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Microdomains of Intracellular Ca²⁺: Molecular Determinants and Functional Consequences

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

ABSTRACT

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

1. The SERCAs

2. Ca2+ release channels

3. Luminal Ca2+ binding proteins

D. Generation of Ca2+ Microdomains by RyRs

1. The Ca2+ sparks

2. Generating a global Ca2+ signal

3. The regulation of RyR activity in vivo

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

1. Ca2+ effects within mitochondria

2. Effect of mitochondrial Ca2+ homeostasis on cellular Ca2+ signals

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

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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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 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 expressed 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 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 receptor-operated 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) receptor 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 µm²/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 membrane 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 µm² 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 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.

	II. PLASMA MEMBRANE CALCIUM CHANNELS

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A. Generation of Subplasma Membrane Ca²⁺ 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 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 µM 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 µM 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 µM 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 µM (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 µM 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 µM 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, 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 µM (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 µM 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 µM. Higher levels of Ca²⁺ in the subplasma membrane region (compared with 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 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 bulk cytoplasm, both at rest (80 vs. 30 nM, respectively) and upon stimulation (well above 1 µM 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 µM, respectively (284).

View larger version (28K): [in this window] [in a new window]   FIG. 1. [Ca2+] 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).]

View larger version (35K): [in this window] [in a new window]   FIG. 2. Changes of [Ca2+] 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).]

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, 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.

View larger version (49K): [in this window] [in a new window]   FIG. 3. Localized Ca2+ changes in pancreatic acinar cells. Effects of cADPR (A), inositol 1,4,5-trisphosphate (IP3) (B), and cADPR + IP3 (C) on Ca2+-activated Cl current and localized increases of [Ca2+] at the apical pole in pancreatic acinar cells. [From Cancela et al. (43).]

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 diffusion 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).

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 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 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⁺-insensitive 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).

	III. THE ENDOPLASMIC AND THE SARCOPLASMIC RETICULUM

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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 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 qualitative 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.

B. Membrane and Luminal Continuity

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 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).

C. Molecular Heterogeneity

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 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

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 specific 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 RyR-dependent 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 protein-protein 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 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 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 metabotropic 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 µm) as well as macroscopic (10 µm) 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: 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 Ca²⁺ 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 (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.

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 µm 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.

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