Retrograde activation of store-operated calcium channel

Cell Calcium 33 (2003) 375–384

Retrograde activation of store-operated calcium channel Jianjie Ma∗ , Zui Pan Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA Received 5 February 2003; accepted 10 February 2003

Abstract Store-operated Ca2+ entry represents an important mechanism for refilling of a depleted intracellular-reticulum Ca2+ store following sustained activation of the IP3 receptor or ryanodine receptor RyR/Ca2+ release channel in the endoplasmic/sarcoplasmic reticulum (ER/SR). Recent studies have demonstrated the existence of store-operated Ca2+ channel (SOC) in muscle cells, whose activation process appears to be coupled to conformational changes of the RyR. Regulation of the plasma membrane (PM)-resided SOC by the SR-located RyR requires an integrity of the junctional membrane structure between SR and PM. Proteins that interact with RyR or influence the Ca2+ buffering capacity in the ER or SR lumen also participate in the activation process of SOC. Calsequestrin (CSQ) and calreticulin (CRT) are SR/ER-resident proteins, with highly negative charged regions at the carboxyl-terminal end that exhibit high buffering capacity for luminal Ca2+ . CSQ and CRT not only modulate the intracellular Ca2+ release process but also might provide retrograde signals to regulate the function of SOC. The functional interplay between CSQ, RyR and SOC may serve essential roles of Ca2+ signaling in muscle contraction and development. A tight link between the expression of CRT and operation of SOC exist in certain cancer cells, where the reduced sensitivity to apoptosis may correlate with the altered function of SOC. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Sarcoplasmic reticulum; Ryanodine receptor; TRP; Excitation–contraction coupling; Apoptosis; Ca2+ homeostasis; Junctophilin; Muscle fatigue; Calsequestrin; Calreticulin

1. Introduction Calcium is an important second messenger that plays an integral role in a wide variety of biological processes, including signal transduction and enzymatic regulation, gene expression and protein trafficking, cell proliferation and apoptosis, and the coordination of muscle excitation–contraction (E–C) coupling [1]. In general, there are two sources of this signaling ion in the cell: channels in the plasma membrane (PM) that open to allow external Ca2+ to flow into the cytoplasm, and internal stores in the form of endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) that release Ca2+ into the cytosol. The effective coupling of extracellular Ca2+ entry and intracellular Ca2+ release is often mediated by an intimate interaction between protein components on the PM and ER/SR. At the cellular level, Ca2+ can be either a life or a death signal, as changes in cytosolic-free Ca2+ concentration and intracellular Ca2+ content can control cell growth and

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author. Tel.: +1-732-235-4494; fax: +1-732-235-4483. E-mail address: [email protected] (J. Ma).

proliferation or induce programmed cell death [2,3]. As a molecular signal that initiates the contractile events of heart and skeletal muscles, a precise spatial and temporal encoding of Ca2+ signal is achieved through cross-talk between signal detectors and mediators on both PM and SR membranes, a cascade of coordinated events that often involve both orthograde and retrograde protein–protein interactions [4,5]. The internal Ca2+ stores, located in the SR of muscle cells and ER of non-muscle cells, have a limited capacity; it must be regularly replenished through the entry of Ca2+ from the external environment. Depletion of these intracellular-reticulum Ca2+ stores, following activation of ryanodine receptor (RyR) or IP3 receptor or other Ca2+ release mechanisms, triggers Ca2+ entry from the external environment through a process known as capacitative Ca2+ entry via activation of store-operated Ca2+ channels (SOC) located in the PM [6,7]. Research into the molecular and cellular function of store-operated Ca2+ entry (SOCE) has been carried out primarily in non-excitable cells (i.e. lymphocytes, mast cells, etc.) [8–10], and to some extent in smooth muscle cells [11]. In recent years, the SOCE pathway has received great interest not only because of its unusual nature as retrograde signaling, but also due to its

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wide occurrence in both excitable and non-excitable cells and its possible role in physiological and pathophysiological situations. More recently, the SOCE pathway has been identified in muscle cells, and its physiological functions are being recognized [12–14]. This review article will focus on four topics related to the function and regulation of SOC. First, we will introduce the concept of E–C coupling and outline some of the relevant studies dealing with the orthograde and retrograde Ca2+ signaling processes in muscle cells. Second, we will introduce the concept of SOCE in skeletal muscle and discuss its possible regulation by the spatial coupling between PM and SR and conformational changes of RyR. The putative roles of SOC in muscle development and fatigue will be discussed as well. Third, we will summarize some of the recent studies on the retrograde regulation of SOC via signals originating from the SR lumen mediated by calsequestrin (CSQ) and calreticulin (CRT). Finally, the participation of SOC in programmed cell death will be discussed.

2. Orthograde and retrograde Ca2+ signaling in muscle cells Perhaps the best-studied example of Ca2+ signaling is that defined by membrane-excitation induced Ca2+ release in skeletal muscle [15,16]. Here the initiation of myofilament contraction is triggered mainly by the release of Ca2+ from SR, a process that is under tight control by the excitation status of the PM. This so-called E–C coupling phenomenon takes place in a highly specialized junctional region that arise from close proximity between PM and SR. More precisely, transverse tubular invaginations of the PM touch the terminal cisternae of SR to form a unique anatomical structure known as the triad junction [17,18]. This triad junction not only ensures efficient spatial coupling between voltage sensor of the PM and Ca2+ release channel of the SR, but also enables well-coordinated temporal control of Ca2+ movements. We now know that the dihydropyridine receptors (DHPR) located on the PM are voltage-gated/L-type Ca2+ channels, as well as voltage sensors of the PM that gate opening of the RyR/Ca2+ release channel [16,19,20]. The RyR, in addition to being the conducting unit of a Ca2+ release channel [21,22], provides the structural link with the DHPR/voltage sensor [23] and may also participate in the regulation of SOCE [13,24]. The modulatory or transducing function of RyR is presumably achieved by a large cytoplasmic structure that accounts for close to 90% of the whole molecular mass of RyR [25,26]. In skeletal muscle, DHPRs located in the transverse tubule membrane function mainly as the voltage sensor which sends an orthograde signal to control opening of the RyR/Ca2+ release channel [19,20] (see Fig. 1A). The operation of voltage-induced Ca2+ release (VICR) appears to involve a direct physical interaction between DHPR and RyR in the triad junction of skeletal muscle, without requiring the

transmembrane movement of extracellular Ca2+ [27]. In the heart muscle, the DHPR is encoded by a gene that is different from that in skeletal muscle and the resulting L-type Ca2+ channel has fast kinetics of activation [28]. Therefore, membrane depolarization-induced activation of the L-type Ca2+ channel initiates rapid influx of Ca2+ from extracellular space, which in turn triggers opening of the RyR channel via Ca2+ -induced Ca2+ release (CICR) process in cardiac myocytes [29] (see Fig. 1B). Peripheral coupling between PM and SR in the form of dyad junction mediates efficient communication from L-type Ca2+ channel to RyR in the cardiac muscle. As a voltage-gated Ca2+ channel, the DHPR of skeletal muscle has slow activation kinetics, and does not support Ca2+ influx under normal physiological conditions [30]. The twitch force in skeletal muscle is triggered mainly by the acute release of Ca2+ from the SR, primarily via VICR, and secondarily amplified by CICR through activation of neighboring RyRs not directly coupled to DHPR [31]. Under repetitive stimulation condition (e.g. muscle exercise and fatigue), however, the entry of extracellular Ca2+ may be required to compensate for the sustained demand of elevated cytosolic [Ca2+ ], and also for the replenishment of a diminished SR Ca2+ store. Another physiological function or requirement for extracellular Ca2+ entry in skeletal muscle exist in the early developmental stages of myogenesis. The embryonic skeletal muscle does not have well-developed transverse tubule network and well-coordinated triad junction structure [32], which sets a hindrance to the operation of VICR. Therefore, alternative pathway other than the voltage-dependent Ca2+ entry must exist in skeletal muscle to regulate the movement of extracellular Ca2+ influx. SOCE may represent one of such Ca2+ entry mechanisms. Another unique feature of skeletal muscle is that not only an orthograde signal from DHPR to the RyR is required for E–C coupling, but also a retrograde signal from RyR to the DHPR regulates the function of DHPR as a Ca2+ channel [4] (see Fig. 1A). This finding was based largely on experiments with cultured skeletal myotubes derived from mutant mice lacking the skeletal isoform of RyR, ryr1(−/−) [33,34]. Compared with normal myotubes, the ryr1(−/−) myotubes exhibit a dramatic reduction in the density of DHPR-mediated Ca2+ current. Expression of the skeletal RyR in the ryr1(−/−) myotube enhances voltage-dependent Ca2+ current without apparent changes in the density of DHPR on the PM. These observations led to the conclusion that RyR promotes the Ca2+ channel function of DHPR. This ability seems to be a unique property of skeletal muscle since the cardiac isoform of RyR is incapable of either restoring E–C coupling or rescuing the DHPR/Ca2+ channel function in the ryr1(−/−) myotubes [35]. Thus, the signaling between the DHPR and RyR appears to be bi-directional in skeletal muscle, such that the channel activity associated with each protein is strongly dependent upon their intermolecular interaction.

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Fig. 1. Molecular components of orthograde and retrograde Ca2+ signaling in muscle cells. (A) In skeletal muscle, transverse tubule (T-tubule) invagination of the PM touches the SR. The close PM–SR junctional structure enables direct control of the RyR1/Ca2+ release channel by the DHPR via VICR. RyR1 can also influence the function of DHPR as a voltage-gated Ca2+ channel in the retrograde direction. (B) In cardiac muscle, Ca2+ entry through DHPR triggers opening of the RyR2/Ca2+ release channel via CICR. (C) Activation of SOCE in skeletal muscle is analogous to the retrograde signaling process shown in (A), whereby conformational changes of RyR presumably gates opening of SOC via direct contact interaction. CSQ and CRT are SR luminal Ca2+ binding proteins, which could influence the activation of SOCE by either altering the function of RyR or altering the Ca2+ homeostasis inside the SR lumen. (D) JP and MG29 are essential protein components of the junctional membrane structure between PM and SR (left panel). Disruption of JP or MG29 expression results in uncoupling of PM with SR, leading to dysfunction of VICR, CICR and SOCE (right panel).

3. Conformation-dependent and RyR-mediated activation of SOC Since the initial concept of capacitative or SOCE introduced by Putney in 1986 [36], the research field has gone through a burst of activities largely due to the discovery of the transient receptor potential (TRP) gene, a molecular candidate for SOC. The TRP superfamily are now classified according to their sequence homology and functional diversity into four subfamilies: TRPV, TRPM, TRPP, and TRPML (for review see Refs. [7,37–39]). Extensive structure–function studies have shown that although most of the TRP proteins encode channels that are selective for cations, not all of which are regulated by intracellular Ca2+ store depletion [7]. It has been postulated that the true identity of SOC may actually be a heteromultimeric molecular complex that spans the junctional membrane of ER and PM with separate Ca2+ -sensing and Ca2+ -conducting domains. More detailed

discussions related to the topic of TRP can be found in other review articles in this special issue of Cell Calcium. Majority of the researches into the cellular and molecular function of SOC has been performed primarily in non-excitable cells. Although accumulative evidence calls for the need for a non-voltage-dependent Ca2+ entry in skeletal muscle, functional evidence supporting the existence of SOC in skeletal muscle are just beginning to emerge recently. The first direct demonstration of SOCE in skeletal muscle was provided by Kurebayashi and Ogawa [12]. Using skeletal muscle fibers isolated from adult mice, they showed that depletion of the SR Ca2+ stores by repetitive treatments with high-K+ solutions in combination with inhibitors of the SERCA Ca2+ pump resulted in activation of extracellular Ca2+ entry. This component of SOCE in skeletal muscle was sensitive to blockade by Ni2+ , resistant to nifedipine, and suppressed by PM depolarization. Moreover, this SOCE pathway is sufficient to refill the depleted

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Fig. 2. Graded activation of SOCE in skeletal muscle. Quenching of Fura-2 fluorescence by the influx of Mn2+ (0.5 mM) through SOC was measured at an excitation wavelength of 360 nm and emission wavelength of 510 nm. Individual skeletal muscle myotubes were treated with either 10 mM caffeine (A), or 10 mM caffeine plus 1 ␮M ryanodine (B), or 10 ␮M thapsigargin (C). These treatments created different degrees of depletion of Ca2+ stores from the SR, which were followed by graded activation of SOCE as indicated by the different slope of Fura-2 quenching by Mn2+ .

SR Ca2+ store within several minutes in skeletal muscle fibers. The coupling mechanism of SOCE remains elusive and has been proposed to involve the diffusion of a soluble messenger [40], direct interaction between RyR or IP3 receptors and SOC channels [41], or a secretion-like docking mechanism [42]. Although evidence indicates that physical docking of ER with PM is involved in the activation of SOC, the molecules and/or signals that couple ER/SR Ca2+ depletion to opening of SOC have yet to be identified (see Fig. 1C). During the past few years, increasing evidence has accumulated supporting the conformational coupling hypothesis, owing mainly to studies conducted on TRP channels. Conformation coupling between SOC and RyR or IP3 receptors is analogous to the retrograde regulation of DHPR function by RyR in skeletal muscle [4,5]. The RyR-mediated intracellular Ca2+ release mechanism is not limited to muscle cells, as many non-muscle cells, including neurons [43], lymphocytes [44,45], and epithelial cells also express RyRs [46]. It has been shown that Ca2+ release by activation of RyRs in non-muscle cells activates SOCE in a manner similar to Ca2+ release from the IP3 -sensitive stores. The functional coupling between RyR and SOC, if it exists, is likely to be influenced by the structural interaction between PM and SR. In the case of muscle cells, those proteins participate in the formation of dyad and triad junction, for example mitsugumins (MG) and junctophilins (JP) may modulate the interaction between RyR and SOC and the overall Ca2+ signaling process (see Fig. 1D). To test the putative regulation of SOC by RyR in muscle cells, we used primary cultured myotubes derived from neonates of the ryr1(−/−)ryr3(−/−) and ryr1(+/−)ryr3(−/−) mice. It is known that deletion of either RyR1 or RyR3 does not disrupt the triad junction structure [47]. Therefore, the ryr1(−/−)ryr3(−/−) and ryr1(+/−)ryr3(−/−) mice offer unique models to examine the potential regulation of SOC by RyR1, the main isoform

of RyR present in skeletal muscle. The ryr1(+/−)ryr3(−/−) myotubes responded to caffeine stimulation with rapid release of Ca2+ from SR. Following this transient SR Ca2+ depletion SOCE became evident as indicated by the quenching of Fura-2 fluorescence by Mn2+ (see Fig. 2A). Simultaneous application of caffeine and ryanodine, ligands that induce permanent opening of the RyR/Ca2+ release channel, resulted in SR Ca2+ release similar to caffeine alone, but the extent of SOCE was significantly larger (Fig. 2B). Thapsigargin, a potent inhibitor of the SR Ca2+ pump, produced sustained depletion of Ca2+ from SR and induced even greater activation of SOCE in ryr1(+/−)ryr3(−/−) cells (Fig. 2C). Near complete inhibition of SOCE in ryr1(+/−)ryr3(−/−) cells was observed upon addition of 20 ␮M SKF 96365, a known blocker of SOC. The ryr1(−/−)ryr3(−/−) myotubes, lacking both ryr1 and ryr3, failed to respond to caffeine or ryanodine, but contained thapsigargin-sensitive SR Ca2+ store similar to that present in ryr1(+/−)ryr3(−/−) cells (see Ref. [13]). SOC activity in ryr1(−/−)ryr3(−/−) cells, however, was significantly smaller than that in ryr1(+/−)ryr3(−/−) cells. After depletion of SR Ca2+ with thapsigargin, ryr1(−/−)ryr3(−/−) cells exhibited a residual component of SOCE (∼40% of that present in ryr1(+/−)ryr3(−/−) cells), which was partially sensitive to inhibition by SKF 96365 [13]. These data provide direct evidence for a RyR-coupled activation of SOC in skeletal muscle. Based on the different degree of SOCE triggered by caffeine, caffeine and ryanodine, or thapsigargin in ryr1(+/−)ryr3(−/−) cells, one can conclude that SOCs in skeletal muscle can function in a graded manner depending on the SR Ca2+ content or the conformation of RyR [13]. Our more recent studies have identified a developmental change of SOC in prenatal skeletal muscle (Collet and Ma, unpublished data), which may be a consequence of altered PM–SR junctional structure or altered expression of related Ca2+ -regulatory proteins associated with maturation of muscle cells.

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4. Junctional membrane structure and SOCE Because of the necessity of maintaining a close-spatial contact between PM and SR for the efficient operation of VICR, CICR as wells as SOCE, finding the “link” or “glue” between the PM and ER/SR has been a real challenge to understand the cellular and molecular mechanism of E–C coupling in muscle cells. In addition to DHPRs and RyRs, other proteins of the PM–SR junction also play critical roles in muscle E–C coupling [48]. Transgenic mice that lack expression of either DHPRs or RyRs still form seemingly normal triad junctions, indicating that structural components other than the DHPR/RyR interaction are needed for a close apposition of PM and SR membranes [49,50]. Heterologous cells transfected with both DHPR and RyR cDNAs showed neither Ca2+ release in response to membrane depolarization, nor close association between the PM and ER [51,52]. Thus, the DHPR/RyR interaction is neither necessary nor sufficient for the formation of triad and dyad junctions. Although several transmembrane proteins with no established physiological roles have been identified as components of triad junctions, none appears to be a candidate molecule for mediating the physiological coupling of the junctional complex. Since the gap size between PM and SR is reduced when the triad lacked RyRs (from ∼12 nm in normal muscle to ∼7 nm in ryr1(−/−)ryr3(−) muscle) [47], the structural link between the PM and SR is likely to be flexible or elastic. Using a combination of monoclonal antibody immunocytochemistry and cDNA library screening techniques, Takeshima and colleagues have recently discovered a group of novel membrane proteins termed MG and JP that in skeletal and cardiac muscle are exclusively localized to the triad and dyad junctions, respectively [53,54]. Mitsugumin29 (MG29) is a synaptophysin family member protein localized specifically in the triad junction of skeletal muscle, and to a lesser extent also present in the tubular membranes of the kidney [53]. In mature skeletal muscle, MG29 is present predominantly on the transverse tubule invagination of PM [55] (see Fig. 1D). Morphological and functional studies revealed important physiological function of MG29 in both refinement of the membrane structure and effective E–C coupling in skeletal muscle. Abnormalities of membrane ultrastructure around the triad junction were detected in skeletal muscle from the mg29(−/−) mice: the transverse tubules were swollen and the SR networks were poorly formed with vacuolated and fragmented structures, leading to misalignment of triad junctions [56]. In the mg29(−/−) muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Unlike the normal skeletal muscle whose E–C coupling machinery does not rely on the entry of extracellular Ca2+ , skeletal muscle from the mutant mg29(−/−) mice exhibits clear dependence on extracellular Ca2+ for contraction. As MG29 is involved in the coupling between PM and SR, presumably the expression of MG29 will have functional impact on the RyR-mediated activation of SOC in muscle cells.

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A distinct phenotype of the mg29(−/−) mice is their failure to complete an endurance test, that is, their skeletal muscles exhibit increased susceptibility to fatigue [57]. Quantitative measurements of intracellular [Ca2+ ]i with Fura-2 fluorescent indicator in individual myotubes derived from different mutant mice enabled us to identify a defective regulation of SOC in mg29(−/−) cells, and begin to define the mechanism of SOC operation in skeletal muscle [13]. Specifically, we found that SOCE is severely compromised in mg29(−/−) cells, and the defective function of SOCE may underlie the phenotypic changes of muscle performance in response to repetitive stimulation. Fatigue is an important functional property of skeletal muscle, and is defined as a reversible decrease in the isometric contractile force in response to an increase in the frequency or duration of stimulation. Experiments with intact muscle fiber show that the size of the Ca2+ store declines during fatigue and recovers upon rest [58]. Although the twitch force initiated by a single electrical stimulation does not depend on the movement of extracellular Ca2+ , the fatigue pattern of isolated muscle fibers shows a clear dependence on extracellular Ca2+ . Changing the bath solution [Ca2+ ]o from 2 to 0 mM significantly increased the fatigability of the muscle, as indicated by the reduction in the sustained force output at the end of the tetanic stimulation (see Ref. [13]). The reduction of SR Ca2+ stores associated with muscle fatigue can trigger the activation of SOCE, which could play a role in the overall Ca2+ handling properties of skeletal muscle. Indeed, the mg29(+/+) and mg29(−/−) muscles showed comparable responses to fatigue stimulation at 0 [Ca2+ ]o or after inhibition of SOC with SKF-96365. This suggests that the increased susceptibility to fatigue stimulation is likely a consequence of dysfunction of SOC in mg29(−/−) muscle cells. JPs are novel membrane proteins with unique structural motifs that are capable of initiating and maintaining a tight PM–SR junctional membrane structure in excitable cells [54] (see Fig. 1D). JP contains a single transmembrane segment at the carboxyl-terminal end that inserts into the SR membrane, and a large cytoplasmic region with repeated motifs of 14 amino acid residues termed “membrane occupation and recognition nexus” or MORN motifs. The MORN motifs exhibit selective binding affinity to PM. When expressed in non-muscle cultured cells, JP induced formation of junctional ER-PM complexes, with a gap size of ∼7 nm. In normal skeletal muscle myotubes, the junctional gap is ∼12 nm in the presence of RyR, but narrows to 7 nm in RyR-deficient myotubes [47]. These observations imply some elasticity in JP, depending on presence or absence of RyR in the juctional membrane complexes. Indeed, sequence alignments of the different JP isoforms reveals a highly conserved alpha-helical region after the MORN motif that could presumably mediate the elastic property of JP [59]. In the heart, JP2, the cardiac isoform of JP, appears to be essential for dyad junction formation because disruption of its expression in mice produces embryonic lethality, as a result of defective junctional membrane coupling and

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unsynchronized intracellular Ca2+ transients [54]. The deficient junctional membrane structure and abnormal intracellular Ca2+ transients observed in cardiac myocytes of the jp2(−/−) mice resemble some of the functional changes associated with diseased human heart, that is, widening of the dyadic membrane cleft and reduced efficiency of intracellular Ca2+ release measured in congestive heart failure (CHF) [60]. Although the pathogenesis of CHF is probably multifactorial, alterations in E–C coupling are a central finding in all animal models of CHF and in failing human heart. Presumably, mutations or altered expression of JPs might induce human diseases by affecting the Ca2+ signaling of excitable cells. The jp1(−/−) mice showed no milk sucking capability because of weak contractility of the jaw muscle, and died within 5–12 h after birth [61]. Ultrastructural analysis revealed that triad junctions were reduced in number, and that the SR network was often structurally abnormal in skeletal muscles of the jp1(−/−) mice. The mutant muscle developed less contractile force evoked by low-frequency electrical stimuli and showed abnormal sensitivities to extracellular Ca2+ . These data indicate that JP1 contributes to the construction of triad junctions and is essential for the efficiency of signal conversion during E–C coupling in skeletal muscle. Regarding the cellular and molecular function of JP in muscle Ca2+ signaling, several key questions remain: first, how do the different structural domains of JP mediate the elastic coupling of the PM–SR junction? Second, what is the impact of changes in PM–SR coupling on the operation of VICR, CICR and SOCE? Understanding these questions will undoubtedly provide valuable clues to the molecular mechanism of Ca2+ signaling in excitable cells.

5. Retrograde regulation of SOC by CSQ and CRT CSQ is a SR-resident protein in muscle cells whose primary known function is to buffer Ca2+ in the lumen of SR. It binds Ca2+ with high capacity (40–50 mol Ca2+ /mol of CSQ) and moderate affinity (Kd ∼ 1 mM) [62]. Recent studies have shown, however, that CSQ participates in the active Ca2+ release process from SR not simply by being a passive Ca2+ storage protein, but also by actively modulating the function of the RyR/Ca2+ release channel. The carboxyl-terminus of CSQ contains an aspartate-rich region (asp, a.a. 354–367) (see Fig. 4), which functions as a major Ca2+ binding motif [63], and also interacts with triadin or junctin, proteins of the SR membrane complexed to RyR with unclear roles in the operation of E–C coupling [64,65]. A different region of CSQ (junc, a.a. 86–191) has been previously suggested to bind to junctin and triadin. The functional significance of these CSQ regions in muscle Ca2+ signaling has not been examined. We tested the hypothesis that the RyR might receive information on the state of SR Ca2+ depletion via a direct

retrograde signal from CSQ, and thereby modulate both RyR-mediated Ca2+ release and RyR-mediated SOCE [66]. We found that overexpression of CSQ not only enhances active Ca2+ release through the RyR, but also suppresses SOCE. The CSQ-mediated inhibition of SOCE appears to involve the asp-rich region of CSQ, since the inhibitory effect was only observed with wt-CSQ and junc-CSQ, but not with asp-CSQ. These data provide the first direct evidence for regulation of SOCE, a cell surface membrane function, through the luminal side of the SR membrane. Although the gene(s) responsible for SOC have yet to be identified, and the exact nature of signal transduction involved in the activation of SOC remains largely unknown, our data indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. These interactions are possibly taking place at the junctional site of the SR. It will be interesting, therefore, to see how the absence of CSQ in a knockout model would affect the function of SOC in skeletal muscle. The presence of exogenously expressed CSQ in the SR lumen adds extra Ca2+ buffering capacity, and increases the driving force for Ca2+ movement across the SR membrane. To test whether the CSQ mediated inhibition of SOCE is due to changes in the Ca2+ buffering capacity in the SR or it might reflect altered intermolecular interactions in the junctional complex of RyR, the following two sets of experiments were performed. First, thapsigargin was used to completely deplete in SR Ca2+ stores with cells bathed in a Ca2+ -free medium. Second, a high affinity Ca2+ buffer, BAPTA-AM, was used to control the changes in cytosolic Ca2+ . Under both conditions, overexpression of wt-CSQ and junc-CSQ in C2C12 cells resulted in significant reduction in the rate of Fura-2 fluorescence quenching by Mn2+ when the SR is completely depleted of Ca2+ (Fig. 3). On average, ∼10-fold reduction in Mn2+ -influx rate was observed in cells overexpressing wt-CSQ and junc-CSQ compared with control and cells overexpressing asp-CSQ. These experiments ruled out the possibility that the reduction of SOCE seen with overexpression of wt-CSQ and junc-CSQ results from an incomplete depletion of SR Ca2+ stores, or due to potential changes in myoplasmic [Ca2+ ]i . It is likely that CSQ can regulate the function of the PM-located SOC likely via retrograde interaction with the junctional protein complex in the SR. In non-muscle tissues, a major Ca2+ binding protein in ER lumen is CRT, although it was first isolated from the muscle cells [67]. CRT is a highly conserved ER chaperone protein with multifunctional roles, and is present in every cell types of the higher organisms except erythrocytes [68]. Examination of the primary amino acid sequence of CRT reveals that, similar to CSQ, it too contains a highly negatively charged region at its carboxyl terminus (Fig. 4). This very acidic domain can bind Ca2+ with a relatively high capacity and low affinity (Kd ∼ 2 mM; Bmax ∼ 25 mol of Ca2+ /mol of protein). The conservation of this negatively charged

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Fig. 3. Inhibition of SOCE by overexpression of CSQ in skeletal muscle. Full-length (wt-CSQ) and two deletion mutants of CSQ, one lacking the aspartate-rich region (asp-CSQ, a.a. 354–367) and the other lacking the junction binding motif (junc-CSQ, a.a. 86–191), were overexpressed in C2C12 skeletal muscle myotubes, respectively. Individual C2C12 cells were treated with 10 ␮M thapsigargin and incubated with 50 ␮M BAPTA-AM, with 0 [Ca2+ ] present in the bath solution. These treatments resulted in complete depletion of the intracellular Ca2+ store. Under these conditions, prominent inhibitions of SOCE, measured with Mn-quenching of Fura-2 fluorescence, were observed with cells overexpressing wt-CSQ (B) and junc-CSQ (C), but not asp-CSQ (D), compared with the controls (A). The results suggest that the inhibitory effect of CSQ on SOCE likely involves the aspartate-rich Ca2+ binding domains of CSQ, which cannot be solely due to the enhanced Ca2+ buffering capacity in the SR.

Fig. 4. Conservation of negatively charged carboxyl termini in CSQ and CRT. The primary amino acid sequence of CSQ from rabbit skeletal muscle contains 28 negatively charged aspartate (D) or glutamate (E) residues at the carboxyl-terminal end. Similarly, CRT from rabbit skeletal muscle also contains 36 negatively charged residues close to the carboxyl-terminal end followed by the KDEL ER retrieval signal. These negatively charged residues form the binding sites for luminal Ca2+ , and could participate in regulation of SOCE and CICR.

region of the carboxyl terminus of both of these SR/ER Ca2+ binding proteins supports a significant functional role for CSQ and CRT. Indeed, several studies have reported that overexpression of CRT attenuated SOCE in various cell types [69–72]. For example, altered ER Ca2+ response and reduced SOCE were observed in cells overexpressing the full-length and amino terminal-truncated CRT but not in cells overexpressing calnexin, a homologous chaperone protein with the similar proline-rich region of CRT. These data indicate that the effect of CRT on SOCE was likely mediated by its negatively charged carboxyl-terminal domain. A major controversy exists in the literature on the mechanisms of modulation of SOCE by CRT in relationship to its Ca2+ buffering capacity. Some showed that overexpression of CRT in mouse fibroblast cells inhibited SOCE even at times when the cells were completely depleted of their ER Ca2+ storages by thapsigargin [71]. These studies are consistent with our results with CSQ-mediated regulation of SOCE in muscle cells, suggesting that the presence of high concentrations of CSQ and CRT do not simply attenuate SOCE by their enhanced luminal Ca2+ buffering capacity. Others, however, demonstrated that overexpression of CRT in RBL-1 cells had no significant effects on SOC function

based on whole-cell patch-clamp studies, when the cellular ER Ca2+ stores were rapidly depleted by ionomycin or high concentration of IP3 [72]. This result suggests that the CRT-mediated inhibition of SOC currents in these cells could be interpreted by a delayed Ca2+ release process across the ER membrane. It is likely that the differential effects of CRT on SOCE may be cell specific. Regarding the cellular and molecular mechanism of CRT regulation of SOCE, further investigations are required.

6. Involvement of Ca2+ flux and SOCE in apoptosis A growing body of evidences has suggested a pivotal role for Ca2+ in apoptosis, a programmed cell death that is essential for proper development and the maintenance of tissue homeostasis [2,3]. Numerous apoptotic stimuli, including growth factor withdrawal, excessive nitric oxide production, anti-cancer drugs, hormonal stimulation and activation of surface antigen receptors are known to alter the concentration of Ca2+ in the cytosol and the storage of Ca2+ in the ER. Compounds such as Ca2+ ionophores and thapsigargin have been shown to trigger apoptosis in a

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variety of cells [73,74]. Moreover, the anti-apoptosis effects of Bcl-2 have been shown to correlate with changes in ER Ca2+ stores [75]. In the Ca2+ signaling process of apoptosis, ER as the main intracellular Ca2+ store, appears to be a central player. The mitochondria also take up and release Ca2+ very efficiently and are often intimately located close to ER Ca2+ release sites. Thus, the panoramic picture of Ca2+ signaling in apoptosis consists of Ca2+ release from ER, depletion of ER Ca2+ stores, and subsequent activation of SOCE. These Ca2+ movements will result in excess mitochondria Ca2+ uptake and elevation of cytosolic Ca2+ . Such altered Ca2+ homeostasis can be the trigger for apoptosis. We tested whether the depletion of ER Ca2+ stores or the elevation of cytosolic Ca2+ is the trigger for apoptosis in heterologous cells stably transfected with the RyR cDNA [76]. Using a combination of ryanodine and caffeine, one can lock the RyR/Ca2+ release channel in a permanently open state. This causes complete depletion of the ER Ca2+ stores. In cells bathed with a Ca2+ -free medium or pretreated with an intracellular Ca2+ chelator (BAPTA-AM) to eliminate the global elevation of cytosolic Ca2+ , depletion of ER Ca2+ stores with caffeine and ryanodine still induced apoptotic cell death. This result indicates that depletion of ER Ca2+ stores rather than elevation of cytosolic Ca2+ is the trigger for apoptosis in these cells. Other investigators have identified similar phenomenon with other cell types. For example, thapsigargin induces apoptosis in LNCaP human prostate cancer cells and S49 mouse lymphoma cells, primarily due to the depletion of ER Ca2+ stores and not related to elevation of cytosolic Ca2+ [77,78]. ER Ca2+ stores can also be the target of other signaling molecules, such as nitric oxide, which induced apoptotic cell death through depletion of ER Ca2+ stores and ER stresses [79]. Furthermore, the effects of certain pro-apoptotic proteins on programmed cell death are correlated with their influence on ER Ca2+ homeostasis [80,81]. However, in some cases, it seems that Ca2+ store depletion by itself is not sufficient to induce apoptosis and SOCE is the alternative target. In contrary to androgen-sensitive prostate cancer cell line LNCaP, in many androgen-insensitive prostate cancer cell lines, such as TSU-Pr1, DU-145, AT-3, PC-3 cells and LNCaP overexpressing Bcl-2 protein, Ca2+ store depletion per se is no longer sufficient to induce apoptosis [74,82,83]. Additional sustained SOCE is required for the initiation of apoptosis. CRT expression seems to be associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in androgen-insensitive cells for the down-regulation of CRT by anti-sense oligonucleotide treatment reverses the androgen-induced resistance to A23187 [74]. Recent studies indicated the important role of SOCE in the regulation of phosphatidylserine transbilayer migration, an early indicator of cells undergoing apoptosis. Incubation of cells with SKF-96365, or miconazole, inhibitors of SOCE, is shown to reduce the degree of phosphatidylserine externalization triggered by intracellular Ca2+ depletions [84]. Further studies to clarify the role of

SOC in apoptosis and the mechanism of SOC operation will provide us a novel approach to the treatment and chemoprevention of cancer by targeting the Ca2+ signaling processes.

7. Concluding remarks SOCE represents an important link between the extracellular Ca2+ reservoir and intracellular Ca2+ storage. This pathway appears to be ubiquitously present in both excitable and non-excitable cells. As this signal transduction event involves two separate membranes, a tight spatial coupling between protein components on the PM and intracellular membrane is essential for the operation of this unique Ca2+ signaling pathway. MG and JP are key protein components that regulate the PM–SR coupling, and may play important roles in the gating process of the SOC. CSQ and CRT are Ca2+ binding proteins present in the intracellular organelles, and presumably also participate in the graded activation of the SOC. Tight control of the SOCE is essential for a variety of cellular processes, including cell proliferation and programmed cell death, coordinated control of gene expression and enzymatic function, and the biogenesis and contractile properties of muscle cells.

References [1] E. Carafoli, Calcium signaling: a tale for all seasons, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 1115–1122. [2] P. Nicotera, S. Orrenius, The role of calcium in apoptosis, Cell Calcium 23 (1998) 173–180. [3] D.J. McConkey, The role of calcium in the regulation of apoptosis, Scanning Microsc. 10 (1996) 777–793. [4] R.T. Dirksen, Bi-directional coupling between dihydropyridine receptors and ryanodine receptors, Front Biosci. 7 (2002) d659–d670. [5] J. Ma, Z. Pan, Junctional membrane structure and store operated calcium entry in muscle cells, Front Biosci. 8 (2003) D242–D255. [6] J.W. Putney Jr., L.M. Broad, F.J. Braun, J.P. Lievremont, G.S. Bird, Mechanisms of capacitative calcium entry, J. Cell Sci. 114 (Pt. 12) (2001) 2223–2229. [7] R. Vennekens, T. Voets, R.J. Bindels, G. Droogmans, B. Nilius, Current understanding of mammalian TRP homologues, Cell Calcium 31 (2002) 253–264. [8] A. Zweifach, R.S. Lewis, Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 6295–6299. [9] M. Hoth, R. Penner, Depletion of intracellular calcium stores activates a calcium current in mast cells, Nature 355 (1992) 353–355. [10] C. Fasolato, B. Innocenti, T. Pozzan, Receptor-activated Ca2+ influx: how many mechanisms for how many channels? Trends Pharmacol. Sci. 15 (1994) 77–83. [11] A. Gibson, I. McFadzean, P. Wallace, C.P. Wayman, Capacitative Ca2+ entry and the regulation of smooth muscle tone, Trends Pharmacol. Sci. 19 (1998) 266–269. [12] N. Kurebayashi, Y. Ogawa, Depletion of Ca2+ in the sarcoplasmic reticulum stimulates Ca2+ entry into mouse skeletal muscle fibres, J. Physiol. 533 (2001) 185–199. [13] Z. Pan, D.M. Yang, R.Y. Nagaraj, T.A. Nosek, H. Takeshima, H. Cheng, J. Ma, Dysfunction of store-operated Ca2+ channel in muscle cells lacking mg29 gene, Nat. Cell Biol. 4 (2002) 379–383.

J. Ma, Z. Pan / Cell Calcium 33 (2003) 375–384 [14] D.L. Hunton, P.A. Lucchesi, Y. Pang, X. Cheng, L.J. Dell’Italia, R.B. Marchase, Capacitative calcium entry contributes to nuclear factor of activated T-cells nuclear translocation and hypertrophy in cardiomyocytes, J. Biol. Chem. 277 (2002) 14266–14273. [15] G. Meissner, X. Lu, Dihydropyridine receptor–ryanodine receptor interactions in skeletal muscle excitation–contraction coupling, Biosci. Rep. 15 (1995) 399–408. [16] M.F. Schneider, Control of calcium release in functioning skeletal muscle fibers, Annu. Rev. Physiol. 56 (1994) 463–484. [17] C. Franzini-Armstrong, A.O. Jorgensen, Structure and development of E–C coupling units in skeletal muscle, Ann. Rev. Physiol. 56 (1994) 509–534. [18] B.E. Flucher, Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad, Dev. Biol. 154 (1992) 245–260. [19] E. Rios, G. Pizzaro, E. Stefani, Charge movement and the nature of signal transduction in skeletal muscle excitation–contraction coupling, Ann. Rev. Physiol. 54 (1992) 109–133. [20] E. Rios, J. Ma, A. Gonzalez, The mechanical hypothesis of excitation–contraction coupling in skeletal muscle, J. Muscle Res. Cell Motil. 12 (1991) 127–135. [21] F.A. Lai, H.P. Erickson, E. Rousseau, Q.Y. Liu, G. Meissner, Purification and reconstitution of the calcium release channel from skeletal muscle, Nature 331 (1988) 315–319. [22] M.B. Bhat, J. Zhao, W. Zang, C.W. Balke, H. Takeshima, W.G. Wier, J. Ma, Caffeine-induced release of intracellular Ca2+ from Chinese hamster ovary cells expressing skeletal muscle ryanodine receptor. Effects on full-length and carboxyl-terminal portion of Ca2+ release channels, J. Gen. Physiol. 110 (1997) 749–762. [23] T. Wagenknecht, R. Grassucci, J. Frank, A. Saito, M. Inui, S. Fleischer, Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum, Nature 338 (1989) 167– 170. [24] K.I. Kiselyov, D.M. Shin, Y. Wang, I.N. Pessah, P.D. Allen, S. Muallem, Gating of store-operated channels by conformational coupling to ryanodine receptors, Mol. Cell 6 (2000) 421–431. [25] M.B. Bhat, J. Zhao, H. Takeshima, J. Ma, Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor, Biophys. J. 73 (1997) 1329–1336. [26] H. Takeshima, S. Nishimura, T. Matsumoto, H. Ishida, K. Kangawa, N. Minamino, H. Matsuo, M. Ueda, M. Hanaoka, T. Hirose, et al., Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor, Nature 339 (1989) 439– 445. [27] G. Brum, E. Rios, E. Stefani, Effects of extracellular calcium on calcium movements of excitation–contraction coupling in frog skeletal muscle fibres, J. Physiol. 398 (1988) 441–473. [28] T. Tanabe, A. Mikami, S. Numa, K.G. Beam, Cardiac-type excitation–contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA, Nature 344 (6265) (1990) 451–453. [29] A. Fabiato, Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum, Am. J. Physiol. 245 (1983) C1–C4. [30] G. Cota, E. Stefani, Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog, J. Gen. Physiol. 94 (1989) 937–951. [31] D. Yang, Z. Pan, H. Takeshima, C. Wu, R.Y. Nagaraj, J. Ma, H. Cheng, RyR3 amplifies RyR1-mediated Ca2+ -induced Ca2+ release in neonatal mammalian skeletal muscle, J. Biol. Chem. 276 (2001) 40210–40214. [32] B.E. Flucher, H. Takekura, C. Franzini-Armstrong, Development of the excitation–contraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils, Dev. Biol. 160 (1993) 135–147. [33] J. Nakai, R.T. Dirksen, H.T. Nguyen, I.N. Pessah, K.G. Beam, P.D. Allen, Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor, Nature 380 (1996) 72–75.

383

[34] A. Fleig, H. Takeshima, R. Penner, Absence of Ca2+ current facilitation in skeletal muscle of transgenic mice lacking the type 1 ryanodine receptor, J. Physiol. 469 (Pt 2) (1996) 339–345. [35] J. Nakai, T. Ogura, F. Protasi, C. Franzini-Armstrong, P.D. Allen, K.G. Beam, Functional nonequality of the cardiac and skeletal ryanodine receptors, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1019– 1022. [36] J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7 (1986) 1–12. [37] B. Minke, B. Cook, TRP channel proteins and signal transduction, Physiol. Rev. 82 (2002) 429–472. [38] C. Zitt, C.R. Halaszovich, A. Luckhoff, The TRP family of cation channels: probing and advancing the concepts on receptor-activated calcium entry, Prog. Neurobiol. 66 (2002) 243–264. [39] C. Montell, L. Birnbaumer, V. Flockerzi, The TRP channels, a remarkably functional family, Cell 108 (2002) 595–598. [40] C. Randriamampita, R.Y. Tsien, Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx, Nature 364 (1993) 809–814. [41] R.F. Irvine, ‘Quantal’ Ca2+ release and the control of Ca2+ entry by inositol phosphates—a possible mechanism, FEBS Lett. 263 (1990) 5–9. [42] R.L. Patterson, D.B. van Rossum, D.L. Gill, Store-operated Ca2+ entry: evidence for a secretion-like coupling model, Cell 98 (1999) 487–499. [43] D. Balschun, D.P. Wolfer, F. Bertocchini, V. Barone, A. Conti, W. Zuschratter, L. Missiaen, H.P. Lipp, J.U. Frey, V. Sorrentino, Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning, EMBO J. 18 (1999) 5264–5273. [44] T. Girard, D. Cavagna, E. Padovan, G. Spagnoli, A. Urwyler, F. Zorzato, S. Treves, B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators, J. Biol. Chem. 276 (2001) 48077–48082. [45] Y. Sei, K.L. Gallagher, A.S. Basile, Skeletal muscle type ryanodine receptor is involved in calcium signaling in human B lymphocytes, J. Biol. Chem. 274 (1999) 5995–6002. [46] B.S. Lee, S. Sessanna, S.G. Laychock, R.P. Rubin, Expression and cellular localization of a modified type 1 ryanodine receptor and L-type channel proteins in non-muscle cells, J. Membr. Biol. 189 (2002) 181–190. [47] T. Ikemoto, S. Komazaki, H. Takeshima, M. Nishi, T. Noda, M. Iino, M. Endo, Functional and morphological features of myocytes from mutant mice lacking both ryanodine receptors type 1 and type 3, J. Physiol. 501 (1997) 305–312. [48] P.S. McPherson, K.P. Campbell, The ryanodine receptor/Ca2+ release channel, J. Biol. Chem. 268 (1993) 13765–13768. [49] C. Franzini-Armstrong, M. Pincon-Raymond, F. Rieger, Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings, Dev. Biol. 146 (1991) 364–376. [50] X.-H. Sun, F. Protasi, M. Takahashi, H. Takeshima, D.G. Ferguson, C. Franzini-Armstrong, Molecular architecture of membranes involved in excitation–contraction coupling of cardiac muscle, J. Cell Biol. 129 (1995) 659–671. [51] H. Takekura, H. Takeshima, S. Nishimura, M. Takahashi, T. Tanabe, V. Flockerzi, F. Hofmann, C. Franzini-Armstrong, Co-expression in CHO cells of two muscle proteins involved in excitation–contraction coupling, J. Muscle Res. Cell Motil. 16 (1995) 465–480. [52] N. Suda, D. Franzius, A. Fleig, S. Nishimura, M. Bodding, M. Hoth, H. Takeshima, R. Penner, Caffeine-induced release of intracellular Ca2+ from Chinese hamster ovary cells expressing skeletal muscle ryanodine receptor, J. Gen. Physiol. 109 (1997) 619–631. [53] H. Takeshima, M. Shimuta, S. Komazaki, K. Ohmi, M. Nishi, M. Iino, A. Miyata, K. Kangawa, Mitsugumin29, a novel synaptophysin family member from the triad junction in skeletal muscle, Biochem. J. 331 (1998) 317–322.

384

J. Ma, Z. Pan / Cell Calcium 33 (2003) 375–384

[54] H. Takeshima, S. Komazaki, M. Nishi, M. Iino, K. Kangawa, Junctophilins: a novel family of junctional membrane proteins, Mol. Cell 6 (2000) 1–22. [55] N.R. Brandt, A.H. Caswell, Localization of mitsugumin 29 to transverse tubules in rabbit skeletal muscle, Arch. Biochem. Biophys. 371 (1999) 348–350. [56] M. Nishi, S. Komazaki, N. Kurebayashi, Y. Ogawa, T. Noda, M. Iino, H. Takeshima, Abnormal features in skeletal muscle from mice lacking mitsugumin29, J. Cell Biol. 147 (1999) 1473–1480. [57] R.Y. Nagaraj, C.M. Nosek, M.A. Brotto, M. Nishi, H. Takeshima, T.M. Nosek, J. Ma, Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene, Physiol. Genomics 4 (2000) 43–49. [58] D.G. Allen, H. Westerblad, Role of phosphate and calcium stores in muscle fatigue, J. Physiol. 536 (2001) 657–665. [59] M. Nishi, K. Hashimoto, K. Kuriyama, S. Komazaki, M. Kano, S. Shibata, H. Takeshima, Motor discoordination in mutant mice lacking junctophilin type 3, Biochem. Biophys. Res. Commun. 292 (2002) 318–324. [60] A.M. Gomez, H.H. Valdivia, H. Cheng, M.R. Lederer, L.F. Santana, M.B. Cannell, S.A. McCune, R.A. Altschuld, W.J. Lederer, Defective excitation–contraction coupling in experimental cardiac hypertrophy and heart failure, Science 276 (1997) 800–806. [61] K. Ito, S. Komazaki, K. Sasamoto, M. Yoshida, M. Nishi, K. Kitamura, H. Takeshima, Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1, J. Cell Biol. 154 (2001) 1059–1067. [62] L. Fliegel, M. Ohnishi, M.R. Carpenter, V.K. Khanna, R.A. Reithmeier, D.H. MacLennan, Amino acid sequence of rabbit fast-twitch skeletal muscle calsequestrin deduced from cDNA and peptide sequencing, Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 1167–1171. [63] D.W. Shin, J. Ma, D.H. Kim, The asp-rich region at the carboxyl-terminus of calsequestrin binds to Ca2+ (2+) and interacts with triadin, FEBS Lett. 486 (2000) 178–182. [64] L. Zhang, J. Kelley, G. Schmeisser, Y.M. Kobayashi, L.R. Jones, Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane, J. Biol. Chem. 272 (1997) 23389–23397. [65] L.R. Jones, L. Zhang, K. Sanborn, A.O. Jorgensen, J. Kelley, Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum, J. Biol. Chem. 270 (1995) 30787– 30796. [66] D.W. Shin, Z. Pan, E.K. Kim, J.M. Lee, M.B. Bhat, J. Parness, D.H. Kim, J. Ma, A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle, J. Biol. Chem. 278 (2002) 3286–3292. [67] L. Fliegel, K. Burns, D.H. MacLennan, R.A. Reithmeier, M. Michalak, Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum, J. Biol. Chem. 264 (1989) 21522–21528. [68] M. Michalak, E.F. Corbett, N. Mesaeli, K. Nakamura, M. Opas, Calreticulin: one protein, one gene, many functions, Biochem. J. 344 (1999) Pt 2281–Pt 2292. [69] S. Arnaudeau, M. Frieden, K. Nakamura, C. Castelbou, M. Michalak, N. Demaurex, Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria, J. Biol. Chem. 277 (2002) 46696–46705.

[70] H. Llewelyn Roderick, D.H. Llewellyn, A.K. Campbell, J.M. Kendall, Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells, Cell Calcium 24 (1998) 253– 262. [71] L. Mery, N. Mesaeli, M. Michalak, M. Opas, D.P. Lew, K.H. Krause, Overexpression of calreticulin increases intracellular Ca2+ storage and decreases store-operated Ca2+ influx, J. Biol. Chem. 271 (1996) 9332–9339. [72] C. Fasolato, P. Pizzo, T. Pozzan, Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1 cells, Mol. Biol. Cell 9 (1998) 1513–1522. [73] S. Jiang, S.C. Chow, P. Nicotera, S. Orrenius, Intracellular Ca2+ signals activate apoptosis in thymocytes: studies using the Ca2+ -ATPase inhibitor thapsigargin, Exp. Cell Res. 212 (1994) 84–92. [74] N. Zhu, Z. Wang, Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells, Cancer Res. 59 (1999) 1896– 1902. [75] H. Wei, W. Wei, D.E. Bredesen, D.C. Perry, Bcl-2 protects against apoptosis in neuronal cell line caused by thapsigargin-induced depletion of intracellular calcium stores, J. Neurochem. 70 (1998) 2305–2314. [76] Z. Pan, D. Damron, A.L. Nieminen, M.B. Bhat, J. Ma, Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor, J. Biol. Chem. 275 (2000) 19978–19984. [77] I.E. Wertz, V.M. Dixit, Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells, J. Biol. Chem. 275 (2000) 11470–11477. [78] X. Bian, F.M. Hughes, Y. Huang, J.A. Cidlowski, J.W. Putney Jr., Role of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells, Am. J. Physiol. 272 (1997) C1241–C1249. [79] S. Oyadomari, K. Takeda, M. Takiguchi, T. Gotoh, M. Matsumoto, I. Wada, S. Akira, E. Araki, M. Mori, Nitric oxide-induced apoptosis in pancreatic cells is mediated by the endoplasmic reticulum stress pathway, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 10845– 10850. [80] L.K. Nutt, A. Pataer, J. Pahler, B. Fang, J. Roth, D.J. McConkey, S.G. Swisher, Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores, J. Biol. Chem. 277 (2002) 9219–9225. [81] Z. Pan, M.B. Bhat, A.L. Nieminen, J. Ma, Synergistic movements of Ca2+ and Bax in cells undergoing apoptosis, J. Biol. Chem. 276 (2001) 32257–32263. [82] Y. Furuya, P. Lundmo, A.D. Short, D.L. Gill, J.T. Isaacs, The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin, Cancer Res. 54 (1994) 6167–6175. [83] F. Vanden Abeele, R. Skryma, Y. Shuba, F. Van Coppenolle, C. Slomianny, M. Roudbaraki, B. Mauroy, F. Wuytack, N. Prevarskaya, Bcl-2-dependent modulation of Ca2+ homeostasis and store-operated channels in prostate cancer cells, Cancer Cell. 1 (2002) 169– 179. [84] C. Kunzelmann-Marche, J.M. Freyssinet, M.C. Mart´ınez, Regulation of phosphatidylserine transbilayer redistribution by store-operated Ca2+ entry: role of actin cytoskeleton, J. Biol. Chem. 276 (2001) 5134–5139.