Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

以Ca-Ca距离为基础的构象空间的可视化研究

蛋白质结构构象呈现明显的规律,研究其在特定构象空间的分布对蛋白质结构预测和模拟具有重要意义。本文以449个非冗余的高分辨串蛋白质结构为材料,以Ga-Ga距离向量代表蛋白质片段。然后利用主成分分析方法,建立蛋白质片段构象空间的可视化构图,并且单个蛋白质分子可以映射到该空间形成一个顺序连接的路径。从而,可以很直观的分析各长度片段（4-9个残基的片段）的分布情况及其内在的连接关系。图形显示了明显的聚集性,以及各种类型片段与二级结构明显的对应关系。     

The Influence of Ca Buffers on Free [Ca] Fluctuations and the Effective Volume of Ca Microdomains

Intracellular calcium (Ca²⁺) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca²⁺ binding proteins (Ca²⁺ buffers) play an important role in regulating Ca²⁺ concentration ([Ca²⁺]). Buffers typically slow [Ca²⁺] temporal dynamics and increase the effective volume of Ca²⁺ domains. Because fluctuations in [Ca²⁺] decrease in proportion to the square-root of a domain’s physical volume, one might conjecture that buffers decrease [Ca²⁺] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca²⁺ signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca²⁺ influx with passive exchange of both Ca²⁺ and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca²⁺ and buffer and use these stochastic differential equations to determine the magnitude of [Ca²⁺] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca²⁺ signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca²⁺] fluctuations during periods of Ca²⁺ influx, whereas stationary (immobile) Ca²⁺ buffers do not. Also contrary to expectations, we find that in the absence of Ca²⁺ influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca²⁺] fluctuations. We derive an analytical formula describing the influence of rapid Ca²⁺ buffers on [Ca²⁺] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca²⁺ buffers alter the dynamics of [Ca²⁺] fluctuations in a nonintuitive manner. The finding that Ca²⁺ buffers do not suppress intrinsic domain [Ca²⁺] fluctuations raises the intriguing question of whether or not [Ca²⁺] fluctuations are a physiologically significant aspect of local Ca²⁺ signaling.     

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.     

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Low-Affinity Ca Indicators Compared in Measurements of Skeletal Muscle Ca Transients

The low-affinity fluorescent Ca²⁺ indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca²⁺ transient (Δ[Ca²⁺]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly track Δ[Ca²⁺]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (ΔF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with furaptra; thus, these indicators do not track Δ[Ca²⁺] with kinetic fidelity. In contrast, the ΔF time course of Mag-fluo-4 was identical to furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Δ[Ca²⁺]. Mag-fluo-4's ΔF has a larger signal/noise ratio than furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Δ[Ca²⁺] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg²⁺, the amplitude of the Mag-fluo-4 signal, and its calibration in Δ[Ca²⁺] units, is likely to be more sensitive to variations in [Mg²⁺] than furaptra's.     

Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations

K-stimulated (voltage-dependent) influx of 45Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain. Influx was terminated at 1 s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D. A., and Blaustein, M. P., 1980, J. Gen. Physiol., 76:709- 728). This influx was blocked by multivalent cations with half- inhibition constants (K1) that clustered in three distinct groups: (a) K1 greater than 1 mM (Mg2+, Sr2+, and Ba2+); (b) K1 = 30-100 microM (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Hg2+); (c) K1 less than 1 micro M (Cd2+, Y3+, La3+ and the trivalent lanthanides, and Pb2+). Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization. The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s; inhibition was similar after 1 s or 30 min of exposure to these ions. The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation. The relative efficacies of the lanthanides as fast channel blockers were compared; there was a decrease in inhibitory potency with decreasing ionic radius. A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations. The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them. Small cations are unable to bind effectively to both anions. The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel.     

Effects of light quality on chlorophyll-forms Ca 684, Ca 690 and Ca 699 of the diatom Phaeodactylum tricornutum

Colored light modifies the relative concentration of chlorophyll-forms of the diatom Phaeodactylum tricornutum compared to white-light control. No change in the ratio carotenoids/chlorophylls was observed after 4 days exposure to green light (max: 530 nm), blue light (max: 470 nm) or red light ( > 650 nm) of same intensity.However, the absorption spectra were modified, the content in Ca 684, Ca 690, Ca 699 forms increased in red and green light cultures and photosynthetic unit size of PS II decreased by 30% in green and blue light cultures.Fluorescence emission and fluorescence excitation spectra according to the Butler and Kitajima method (1975) were carried out for each culture. Ca 669 form was predominant in the two photosystems. The newly appeared far red forms fluoresce at 715 nm like PS I forms.We conclude that these new forms originated in a rearrangement of PS II forms. They do not transmit excitation energy to reaction center of PS I and are disconnected from the other chlorophyll-forms of the photosynthetic antennae.Abbreviations ABS absorption - Ca chlorophyll-complex - chla chlorophyll a - chl c chlorophyll c - chl t total chlorophylls - D.C.M.U. 3-(3, 4 dichlorophenyl) 1-diméthyl-urea - d^v division - F fluorescence - PS I and PS II photosystem I and photosystem II     

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor

Thecoupling mechanism between depletion of Ca²⁺ stores in theendoplasmic reticulum and plasma membrane store-operated ion channelsis fundamental to Ca²⁺ signaling in many cell types and hasyet to be completely elucidated. Using Ca²⁺release-activated Ca²⁺ (CRAC) channels in RBL-2H3 cells asa model system, we have shown that CRAC channels are maintained in theclosed state by an inhibitory factor rather than being opened by theinositol 1,4,5-trisphosphate receptor. This inhibitory role can befulfilled by the Drosophila protein INAD (inactivation-noafter potential D). The action of INAD requires Ca²⁺ andcan be reversed by a diffusible Ca²⁺ influx factor. Thusthe coupling between the depletion of Ca²⁺ stores and theactivation of CRAC channels may involve a mammalian homologue of INADand a low-molecular-weight, diffusible store-depletion signal.

     

Regulation of endogenous and heterologous Ca release-activated Ca currents by pH

Deviations from physiological pH (∼pH 7.2) as well as altered Ca²⁺ signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca²⁺ influx is the store-operated Ca²⁺ entry (SOCE) or Ca²⁺ release-activated Ca²⁺ current (I_CRAC), which is activated upon depletion of intracellular Ca²⁺ stores. We here show that extracellular and intracellular changes in pH regulate both endogenous I_CRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous I_CRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP₃-induced I_CRAC. In the absence of IP₃, external alkalization did not elicit endogenous I_CRAC but was able to activate heterologous I_CRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP₃-induced activation of endogenous and heterologous I_CRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of I_CRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates I_CRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate I_CRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Subpopulation of store-operated Ca2+ channels regulate Ca2+-induced Ca2+ release in non-excitable cells

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.     

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.