the channel and defective channel
function (205). Similarly, it was shown that PKC-␣, by
phosphorylating the inhibitor (I-1) of protein-phosphatase
1 (PP1), affects PLN phosphorylation and hence SR Ca2⫹
loading. As a consequence, PKC-␣, by integrating different
Ca2⫹-dependent signaling routes, guides the functional
derangement of pathological cardiac hypertrophy (35).
The conceptually simpler genetic diseases provide related
examples. Recently, two genetic disorders have been ascribed to mutations in RyRs, a subset of arrhythmogenic
right ventricular dysplasia cases (ARVD2), and stressinduced polymorphic tachycardia (VTSIP), in which, respectively, the prevailing or only clinical manifestation is
a fatal arrhythmia (374). The diseases are caused by different missense mutations, located in the pore forming
region (ARVD2) or in the regions binding the channel
negative modulator FKBP12.6 (ARVD2 and VTSIP). Interestingly, in the latter case, the mutations of the two
diseases differently affect FKBP12.6 binding, decreasing
385
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ROSARIO RIZZUTO AND TULLIO POZZAN
F. Turning Puffs Into Global Signals: Role of Other
Second Messengers
Physiol Rev • VOL
IV. MITOCHONDRIA
A. Stores, Sinks, or Inactive Elements
in Ca2ⴙ Homeostasis?
The capacity of mitochondria to rapidly move Ca2⫹
across their membranes was a relatively early notion in
bioenergetics and cell biology. The principle of energy
conservation in mitochondria (i.e., the translocation by
protein complexes of H⫹ across an ion-impermeable inner membrane) generates a very large H⫹ electrochemical
gradient that can be employed not only by the H⫹-ATPase
for running the endoergonic reaction of ADP phosphorylation, but also to accumulate cations into the matrix.
Indeed, research carried out in the 1960s by various
groups demonstrated that energized mitochondria can
rapidly take up Ca2⫹ from the medium and characterized
the following fundamental principles (for reviews, see
Refs. 75, 304): 1) an electrogenic pathway [i.e., most likely
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Pancreatic acinar cells are an interesting example of
how the site of [Ca2⫹]c increases determines different
cellular effects. Agonist-evoked [Ca2⫹]c signals originate
in the apical pole when Ca2⫹ puffs merge into a larger
[Ca2⫹]c rise (370). These rises can either remain confined
to the apical region or be extended to the whole cell (43,
179). In the former case, the effect is limited to the
activation of the main physiological function of the cell
(secretion of digestive enzymes), in the latter long-term
effects are observed (ranging from activation of gene
expression to cell death) (272, 372). In this complex system, in which different hormonal stimulations, e.g., cholecystokinin (CCK) and ACh, have distinct Ca2⫹ signaling
and functional effects, interesting information has been
acquired.
First of all, the diffusibility of local increases (puffs)
by CICR differs in different portions of the cell. When a
[Ca2⫹]c rise was induced by local uncaging of Ca2⫹, its
capacity of triggering a Ca2⫹ wave differed in the various
portions of this polarized cell (being higher in the apical
region) and required the concerted action of RyRs and
IP3Rs (8). Indeed, when either class of channel was inhibited, the diffusion of the Ca2⫹ wave was blocked. Thus the
apical region, in which receptor clustering (165, 183)
could account for the higher sensitivity, acts as the “pacemaker” of the agonist-evoked [Ca2⫹]c rise. As to the capacity of the [Ca2⫹]c wave to extend to the basal region,
two aspects appear critical. The first is the intrinsic potency of the stimulus (256). Supramaximal stimulation
with agonists always induces a Ca2⫹ wave that reaches
the basal region. As to stimulation with lower, more physiological agonist concentrations, there is a general consensus that the [Ca2⫹]c rise generated by stimulation of
different receptors occurs initially in the apical region,
suggesting that the three types of channels are clustered
in the same stores and activated by the appropriate receptor pathway (40). Local Ca2⫹ rises could be converted
into global responses through the concerted action of
different intracellular messengers (IP3, cADP ribose,
NAADP), in an agonist-specific manner. ACh-evoked
spikes could be efficiently transformed into global signals
by further intracellular addition of NAADP and cADP
ribose, CCK-evoked spikes by IP3 (43). According to Cancela et al. (43), low concentrations of any of these Ca2⫹
mobilizing stimulus elicited only Ca2⫹ elevations localized to the apical pole (see Fig. 3). At variance, however,
some reports indicate that the RyR agonist cADP ribose
and NAADP increased [Ca2⫹]c primarily in the basolateral
portion of the cell (165, 180). This experimental discrepancy has not been solved yet.
Finally, ACh-triggered cADP ribose synthesis was
eliminated in CD38 knock-out animals, and this resulted
in different alteration of the Ca2⫹ signaling at low and
high agonist concentrations, respectively (102). Thus conceptually different mechanisms (membrane receptor clustering, molecular diversity of intracellular stores, crossregulation of second messenger biosynthesis) appear to
control the generation and diffusion of intracellular Ca2⫹
signals in polarized cells. This adds a level of complexity
to the system and a further mechanism for the integration
of different receptor pathways into a common signaling
route.
The extension from the apical to the basal region of
the cell is then controlled by another checkpoint, i.e., the
clustering of mitochondria, acting as fixed Ca2⫹ buffers,
at the boundary between the two cell domains. As described in more detail in section IV, efficient uptake by
tightly packed mitochondria represents a “firewall” transiently blocking the Ca2⫹ wave (372). To proceed, the
Ca2⫹ wave must therefore overwhelm their uptake capacity and initiate a CICR-mediated wave downstream of the
barrier. Overall, this biological system provides a clear
example of how the concepts identified, and clarified, in
simpler models (the occurrence of elementary IP3R-dependent events, the puffs, and the different modes for
extending them to large cell domains, or the whole cell)
have a precise physiological application in cell types in
which different modes of Ca2⫹ signaling exert different
functional effects. The elucidation of the regulatory mechanisms involved will provide new pharmacological clues
for addressing pathological conditions (in the case of the
pancreas, the medical emergency of acute pancreatitis)
caused by deranged Ca2⫹ signals. On this topic, recent
interesting work has provided insight into the pathogenesis of pancreatic damage induced by alcohol (62) or bile
reflux (160, 392).
MICRODOMAINS OF INTRACELLULAR CALCIUM
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ical work demonstrated that important mitochondrial
metabolic enzymes are regulated by Ca2⫹, thus suggesting
fluctuations of matrix Ca2⫹ concentration and fine tuning
of the enzymes in physiological conditions (206). However, given that no direct experimental evidence could
support the notion that the Ca2⫹ concentration in the
mitochondrial matrix ([Ca2⫹]m) rapidly changes upon cell
stimulation, this observation did not modify the general
perception of mitochondria as relatively inactive bystanders in the complex scene of cellular Ca2⫹ homeostasis.
This situation was completely reversed by the direct
demonstration of mitochondrial Ca2⫹ uptake in intact
cells, and by the appreciation of the importance in this
process of subcellular heterogeneity in Ca2⫹ levels, the
theme of the current review. This became possible when
tools were developed that allowed the selective measurement of [Ca2⫹]m in living cells. This was first achieved by
targeting to mitochondria a Ca2⫹-sensitive photoprotein,
aequorin (311), and allowed us to demonstrate that a
rapid [Ca2⫹]m peak, reaching values well above those of
the bulk cytosol, parallels the [Ca2⫹] rise evoked in the
cytoplasm by cell stimulation (305). Similar conclusions
could be reached also with fluorescent indicators, such as
the positively charged Ca2⫹ indicator rhod 2 (that accumulates within the organelle) (30, 74) and the more recently developed GFP-based fluorescent indicators (89,
90, 239). With the latter probes, endowed with a much
stronger signal than the photoprotein, single-cell imaging
of organelle Ca2⫹ can be carried out (90, 360). Thus it is
possible to match the accurate estimates of [Ca2⫹]m values, obtained with the photoprotein, with detailed spatiotemporal analyses of [Ca2⫹]m transients (see Fig. 5). With
these tools in hands, not only the notion was confirmed
that mitochondria promptly respond to cytosolic [Ca2⫹]
rises, but also that the [Ca2⫹]c oscillations, the typical
response to agonists of many cell types, are paralleled by
rapid spiking of [Ca2⫹]m, thus providing a frequency-mediated signal specifically decoded within the mitochondria, as clearly shown in hepatocytes (133), cardiomyocytes (313), and HeLa cells (90).
A critical, and still somewhat controversial, issue
was why mitochondria in situ behave so differently from
what is expected based on the properties of their Ca2⫹
transporters established in vitro? In other words, why
does [Ca2⫹]m rise, in a few seconds, to values above 10
␮M (in some cell types up to 500 ␮M) in response to
[Ca2⫹]c elevations that rarely exceed 2–3 ␮M, given that in
the latter conditions mitochondrial Ca2⫹ accumulation
should be very slow? A simple explanation would have
been that the affinity of the transporters within cells is
much higher than previously reported. However, direct
perfusion of permeabilized cells with buffered [Ca2⫹] similar to those measured in the cytoplasm of stimulated cells
induced a relatively inefficient Ca2⫹ loading of mitochondria (thus confirming the notion of a low-affinity uptake
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a channel, as was recently directly demonstrated (161)]
that acts to equilibrate Ca2⫹ with its electrochemical gradient, and thus accumulates the cation into the matrix,
and 2) two exchangers (with H⫹ and Na⫹, mostly expressed in nonexcitable and excitable cells, respectively),
that utilize the electrochemical gradient of the monovalent cations to prevent the attainment of electrical equilibrium (that would imply, for a mitochondrial membrane
potential, ⌬⌿m, of 180 mV and a cytosolic Ca2⫹ concentration of 0.1 ␮M, accumulation of Ca2⫹ into the matrix up
to 0.1 M). It was thus logical to assume that mitochondria
were loaded with Ca2⫹, possibly releasing it in a number
of physiological and/or pathological conditions. In other
words, mitochondria were very promising candidates for
the role of rapidly mobilizable Ca2⫹ stores.
Experimental evidence obtained in the 1980s clearly
disproved this possibility. In those years, while Ca2⫹
emerged as the ubiquitous, fundamental second messenger known to every biology student, it became immediately evident that mitochondria were not the active store
in these signaling pathways. Indeed, the messenger
shown to be produced upon stimulation of G proteincoupled or growth factor receptors, IP3, acts on ion channels located in the ER (351). Moreover, the latter organelle (and not mitochondria) was shown to contain the
molecular elements of a Ca2⫹ store: a pump (to accumulate Ca2⫹ against electrochemical gradient), a channel (to
rapidly release it), and buffering proteins (to increase the
total amount of ion that can be stored; for a review, see
Ref. 291). For this reason, the concept of mitochondria as
cellular Ca2⫹ stores was largely dismissed, and most of
the research in calcium signaling focused on the ER (and
its specialized counterpart of muscle cells, the SR). As
mentioned above, cellular and molecular work allowed to
identify different classes (and subtypes) of Ca2⫹ channels
and emphasis was mostly placed on the role of ER/SR
(and its subcompartments) in spatio-temporally patterning the increases in Ca2⫹ concentration occurring upon
cell stimulation.
At the same time, also the possibility that mitochondria receive Ca2⫹ after stimulation, thus acting as Ca2⫹
sinks, appeared dubious. Indeed, the availability of indicators that could be easily loaded into most cell types and
calibrated into accurate [Ca2⫹] estimates (126) allowed us
to verify that in living cells not only resting values (⬃0.1
␮M), but also those briefly reached after physiological
stimulation (1–3 ␮M) are well below the affinity of mitochondrial Ca2⫹ transporters (i.e., those estimated in the
earlier work with isolated organelles). Thus the general
consensus was that mitochondria in most cases of cellular
Ca2⫹ mobilization would receive little, if any, of it, while
substantial organelle loading could be achieved only in
the case of overt Ca2⫹ overload (i.e., in a number of
pathological conditions, such as, for example, excitotoxic
neuronal challenge). In contrast with this view, biochem-
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ROSARIO RIZZUTO AND TULLIO POZZAN
system) (305). Conversely, discharge of Ca2⫹ from the ER
triggered by the direct perfusion of IP3 (thus causing the
opening of the physiological Ca2⫹ release pathway also in
permeabilized cells) induced mitochondrial Ca2⫹ uptake
almost as efficiently as in intact, stimulated cells (305).
This observation led to the proposition that mitochondria,
upon cell stimulation, are exposed to [Ca2⫹] of the microenvironment of the open IP3-gated channel, that are much
higher than those measured in the bulk cytoplasm. In
other words, “privileged,” local signaling between the
Ca2⫹ store (the ER) and mitochondria appeared to be the
Physiol Rev • VOL
key to the participation of this organelle in intracellular
Ca2⫹ homeostasis. In this scheme, mitochondria could be
proposed to act as detectors of the microheterogeneity of
cellular Ca2⫹ signaling, participating in decoding it into
defined cell action, as discussed below. The close proximity of mitochondria and the main intracellular store of
agonist-releasable Ca2⫹, the ER, had been observed by
electron microscopy in fixed samples of several cell types,
also taking advantage of quick-freezing techniques, that
avoid artifacts due to tissue fixation procedures (276). In
our experiments, we could confirm this proximity in in-
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2⫹
FIG. 5. Microheterogeneity of mitochondrial Ca
response. A: schematic view of ratiometric pericam (239; and see Table 1). B: deconvoluted
images of HeLa cells expressing the ratiometric pericam during a histamine challenge. On the left, the color scale represents the fluorescence
excitation ratio, where blue represents low Ca2⫹ and red high Ca2⫹ levels. The pseudocolor image in the top left panel was taken 0.5 s after the
addition of histamine, and the following images were taken 1 s apart. Note the different response of mitochondrial subpopulations at early times
upon stimulus addition and the diffusion of Ca2⫹ at later times within the mitochondrial network. For experimental details, see Reference 360.
[Modified from Szabadkai et al. (360).] C: kinetics of the nuclear and mitochondrial Ca2⫹ rises measured with a ratiometric pericams targeted to the
mitochondrial matrix and to the nucleoplasm. In the top panel, the overall kinetics of the Ca2⫹ response of a single cell of the nucleus and of different
mitochondrial regions; in the bottom panel, the same trace on an expanded time scale. Note the heterogeneities in the lag time between the rise of
[Ca2⫹] in the nucleoplasm and in different mitochondrial populations within the same cell. [From Filippin et al. (90).]
MICRODOMAINS OF INTRACELLULAR CALCIUM
forward and experimentally evaluated in HeLa cells and
hepatocytes? The occurrence of rapid [Ca2⫹]m responses
has been conclusively shown in a wide variety of cell
systems in which Ca2⫹ is released from the ER through
IP3Rs (e.g., L929 fibroblasts, 143B osteosarcoma and primary hepatocytes, to name but a few) (30, 133, 303).
However, this is not an exclusive property of this signaling mechanism, as the same pattern of mitochondrial
Ca2⫹ signaling (a rapid peak and a slower return to basal
values) has been demonstrated in muscle cells (both cardiac and skeletal), in which Ca2⫹ release from the SR
occurs through the ryanodine receptors (36). Particularly
striking is the case of heart cells, in which, using Ca2⫹
probes as diverse as fluorescent dyes and targeted photoproteins, a beat-to-beat relay of [Ca2⫹]c spiking to
[Ca2⫹]m transients could be demonstrated; every [Ca2⫹]c
pulse induced a [Ca2⫹]m response, which returned to
basal values also at high spiking frequencies (313, 361).
Recently Rudolf et al. (321) demonstrated that also in
skeletal muscle in a live mouse mitochondria are capable
of taking up Ca2⫹ during a single muscle twitch (321). As
to the functional consequences, the pacing of cytosolic,
and hence, mitochondrial oscillations (that is under hormonal control) controls in turn the organelle metabolism,
FIG. 6. Mitochondria/ER interaction.
Electron micrographs (A and B) and
three-dimensional reconstruction (C and
D) of the ER/mitochondria interaction in
an hepatocyte. Please note the very close
apposition of the ER (blue in C and D)
and the outer mitochondrial membrane
(pink). In yellow is the inner mitochondrial membrane, and in green is the
three-dimensional reconstruction of the
inner cristae. (Figure kindly provided by
Dr. Carmen Mannella, Albany, NY.)
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tact, living cells, in which the two organelles were labeled
with specifically targeted GFP mutants (GFP for the ER,
erGFP, and the blue variant for mitochondria, mtBFP),
and resolved with a high-resolution imaging system (optimized for fast acquisition and equipped with software
for deconvolution and three-dimensional rendering of the
image). Figure 6 shows EM images of ER-mitochondria
close apposition and three-dimensional reconstruction of
such images. In keeping with the idea of “local” Ca2⫹
cross-talk between the mitochondria and the ER, we recently demonstrated, through fast single-cell imaging of
mitochondrial [Ca2⫹]m with targeted Ca2⫹-sensitive GFPs
(pericams and cameleons), that [Ca2⫹]m increases originate from a discrete number of sites and rapidly diffuse
through the mitochondrial network. Indeed, fragmentation of the network induced by overexpressing components of the organelle fission machinery, such as the
dynamin-related protein 1 (drp-1), greatly reduces the
global amplitude of the agonist-induced [Ca2⫹]m elevation
(360).
A first obvious question is how general is the concept
of fast mitochondrial responses to physiological Ca2⫹
signals, allowed by the “sensing” of microdomains close
to the mouth of Ca2⫹ channels, the concept originally put
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ROSARIO RIZZUTO AND TULLIO POZZAN
Physiol Rev • VOL
mitochondria (in turn needed for efficient vesicle release)
requires exposure to a local [Ca2⫹] ⬎5 ␮M, a value that is
reached upon opening of P/Q-type, but not L-type voltagegated channels (228). Two concepts thus emerge: 1) the
Ca2⫹ concentration in the microdomain sensed by mitochondria is at least one order of magnitude higher than in
the bulk cytosol, and 2) the sensitivity to microdomains
(rapidly declining with the distance from the channel)
enhances the selectivity of different signaling routes, as it
allows mitochondria to discriminate, based on their localization, the opening of channels giving rise to comparable
bulk responses.
As to the second issue (i.e., the responses to the
small [Ca2⫹] increases), it should be remembered that
mitochondrial Ca2⫹ uptake occurs also at submicromolar
Ca2⫹ levels. Even in resting conditions, a small amount of
Ca2⫹ is accumulated in the organelle, which is released by
dissipation of the mitochondrial membrane potential and
causes a small, but detectable, [Ca2⫹] rise in the cytoplasm and a decrease in the matrix Ca2⫹ concentration.
Thus, in principle, a sustained elevation of [Ca2⫹]c should
always cause a [Ca2⫹]m rise. This concept (and its physiological relevance) was conclusively demonstrated in
steroid-producing cells (adrenal glomerulosa and luteal
primary cultures). In both glomerulosa and luteal cells,
[Ca2⫹]m increases were detected by perfusing permeabilized cells with [Ca2⫹] ⬍0.2 ␮M and by triggering capacitative Ca2⫹ influx in intact cells (a procedure that generated a [Ca2⫹]c rise ⬍0.3 ␮M). Under those conditions, an
increase of mitochondrial NADH production, a Ca2⫹-dependent matrix reaction, was also detected (350, 359).
This provides a clear example of the biological significance of mitochondrial Ca2⫹ responses to “bulk” cytosolic rises: Ca2⫹-dependent mitochondrial NADH production provides, through the activity of transhydrogenases,
the NADPH necessary for the side chain cleavage of cholesterol (the first, rate-limiting step of steroid hormone
biosynthesis). Mitochondrial responses to “bulk” [Ca2⫹]c
rises are not limited however to steroid-producing cells.
An additional example is provided by ECV304 endothelial
cells, in which stimulation with ATP causes a prolonged
[Ca2⫹]c elevation (a plateau of 0.7 ␮M that is maintained
throughout agonist application), that is paralleled by a
sustained [Ca2⫹]m rise of comparable amplitude (172). As
to the exact correlation between [Ca2⫹]c and [Ca2⫹]m
levels, and the establishment of a “threshold” for mitochondrial Ca2⫹ responsiveness, information can be obtained from the work by Bootman and co-workers in HeLa
cells (57) who exposed mitochondria to [Ca2⫹] increases
of different amplitude and source. Mitochondrial Ca2⫹
uptake was slow at 2–300 nM and steeply increased ⬎400
nM, and IP3-dependent Ca2⫹ release from the ER was
more efficient than any other source, favoring the concept
of privileged Ca2⫹ communication between the two organelles.
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thus tuning ATP production to the increased needs of a
rapidly contracting muscle, as discussed below.
What about neurons and other cell types in which
influx through plasma membrane channels represents the
prevailing mechanism for generating a [Ca2⫹]c rise? Also
in these cells, a rapid [Ca2⫹]m rise parallels the localized
or global Ca2⫹ signal, and a number of experimental
observations indicate that exposure to local domains of
high [Ca2⫹] generated in proximity of the opening channels accounts for the efficiency of the mitochondrial response. Specifically, X-ray microanalysis of sympathetic
neurons demonstrated substantial Ca2⫹ uptake into mitochondria during depolarization-induced Ca2⫹ signals and
a marked heterogeneity between individual organelles,
with subplasmalemmal mitochondria undergoing the largest increases (285). In hippocampal neurons, opening of
NMDA-gated channels induced in a subset of mitochondria a Ca2⫹ uptake large enough to completely bleach its
photoprotein probe content, which could be replenished
by diffusion from neighboring mitochondria in a 30-min
recovery period (14). In adrenal chromaffin cells, it was
demonstrated that mitochondria clustered at neurotransmitter release sites are exposed to high [Ca2⫹] upon opening of both plasma membrane and ER channels, thus
implying the existence of highly structured signaling domains, and undergo the largest [Ca2⫹]m responses so far
documented (up to 0.5 mM). In turn, mitochondrial Ca2⫹
uptake modulates the availability of Ca2⫹ for exocytosis,
and indeed, uncouplers greatly enhance catecholamine
secretion (229). Interestingly, rapid responses to subplasma membrane [Ca2⫹] microdomains have been documented also in nonexcitable cells; specifically, mitochondria were shown to sense Ca2⫹ gradients near CRAC
channels. This process, by clearing Ca2⫹ in the proximity
of the channel, has been shown to modulate the Ca2⫹dependent inactivation of the channels and thus determine the net influx of Ca2⫹ and the ensuing biological
response (118, 146).
Two issues remain to be assessed: 1) what is the
magnitude of the Ca2⫹ rise to which mitochondria are
exposed in the microdomain of the above-mentioned examples, and 2) is a rapid, pulsatile increase dependent on
microdomains always necessary, or conversely in some
cases mitochondria can sense the bulk cytosolic increases, and the consequent slow [Ca2⫹]m increases account for the biological response? On the first issue, work
was carried out by various groups using different Ca2⫹
probes and cell models and a coherent set of estimates
was obtained. In permeabilized RBL-2H3 and H9c2 cells,
maximal rates of mitochondrial Ca2⫹ uptake were achieved
by exposing the organelle to ⬃16 and ⬎50 ␮M, respectively (63, 361); in the latter cell type, a rate of uptake
comparable to that observed in intact cells upon maximal
stimulation of RyRs was achieved by perfusing 30 ␮M
Ca2⫹. Finally, in chromaffin cells, rapid Ca2⫹ uptake into
MICRODOMAINS OF INTRACELLULAR CALCIUM
B. Mitochondrial Heterogeneity in Ca2ⴙ Handling
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important and intensely studied processes in neurobiology: synaptic plasticity. In two different experimental
systems (crayfish and Xenopus neuromuscular junctions),
Ca2⫹ efflux from mitochondria localized in the synapse
appears to contribute in part to posttetanic potentiation
(PTP) of neurotransmitter release (366, 395). In the latter
case, PTP was observed also if tetanic stimulation was
applied in Ca2⫹-free media, thus suggesting that mitochondria are, at least partially, loaded with Ca2⫹ also in
resting situation, and thus act as a bona fide pool of
releasable Ca2⫹. Apart from neurons, polarized epithelial
cells provide another clear example of highly organized
mitochondrial distribution and Ca2⫹ responsiveness, with
direct implication for the physiological regulation of cellular function. Specifically, in pancreatic acinar cells, in
which the Ca2⫹ response to a low-dose agonist stimulation is restricted to the apical pole (where it causes
granule secretion) by the action of the largest group of
mitochondria, clustered between the apical and basolateral portions of the cell and acting as a barrier to the
spread of the Ca2⫹ signal (372). Only stimuli that can
overcome this mitochondrial “firewall” induce a global
Ca2⫹ response, and then the functional effects radically
change: the nucleus is reached by the [Ca2⫹]c rise, with
consequent modification of the gene expression profile
and thus long-term alterations of cellular activity. A conceptually related mechanism has been shown to occur in
parotid acinar cells. In these cells, however, fluid secretion requires the activation of both Ca2⫹-dependent Cl⫺
channels of the apical membrane and Ca2⫹-dependent K⫹
channels of the basolateral membrane (to maintain hyperpolarization and hence the driving force for Cl⫺ movement). Thus, not surprisingly, no restriction of the [Ca2⫹]c
signal to the apical pole is observed, nor existence of a
perigranular mitochondrial belt. Conversely, mitochondria are clustered around the nucleus, where they delay
the propagation of the Ca2⫹ wave as well as probably
provide a source for ATP needed in various transport or
enzymatic functions occurring in this domain (39).
C. Functional Role of Mitochondrial Ca2ⴙ Uptake
What is the function of mitochondrial Ca2⫹ uptake,
and how does it correlate with the microheterogeneity of
Ca2⫹ signaling? Given the focus of this review, we do not
discuss in detail the physiological consequences of mitochondrial Ca2⫹ uptake. A brief overview, however, gives
some insight into the potential targets of the different
types of organellar Ca2⫹ signals, described above.
1. Ca2⫹ effects within mitochondria
Three key metabolic enzymes (the pyruvate, ␣-ketoglutarate, and isocitrate dehydrogenases) are activated by
Ca2⫹, by different mechanisms: in the case of pyruvate
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If microdomains are critical determinants of mitochondrial Ca2⫹ responsiveness, it can be argued that only
some mitochondria (or domains of a continuous network)
are appropriately placed to “sense” the high [Ca2⫹]c domains and thus undergo large-amplitude [Ca2⫹]m increases. Thus one would expect that significant heterogeneity in Ca2⫹ uptake should be detected within the mitochondrial population of the cell. This seems the case in
most cell types, although there are clear differences in the
size of the rapidly responding mitochondrial pool. In HeLa
and chromaffin cells, this information was achieved taking advantage of the intrinsic properties of the photoprotein aequorin, that is “consumed” during the experiment,
since it can emit only one photon in the Ca2⫹-dependent
reaction (229, 303). Thus highly responding mitochondria
rapidly exhaust their pool of active photoprotein and in
consecutive stimulations the Ca2⫹ rise essentially reflects
only the activity of “low-responder” organelles. Interestingly, a similar estimate was made in the two cell models:
⬃30% in HeLa cells and 20 – 40% in chromaffin cells. Conversely, in other cell types, most mitochondria rapidly
respond to agonist-induced Ca2⫹ signals. This was conclusively shown in skeletal myotubes, both with the aequorin approach (36) and by measuring [Ca2⫹]m with rhod
2 in permeabilized cells, comparing RyR-mediated responses with maximal uptake due to perfusion of high
[Ca2⫹] (361). Similar data were obtained in RBL-2H3 cells,
in which saturation of mitochondrial Ca2⫹ uptake was
observed during IP3-induced Ca2⫹ release from the ER
(63). A conclusive explanation for these differences is
difficult, given that only recently tools have become available for directly imaging the [Ca2⫹]m hot spots with good
spatial and temporal resolution. It is however reasonable
to assume that the density of ER/SR-mitochondria juxtaposition, and the clustering of Ca2⫹ release channels (as
well as of the yet unidentified mitochondrial Ca2⫹ transporters) will prove to play a major role.
A second, more macroscopic mechanism for mitochondrial heterogeneity, however, was clearly shown to
be operative in those cells in which specialized functions
pose strict requirements to cell morphology, and intracellular distribution of organelles and signaling mechanisms.
Thus mitochondrial distribution to portions of the cell
with radically different functions (and channel repertoires) may cause per se an intrinsic major heterogeneity
in organelle response. Neurons are an obvious example of
complex cell architecture and highly localized signals.
Not surprisingly, numerous reports have appeared on the
selective involvement of resident mitochondria in spatially restricted Ca2⫹ signals. While we refer to specific
reviews for a detailed coverage of this broad topic (100),
we simply point out the functional significance of such an
organization with an example regarding one of the most
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ROSARIO RIZZUTO AND TULLIO POZZAN
2. Effect of mitochondrial Ca2⫹ homeostasis
on cellular Ca2⫹ signals
Mitochondria, distinctly from cytosolic proteins, are
highly sophisticated, “tunable” buffers that vary their activity in different phases and functional states of the cell;
indeed, their number, shape, and distribution (147, 158)
and most likely their responsiveness to Ca2⫹ (232, 282)
are controlled by converging signaling pathways. This
Ca2⫹ buffering activity influences cytosolic Ca2⫹ signals
in two conceptually different ways, i.e., 1) by acting as
high-capacity sinks placed on the way of a propagating
Ca2⫹ wave and 2) by clearing Ca2⫹ in restricted microdomains (such as the microenvironment of a Ca2⫹ channel).
In the first case, spatial clusters of mitochondria have
been demonstrated to isolate functionally distinct domains of polarized cells, namely, a mitochondrial “firewall” was shown to prevent the spread of Ca2⫹ signals
from the apical (secretory) region of pancreatic acinar
Physiol Rev • VOL
cell from the basolateral region, containing the nucleus
(372). Similarly, neuronal mitochondria have been shown
to buffer [Ca2⫹] increases in defined cellular regions, e.g.,
the presynaptic motoneuron ending (66). As to the second
case, a thoroughly investigated example is the regulation
of Ca2⫹ release through IP3Rs. In Xenopus oocytes, the
energization state (and thus the capacity to accumulate
Ca2⫹) was shown to modify the propagating Ca2⫹ waves
induced by IP3 (156). In permeabilized blowfly salivary
glands, it was observed that perfusion of IP3 induced ER
[Ca2⫹] oscillations, the frequency of which increased with
the dose of IP3. Such an effect was observed only upon
energization of mitochondria, implying a primary role of
these organelles in regulating the Ca2⫹ microdomain in
the proximity of IP3Rs and thus the oscillatory pace of
stimulated cells (405). In mammals, this effect has been
seen in many cell systems, including hepatocytes, HeLa
cells, astrocytes, and BHK cells. As to the cellular consequence, very different effects were observed given the
bell-shaped sensitivity of IP3Rs to Ca2⫹ concentration on
the cytosolic side. In astrocytes and hepatocytes, cytosolic excitability appeared enhanced when mitochondrial
Ca2⫹ uptake was inhibited, indicating that mitochondrial
clearance of the Ca2⫹ microdomain reduced the positive
Ca2⫹ feedback on the IP3R and/or buffered substantial
Ca2⫹ loads (30, 132, 338). Conversely, in BHK cells, inhibition of mitochondrial Ca2⫹ uptake resulted in reduction
of ER Ca2⫹ release (169), thus indicating that mitochondria prevent the Ca2⫹-dependent inactivation of the channel.
Recently, a provocative new hypothesis, also related
to mitochondrial regulation of Ca2⫹ microdomains, was
put forward on the basis of studies carried out on the
postsynaptic ending of the neuromuscular junction. Earlier investigations showed that slower release of Ca2⫹
from these mitochondria (through the Na⫹/Ca2⫹ exchanger) can maintain relatively high cytosolic Ca2⫹ for a
period following intense stimulation, thus participating in
the phenomenon of posttetanic potentiation (66, 366). In
a later paper, the contribution of mitochondrial Ca2⫹
release to posttetanic potentiation of neuromuscular
junctions was observed also in Ca2⫹-free medium (395).
These results suggest that Ca2⫹ release from mitochondria
through the Na⫹/Ca2⫹ exchanger, triggered by plasma membrane depolarization, may induce during the action potential
a primary release of Ca2⫹ from mitochondria, rather than
causing the reextrusion of Ca2⫹ loaded during the action
potential. In this case, the mitochondria would act as a
bona fide Ca2⫹ store that is loaded with Ca2⫹ at rest and
releases it upon cell stimulation. Obviously, the general
relevance of this observation must be confirmed, given
that in the vast majority of cells with a variety of experimental approaches, i.e., measurement of free Ca2⫹ with
aequorin, GFP-based probes or dyes (74, 90, 303), and of
total Ca2⫹ by X-ray microanalysis (285) or electron energy
loss imaging (276), mitochondria were demonstrated to
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dehydrogenase through a Ca2⫹-dependent dephosphorylation step, in the latter two cases through the direct
binding of Ca2⫹ to the enzyme complex (134, 206). Recently, also some metabolite transporters were shown to
be regulated by Ca2⫹ and also participate in the enhancement of aerobic metabolism upon cell stimulation (171).
One issue remains to be clarified: why do [Ca2⫹]m rises
reach values up to 500 ␮M, given the matrix dehydrogenases are activated by [Ca2⫹] in the low micromolar range
(a similar K0.5 of 0.5–2 ␮M was reported for the three
enzymes). Several explanations could be given. The first is
that most mitochondrial Ca2⫹ concentration ([Ca2⫹]m)
estimates were obtained in prolonged, supramaximal
stimulations with Ca2⫹-mobilizing agonists, and it is likely
that these conditions do not fully mimic the response to
physiological challenges, that give rise to smaller and
more transient [Ca2⫹]c increases. The second is that a
large [Ca2⫹]m response could be [together with other
mechanisms, such as the enzymatic delay in the rephosphorylation of the pyruvate dehydrogenase (PDH) complex] a route to extend the metabolic activation well
beyond the duration of the [Ca2⫹]c rise (133, 157, 312).
Mitochondrial Ca2⫹ overload and/or the combined
action of apoptotic agents or pathophysiological conditions (e.g., oxidative stress, Refs. 153, 154) may induce a
totally different effect (i.e., a profound alteration of organelle structure and function, Refs. 281, 362) leading to
cell death by necrosis and apoptosis. The reduction of ER
Ca2⫹ loading, such as that caused by Bcl-2, reduces mitochondrial Ca2⫹ uptake and thus the efficacy of apoptotic stimuli (16, 280, 281). Conversely, both Bax and other
proapoptotic proteins (albeit with different mechanisms)
enhance mitochondrial Ca2⫹ loading, and thus the sensitivity of cells to apoptosis (252, 253; for recent reviews,
see Refs. 255, 308, 328).
MICRODOMAINS OF INTRACELLULAR CALCIUM
contain at rest very little calcium and maintain a matrix
[Ca2⫹] similar to that of the bulk cytosol.
V. OTHER INTRACELLULAR CALCIUM STORES
A. Endosomes
Although intensely studied from many points of view,
the literature concerning Ca2⫹ handling by endocytic vesicles is very scarce. A careful analysis of their Ca2⫹ handling properties has been carried out by Petersen’s group
(114) a few years ago, and the results of this study appear
clear cut and thus far undisputed. In particular, the authors showed that their Ca2⫹ content depends on the
inclusion, during vesicle formation, of extracellular medium in their lumen, while upon endocytosis Ca2⫹ is
rapidly lost into the cytoplasm, concomitant to (or because of) endosome acidification. The author also concluded that in cells with active endocytic activity, the
Ca2⫹ taken up with endocytosis may account for a substantial part of the Ca2⫹ influx rate under resting conditions; Pryor et al. (296) concluded that intraorganellar
Ca2⫹ plays a key role in late endosome-lysosome heterotypic fusion.
Physiol Rev • VOL
B. Golgi Apparatus
The Golgi is well known to be highly heterogeneous
in terms of morphology, protein composition, and functions (170, 224). Similarly heterogeneous appears its Ca2⫹
handling mechanisms. Indeed, Ca2⫹ uptake in the cis- and
intermediate Golgi is mediated by the SERCA pumps of
the ER/SR while in the trans-Golgi the existence of intracellular Ca2⫹ pumps other than the classical SERCAs is
now well documented. Specifically, much information has
recently been obtained on the mammalian homolog of
PMR1, the Mn2⫹/Ca2⫹-dependent ATPase of Saccharomyces cerevisiae (322). Its mammalian homolog was identified (128) and investigated, providing new interesting insight into the signaling properties of the Golgi apparatus
(19, 380) and its dysfunction in a human genetic disease
(for a detailed coverage of this topic, see Ref. 379). In
brief, this pump comprises a group of related P-type
ion-motive ATPases (two independent genes, ATPC1 and
ATPC2, have been identified, and ATPC1 was shown to
encode four different splice variants). Evolutionary analysis of the PMR-1-related ATPases (expressed in yeast,
worms, insects, and mammals and closely resembling
some bacterial ATPases) suggests that they represent the
most ancient class of Ca2⫹-ATPases. As to their function,
these ATPases markedly differ from the SERCAs in two
major aspects: 1) they transport equally well Ca2⫹ and
Mn2⫹, and 2) they are located in the Golgi stacks (where
they coexist with SERCAs), the trans-Golgi network, and
the secretory vesicles. Based on the latter property, the
mammalian pump is now referred to as SPCA (secretory
pathway Ca2⫹-ATPase). As to the roles in cell physiology,
two different functions can be envisaged. The first is to
transport Mn2⫹ across endomembranes, thus accounting
for the high [Mn2⫹] of the Golgi and post-Golgi compartments. This allows one to introduce into the lumen Mn2⫹
to be utilized by resident enzymes (such as lactate synthase, that requires Mn2⫹ for activity). Indeed, in lactation, SPCA was shown to be overexpressed. In addition, it
may allow one to prevent cellular Mn2⫹ overload, which
has been implicated in the pathogenesis of neurodegenerative disorders, such as Parkinson’s disease (143). The
second function, shared with the SERCA in the Golgi
apparatus and entirely carried out by SPCA in the later
compartments, is that of accumulating Ca2⫹ into the organelle (see Fig. 7). In this case, much remains to be
understood on the specific relevance of this Ca2⫹ pump,
and in general of the post-ER Ca2⫹ stores. Two nonmutually exclusive roles can be envisioned. The first is that
these stores cooperate with the larger ER compartment in
setting up the fundamental properties of cellular Ca2⫹
signaling. In support of this notion, transfected SPCA was
shown to sustain baseline [Ca2⫹]c oscillations in normal
conditions and after inhibition of SERCAs by thapsigargin
(216). The data indicate that after ER Ca2⫹ discharge,
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Although by far the best-characterized intracellular
compartments endowed with the capacity of accumulating and releasing Ca2⫹ are the ER/SR and mitochondria,
other vesicular compartments of the cells are known to
accumulate Ca2⫹ (the Golgi apparatus, secretory granules, lysosomes, and endocytic vesicles) and to be capable of releasing it under some conditions. To our knowledge nothing is yet known about the Ca2⫹ handling capacity of peroxisomes, and thus these organelles will not
be further discussed.
Unlike the ER and mitochondria that are usually
rather homogeneously distributed within the cytoplasm,
most of these organelles are often highly localized within
cells, and accordingly, their capacity to take up or release
Ca2⫹ in selected regions of the cells may be ideally suited
to generate functionally relevant local microdomains of
Ca2⫹. Several lines of evidence support the notion that the
Ca2⫹ uptake and release mechanisms of these organelles
may be molecularly distinct from that of the ER/SR and
therefore at least potentially the differential activation of
the classical Ca2⫹ stores or of these other stores may be
a tool to selectively and locally activate specific cell functions.
The mechanisms of Ca2⫹ homeostasis by these organelles, however, are far less well understood than that
of the ER/SR and of mitochondria, and accordingly, at the
moment most conclusions must be based on indirect
evidence or mere speculation.
393
394
ROSARIO RIZZUTO AND TULLIO POZZAN
7. [Ca2⫹] changes within the ER and Golgi lumens.
A and B: immunocytochemical localization of recombinant
aequorins targeted to the ER and Golgi lumens in HeLa
cells. Please note the typical perinuclear localization of the
signal and the absence of visible staining of other structures
in B. C and D: kinetic changes of the [Ca2⫹] concentration
measured with the ER and Golgi located aequorins upon
addition of ATP (Pozzan and Rizzuto, unpublished data).
The ER and Golgi Ca2⫹ were initially depleted by treatment
with ionomycin in EGTA-containing medium. Where indicated, CaCl2 (1 mM) was added to refill the organelles with
Ca2⫹, and finally, ATP was added to cause IP3 generation
and Ca2⫹ release. For experimental details, see Pinton et al.
(283).
FIG.
Physiol Rev • VOL
sistance to mechanical stress) requires the tight association of the multilayered epithelium, which is in turn dependent on adhesion structures (the desmosomes). Maturation and membrane exposure of scaffolding proteins,
as well as their assembly into the functional units, depends on luminal Ca2⫹-dependent reactions, and this
could account both for the high expression level of SPCA
in these cells and for their high vulnerability to genetic
lesions of this Ca2⫹ pump (309).
C. Secretory Granules
Although often lumped together under this name,
these organelles are clearly quite different in different
cells and thus, not surprisingly, the mechanisms through
which they take up Ca2⫹ from the cytosol are heterogeneous. To date, most direct information is available for
insulin granules, and detailed indirect information is also
available for other secretory compartments such as zymogen and chromaffin granules and synaptic vesicles. As to
insulin granules, after the initial demonstration that they
marginally contribute to Ca2⫹ homeostasis in permeabilized cells (292, 293), detailed studies have been carried
out with selectively localized aequorin leading to the conclusion that they accumulate Ca2⫹ by means of a vanadate-sensitive Ca2⫹-ATPase, possibly SPCA itself (219).
According to these studies, although the lumen of the
granules is notoriously acidic, the H⫹ gradient plays no
role in Ca2⫹ accumulation, in as much as Ca2⫹ uptake by
the granules is unaffected by drugs that collapse such
gradients as the H⫹ pump inhibitor bafilomycin or the
H⫹/Na⫹ ionophore monensin (219). As to zymogen granules, the mechanism for their Ca2⫹ accumulation has not
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Ca2⫹ influx sustains Golgi loading until this store is fully
loaded. At this point, no further uptake can occur, and
[Ca2]c slowly increases. This sensitizes the resident IP3Rs
and causes the release of Ca2⫹ from the organelle. A
second Ca2⫹ spike is thus produced, that ends with the
depletion also of the Golgi store. The reloading of the
organelle, and the reinitiation of the cycle, could support
repetitive [Ca2⫹]c oscillations. In this view, the Golgi apparatus may play the role of the poorly IP3-sensitive Ca2⫹
store, which was proposed to cooperate with the highly
IP3-sensitive ER store in triggering the repetitive baseline
oscillations that follow the initial large transient [Ca2⫹]c
rise caused by agonist stimulation (the two-pool model
put forward by Berridge, Ref. 21). As to later compartments, no significant agonist-dependent Ca2⫹ release was
detected from the portions of the Golgi containing only
SPCA (381). Conversely, RyR-dependent Ca2⫹ release
from SPCA-containing secretory granules was observed
in pancreatic ␤-cells, and it was proposed that local CICR
from docked vesicles could participate in triggering exocytosis (219). The second role for the Ca2⫹ pumping
activity of SPCA is that of maintaining a steadily high
[Ca2⫹] within the lumen, which is needed for the constitutive activity of a number of resident enzymes (such as
those involved in the posttranslational processing and
sorting of secreted proteins). This aspect may bridge
SPCA function to a human disease. Indeed, the autosomal
dominant skin disorder known as Hailey-Hailey disease
(characterized by erosions and lesions at sites of trauma)
is due to mutations of the gene encoding SPCA1 (148). It
may appear surprising that a ubiquitous Ca2⫹ pump of the
Golgi and post-Golgi membranes causes selectively a skin
disease, but the peculiar properties of this tissue may
provide a reasonable explanation. Skin integrity (and re-
MICRODOMAINS OF INTRACELLULAR CALCIUM
D. Lysosomes
As to lysosomes, it should be stressed that in addition
to Ca2⫹ storing organelles, they can behave also as classical, Ca2⫹-sensitive, secretory vesicles. This is true for
bona fide lysosomes (see, for example, Ref. 314) or for
lysosome-related organelles such as azurophil granules of
neutrophils, cytotoxic T-lymphocyte granules, and mast
cell granules. The mechanism for their loading with Ca2⫹
is presently undefined, but clearly it does not depend on
SERCAs.
A Ca2⫹ store of undefined cytological nature has
been functionally identified a few years ago in several cell
types. These stores do not use SERCA for taking up Ca2⫹
and load only when the cytosolic Ca2⫹ level is massively
increased and for prolonged periods of time (286).
E. IP3R and RyR Expression
While it is undisputed that Golgi, lysosomes, and
secretory vesicles contain in their lumen high amounts of
Ca2⫹, much less clear, and debated, is not only whether
indeed they can release their Ca2⫹ into the cytoplasm
under physiological conditions, but also what type of
Ca2⫹ channel is present in their membrane. We consider
here the recent literature in the field pointing out the
important discrepancies still existing.
First of all, what about the expression of the classical
intracellular channels (IP3Rs and RyRs) in these other
stores? The situation appears relatively clear for the Golgi
complex. The cis and presumably the intermediate Golgi
express not only, as mentioned above, the SERCAs, but
also IP3Rs (216, 283) (see Fig. 7), while it is yet unclear
whether functional RyRs are present on these memPhysiol Rev • VOL
branes. Accordingly, these Golgi compartments behave
largely as extension of the ER and local release of Ca2⫹
during activation may be of importance for some specific
functions localized therein (see below). The trans-Golgi,
on the other hand, apparently only expresses the SPCA
pump, and thus far no evidence has yet been obtained
indicating that physiological agonists can mobilize Ca2⫹
from its lumen (217, 380).
Far more complex is the situation of the secretory
compartment. Petersen and co-workers (115) provided
initial functional evidence that zymogen granules from
the exocrine pancreas are indeed sensitive to IP3, but
other studies (401) have argued against this conclusion.
Along the same line, Nguyen et al. (248) have suggested
that mast cell, histamine-containing, granules do express
IP3Rs, but not SERCAs (248). Both the experiments of
Petersen’s group (115) and those of Nguyen et al. (248)
have been carried out with highly sophisticated image
analysis of fluorescence images carried out in purified
isolated single granules, and thus it is difficult to argue
against the conclusion that IP3 can indeed release Ca2⫹
from these organelles; yet, at least in the pancreatic acinar
cells, there is no doubt that the vast majority of the Ca2⫹
released upon receptor activation comes from ER cisternae intermingled with the zymogen granules (262). However, the possibility that Ca2⫹ released from zymogen
granules plays a key role under pathological conditions
(e.g., in pancreatitis) has been recently suggested (299).
Finally, thus far no conclusive immunocytochemical evidence for the presence of IP3Rs on secretory vesicles of
any type has been provided. The original report (28)
showing expression of IP3Rs in insulin-containing granules has been dismissed later as an artifact of antibody
cross-reactivity (300). The existence of functional IP3Rs
in insulin granules has been more recently clearly excluded also by direct measurement of its luminal Ca2⫹
content that is unaffected by receptor-induced IP3 generation (219). More convincing, yet not conclusive, is the
localization of the IP3Rs on the chromaffin granule membranes (371). The modulation by chromogranin of the
IP3Rs opening probability cannot by itself be considered
an argument in favor of IP3R localization in the granules
given that the latter proteins are present, though at lower
concentration, also in the ER.
It should be also stressed, in addition, that at least the
IP3R1 isoform contains strong ER retention signals in its
transmembrane domains (263), and thus it is difficult to
envisage how it could proceed much further in the secretory pathway than the more proximal cisternae of the
Golgi where the ER retrieval mechanisms are located.
However, as shown above, evidence for the presence of
IP3Rs on the plasma membrane has been provided (in
some cell types), and accordingly IP3Rs, at least in transit
along the secretory pathway, must thus exist.
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been investigated in great detail, but it has been clearly
demonstrated that it does not depend on a thapsigarginsensitive SERCA (115), and a similar conclusion has been
reached for the histamine-containing granules of mast
cells (40, 248). As to chromaffin granules and synaptic
vesicles, the general consensus, based on rather old data,
is that they load via a pH-driven H⫹ (Na⫹)/Ca2⫹ antiport
(40, 94, 166).
The [Ca2⫹] in the lumen of the granules has been
measured both with selectively targeted Ca2⫹ indicators
(insulin and mast cell granules) and by indirect means
(chromaffin granules). The [Ca2⫹] within insulin granules
has been estimated to be ⬃100 –200 ␮M (219), while that
of mast cell and chromaffin cell granules amounts to
⬃10 – 40 ␮M (40, 248). It needs to be stressed that the
difference between the total Ca2⫹ content (tens of millimolar) and the free [Ca2⫹] in the vesicle lumen indicates
the existence of a very strong Ca2⫹ buffer within these
organelles.
395
396
ROSARIO RIZZUTO AND TULLIO POZZAN
F. Channels Other Than the IP3Rs and the RyRs
and Their Subcellular Localization
IP3Rs and RyRs have been cloned, purified, reconstituted in lipid bilayers, recombinantly expressed, and extensively characterized for their subcellular distribution
and functional characteristics. The situation with other
intracellular Ca2⫹ release channels is far less defined and
still rather debated. Over the last few years, much interest
has been devoted to the channels regulated by NAADP.
Up to now, however, these channels not only remain
elusive from the molecular point of view, but their subcellular distribution is highly debated. In sea urchin egg
homogenates, very clear evidence has been provided that
the vesicular compartments that release Ca2⫹ in response
to NAADP are distinct from those sensitive to cADPR or
to IP3. This conclusion has been reinforced by experiments carried out in intact eggs. Most important, the
classical SERCA inhibitor thapsigargin empties the stores
sensitive to IP3 and cADPR, leaving unaffected the response to NAADP. This observation, coupled to the fact
that inhibitors of H⫹-ATPases such as bafilomycin block
the NAADP effect led to the, so far undisputed, conclusion that in the sea urchin egg the NAADP-sensitive comPhysiol Rev • VOL
partment is distinct from the ER and its lumen is acidic
(also defined as “lysosome-like”) (1, 51, 106, 110, 174 –177).
The situation is far less clear in mammalian cells,
where clear-cut evidence by Gerasimenko and colleagues
(112, 113) demonstrates that NAADP not only releases
Ca2⫹ from the same compartment as cADPR and IP3R do,
but also that NAADP acts on the RyRs expressed on the
ER and nuclear envelope. Direct demonstration that
NAADP can activate single RyRs incorporated in lipid
bilayer has been provided recently by Hohenegger et al.
(144). Mitchell et al. (218), on the other hand, provided
very good evidence that NAADP releases Ca2⫹ from insulin granules in B cells of the pancreas, whose lumen is
indeed acidic, but clearly not a “lysosome-like” organelle.
It remains to be established whether this sensitivity to
NAADP depends, as suggested by Gerasimenko and coworkers (112, 113), on the sensitivity of the RyRs to this
second messenger, or whether this is due to the yet
unknown NAADP receptor.
To complicate matters even further, Galione and coworkers (394) very recently showed [using the very same
cells as Gerasimenko and co-workers (112, 113) and
Mitchell et al. (218)] that NAADP acts on Ca2⫹ channels
distinct from RyRs and IP3Rs, and they suggested that the
stores should be identified with lysosome-like organelles.
It should be stressed that acidic “lysosome-like” Ca2⫹
store is an operational definition (based essentially on
pharmacological sensitivity to bafilomycin and other
drugs targeted to acidic compartments), and thus it is not
entirely clear to which cytological entity the reported
sensitivity to NAADP should be attributed. In mammalian
cells we noticed that the distribution of the NAADPsensitive pool described by Yamasaki et al. (394) tends to
be very similar to that of secretory granules, e.g., in the
apical pole in pancreatic acinar cells or diffuse in pancreatic ␤-cell. It is interesting to note another major experimental discrepancy, i.e., that bafilomycin, an inhibitor of
H⫹-ATPases, blocked NAADP-induced Ca2⫹ release from
the lysosome-like stores according to Reference 394, but
it had no effect on Ca2⫹ accumulation/release by insulin
granules according to Reference 218. On the contrary,
Ca2⫹ uptake by insulin granules is blocked by vanadate
(219), an inhibitor of P-type ATPases, and is abolished
also by silencing of the gene encoding SPCA (220).
A clarification of these clearly contradictory findings
is urgently needed. For the time being, we believe that it
is fair to conclude that the existence of NAADP-sensitive
channels pharmacologically distinguishable (and presumably located in different compartments) from the IP3Rs
and RyRs is firmly established in lower eukaryotes, but
still highly debatable in mammalian cells.
NAADP-sensitive channels are not the only noncanonical intracellular Ca2⫹ channels for which experimental evidence has been provided. Considerable interest in
fact has been devoted to the understanding of the nature
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Better documented is the presence of RyRs, in particular in insulin-secreting granules. By means of both
immunocytochemical localization at the EM level, functional studies with selectively targeted Ca2⫹ indicators
and subfractionation experiments, there is solid evidence
supporting the idea that Ca2⫹ can be released from the
insulin granules through the RyRs under physiological
conditions (218, 219). Insulin-secreting granules appear
unique also from other points of view as Ca2⫹ stores: not
only, as mentioned above, do they express RyRs (type 1
and 2), but also SPCA (while most other granule apparently have other type of Ca2⫹ accumulation mechanisms),
and are also sensitive to NAADP (see below).
Finally, as far as lysosomes (or in general acidic
lysosome-like organelles) are concerned, there is no positive evidence for the expression of IP3Rs or RyRs, at least
in mammalian cells, while in some lower eukaryotes (e.g.,
Plasmodia) indirect evidence has been provided indicating that a Ca2⫹-containing, acidic compartment, is sensitive to IP3 (266).
With the exception of insulin granules, the calculated
values for the free Ca2⫹ concentration (10 – 40 ␮M) within
the lumen of the secretory vesicles is about one order of
magnitude lower than in the ER. This suggests that the
contribution of the granules to IP3 (or RyR)-induced bulk
cytoplasmic Ca2⫹ rises may be, anyway, relatively small.
These Ca2⫹ release channels are in fact nonselective cation channels, and thus they transport relatively little Ca2⫹
at these low luminal Ca2⫹ concentrations.
MICRODOMAINS OF INTRACELLULAR CALCIUM
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2⫹
FIG. 8. Heterogeneity of the [Ca ] within a typical mammalian cell. Electron micrograph of an epithelial cell. For each compartment, an
approximate [Ca2⫹] is given, as it results from different experimental approaches. (Electron micrograph kindly provided by Dr. Carlo Tacchetti,
Genova, Italy.)
and role of Ca2⫹ channels regulated by sphingonoids. In
particular, evidence has been provided for the existence
of intracellular Ca2⫹ channels regulated by sphingosine
1-phosphate (S1P) and sphingosinphosphoryl choline
(SPCI). As to the latter, a putative intracellular receptor/
Physiol Rev • VOL
channel, named Scamper, has been cloned, recombinantly
expressed in oocytes and characterized electrophysiologically (199). Most recently, however, these data have been
reevaluated and, based on overexpression data, presumed
membrane topology and functional evidence, it has been
86 • JANUARY 2006 •
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398
ROSARIO RIZZUTO AND TULLIO POZZAN
TABLE
1.
The existence of such alternative Ca2⫹ storage compartments and of their channels could be of major relevance for the generation of localized Ca2⫹ microdomains.
For example, it seems possible that, in the case of both
insulin-containing secretory vesicles and sea urchin lysosomes, the formation of spatially constrained domains of
high Ca2⫹ concentration in the immediate vicinity of an
individual organelle may play a key role either for the fusion
of the secretory vesicle with the plasma membrane or for
heterotypic fusion between lysosomes and endosomes. Furthermore, the possibility of selective activation of Ca2⫹ release pathway from organelles with a specific cellular
location, e.g., the Golgi and secretory granules in highly
polarized cells, may be of central role in local activation
of other signaling pathway distinct from those activated
by the more classical IP3R- or RyR-dependent release.
V. CONCLUDING REMARKS
In the past, the existence and functional significance
of Ca2⫹ microdomains (i.e., localized regions with a
[Ca2⫹] significantly different from that of the bulk cytoplasm) were sometimes seen as working hypotheses,
evoked to explain otherwise mysterious experimental results. This is no longer the case, as Ca2⫹ microdomains
are experimentally verified facts with known biological
consequences, and interwoven with older notions of Ca2⫹
homeostasis (an electron micrograph summarizing the
general conclusions about the microheterogeneity of intracellular Ca2⫹ is presented in Fig. 8). Much remains to
be understood, however. In future work, the availability
of Ca2⫹ probes with high spatial selectivity will be fundamental. Table 1 shows a list of available recombinant
probes that were highly valuable in deciphering Ca2⫹
homeostasis in intracellular organelles. It is easy to predict that this list will rapidly grow with new probes for
monitoring the microenvironment of Ca2⫹ channels
and/or transducers. Exciting roads lie ahead, not only
Recombinant genetically encoded Ca2⫹ indicators
Aequorin
Obelin
Aeq(obelin)/GFP
FIP-CBSM
Cameleons
Pericams
Camgaroos
2G-CaMP
1TN-L15
Cyt
Mit
ERl
TCl
Gl
PM
N
Other
Reference Nos.
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
34, 36–38, 111, 202, 219, 231, 283, 307, 310
201
17, 124
315
68, 89, 221, 222, 240
239, 284
11
242
140
*
*
*
*
*
*
*
*
*
*
*
Aeq, aequorin; Cyt, cytoplasm; Mit, mitochondrial matrix; ERl, ER lumen; TCl, lumen of SR terminal cysternae; Gl, Golgi lumen; PM, plasma
membrane cytoplasmic surface; N, nucleus; other, intermembrane space of mitochondria, cytoplasmic surface of mitochondrial outer membrane,
cytoplasmic surface of the ER, gap junctions, insulin granule lumen. Reference nos. are selected references to original papers describing specifically
targeted versions of these constructs. * Several variants, in terms of primary sequence and/or Ca2⫹ affinity, of the constructs are also available.
Physiol Rev • VOL
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concluded that Scamper is not an intracellular Ca2⫹ channel (327). Last, but not least, Cavalli et al. (47) recently
concluded that Scamper may be located on the t tubules
of cardiac cells, i.e., in a specialized region of the plasma
membrane and not on intracellular stores. Due to these
inconsistencies, it seems fair to conclude that for the
moment there is no convincing evidence for a SPCI-gated
intracellular Ca2⫹ channel.
Less confused, but still far from clarified, is the situation for S1P, which can act both as a primary messenger
acting on a family of G protein-coupled receptors and
intracellularly as a second messenger. A first report was
published in 1990 (116), and scattered data on this topic
have appeared in the most recent literature. Quite recently, however, Nahorski’s group (399) rejuvenated the
field by providing new evidence in support of the notion
that some agonists (e.g., LPA in particular) may mediate
their Ca2⫹-mobilizing action exclusively through S1P production; in their hands, S1P not only releases Ca2⫹ from
stores independently of RyRs and IP3Rs, but also with a
subcellular localization and kinetics quite distinguishable
from that mediated by IP3. It must be also mentioned here
that S1P has been recently also proposed to play a role as
a soluble messenger activating CCE (152).
Finally, there is experimental evidence that is most
often neglected when considering the role of acidic compartments (secretory granules, lysosomes, or lysosomelike organelles) in physiological Ca2⫹ mobilization: this
release, whether due to IP3Rs, RyRs, or any other channel,
must occur in the absence of extracellular Ca2⫹ and
should be insensitive to Ca2⫹ ionophores such as ionomycin or A23187. This is because these ionophores, which
are obligate 2H⫹/Ca2⫹ exchangers, do not release Ca2⫹
from acidic compartments (82, 83). To our knowledge,
with the exception of the insulin granules (219), this
simple test has either not been carried out or has led to
complete inhibition of Ca2⫹ release (thus excluding the
role of acidic compartments from physiological Ca2⫹ mobilization).
MICRODOMAINS OF INTRACELLULAR CALCIUM
ACKNOWLEDGMENTS
We are indebted to P. Magalhães for critically reading the
manuscript and for many suggestions.
Address for reprint requests and other correspondence: T.
Pozzan, Dept. of Biomedical Sciences, Viale G. Colombo 3,
35121 Padua, Italy (e-mail: [email protected]).
GRANTS
The original work by the authors has been supported over
the years by grants from the Italian Ministry of University (Cofin
and Firb), the Italian Association for Cancer Research (AIRC),
Italian Telethon, European Union, Human Science Frontier Program, the Italian Consiglio Nazionale delle Ricerca, and the
Harvard Armenise Foundation.
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