Biophysical characterization of the EF-hand and SAM domain containing Ca2+ sensory region of STIM1 and STIM2

Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER)-membrane associated Ca(2+) sensor which activates store-operated Ca(2+) entry (SOCE). The homologue, STIM2 possesses a high sequence identity to STIM1 ( approximately 61%), while its role in SOCE seems to be distinct from that of STIM1. In order to understand the underlying mechanism for the functional differences between STIM1 and STIM2, we investigated the biophysical properties of the luminal Ca(2+)-binding region which contains an EF-hand motif and a sterile alpha-motif (SAM) domain (hereafter called EF-SAM; residues 58-201 in STIM1 and 149-292 in STIM2). STIM2 EF-SAM has a low apparent Ca(2+)-binding affinity (K(d) approximately 0.5mM), which is similar to that reported for STIM1 EF-SAM. In the presence of Ca(2+), STIM2 EF-SAM is monomeric and well-folded, analogous to what was previously observed for STIM1 EF-SAM. In contrast to apo STIM1 EF-SAM, apo STIM2 EF-SAM is more structurally stable and does not readily aggregate. Our circular dichroism (CD) data demonstrate the existence of a long-lived, well-folded monomeric state for apo STIM2 EF-SAM, together with a less alpha-helical/partially unfolded aggregated state which is detectable only at higher protein concentrations and higher temperatures. Our biophysical studies reveal a structural stability difference in the EF-SAM region between STIM1 and STIM2, which may account for their different biological functions.     

STIM1 knockdown reveals that store-operated Ca2+ channels located close to sarco/endoplasmic Ca2+ ATPases (SERCA) pumps silently refill the endoplasmic reticulum

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.     

Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.     

Relocalization of STIM1 for activation of store-operated Ca(2+) entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca(2+) store

STIM1 (stromal interacting molecule 1), an endoplasmic reticulum (ER) protein that controls store-operated Ca(2+) entry (SOCE), redistributes into punctae at the cell periphery after store depletion. This redistribution is suggested to have a causal role in activation of SOCE. However, whether peripheral STIM1 punctae that are involved in regulation of SOCE are determined by depletion of peripheral or more internal ER has not yet been demonstrated. Here we show that Ca(2+) depletion in subplasma membrane ER is sufficient for peripheral redistribution of STIM1 and activation of SOCE. 1 microM thapsigargin (Tg) induced substantial depletion of intracellular Ca(2+) stores and rapidly activated SOCE. In comparison, 1 nM Tg induced slower, about 60-70% less Ca(2+) depletion but similar SOCE. SOCE was confirmed by measuring I(SOC) in addition to Ca(2+), Mn(2+), and Ba(2+) entry. Importantly, 1 nM Tg caused redistribution of STIM1 only in the ER-plasma membrane junction, whereas 1 microM Tg caused a relatively global relocalization of STIM1 in the cell. During the time taken for STIM1 relocalization and SOCE activation, 1 nM Bodipy-fluorescein Tg primarily labeled the subplasma membrane region, whereas 1 microM Tg labeled the entire cell. The localization of Tg in the subplasma membrane region was associated with depletion of ER in this region and activation of SOCE. Together, these data suggest that peripheral STIM1 relocalization that is causal in regulation of SOCE is determined by the status of [Ca(2+)] in the ER in close proximity to the plasma membrane. Thus, the mechanism involved in regulation of SOCE is contained within the ER-plasma membrane junctional region.     

STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ Entry

The coupling mechanism between endoplasmic reticulum (ER) Ca(2+) stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1-3]. STIM1 was shown to play a crucial role in this coupling process [4-7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca(2+) entry signals.     

Local cytosolic Ca2+ elevations are required for stromal interaction molecule 1 (STIM1) de-oligomerization and termination of store-operated Ca2+ entry

The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.     

STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels.

Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.     

The distinct role of STIM1 and STIM2 in the regulation of store-operated Ca2+ entry and cellular function

Stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum (ER) Ca²⁺ sensors that initiate store-operated Ca ²⁺ entry (SOCE). The roles of STIM1-mediated SOCE in cancer biology have been highlighted in different types of cancer, but that of STIM2 is unknown. By the model of cervical cancer, here we focus on the cooperative regulation of SOCE by STIM proteins and their distinct roles in cellular function. Immunofluorescent stainings of surgical specimens of cervical cancer show that STIM1 and STIM2 are abundant in tumor tissues, but STIM1 is the major ER Ca ²⁺ sensor identified in the invasive front of cancer tissues. STIM1 or STIM2 overexpression in cervical cancer SiHa cells induces an upregulated SOCE. Regarding cellular function, STIM1 and STIM2 are necessary for cell proliferation, whereas STIM1 is the dominant ER Ca ²⁺ sensor involved in cell migration. During SOCE, STIM1 is aggregated and translocated towards the Orai1-containing plasma membrane in association with the microtubule plus-end binding protein EB1. In contrast, STIM2 is constitutively aggregated without significant trafficking or association with microtubules. These results show the distinct role of STIM1 and STIM2 in SOCE and cellular function of cervical cancer cells.     

Morphological and functional aspects of STIM1-dependent assembly and disassembly of store-operated calcium entry complexes

The SOCE (store-operated Ca2+ entry) pathway is a central component of cell signalling that links the Ca2+-filling state of the ER (endoplasmic reticulum) to the activation of Ca2+-permeable channels at the PM (plasma membrane). SOCE channels maintain a high free Ca2+ concentration within the ER lumen required for the proper processing and folding of proteins, and fuel the long-term cellular Ca2+ signals that drive gene expression in immune cells. SOCE is initiated by the oligomerization on the membrane of the ER of STIMs (stromal interaction molecules) whose luminal EF-hand domain switches from globular to an extended conformation as soon as the free Ca2+ concentration within the ER lumen ([Ca2+]ER) decreases below basal levels of ~500 μM. The conformational changes induced by the unbinding of Ca2+ from the STIM1 luminal domain promote the formation of higher-order STIM1 oligomers that move towards the PM and exposes activating domains in STIM1 cytosolic tail that bind to Ca2+ channels of the Orai family at the PM and induce their activation. Both SOCE and STIM1 oligomerization are reversible events, but whether restoring normal [Ca2+]ER levels is sufficient to initiate the deoligomerization of STIM1 and to control the termination of SOCE is not known. The translocation of STIM1 towards the PM involves the formation of specialized compartments derived from the ER that we have characterized at the ultrastructural level and termed the pre-cortical ER, the cortical ER and the thin cortical ER. Pre-cortical ER structures are thin ER tubules enriched in STIM1 extending along microtubules and located deep inside cells. The cortical ER is located in the cell periphery in very close proximity (8-11?nm) to the plasma membrane. The thin cortical ER consists of thinner sections of the cortical ER enriched in STIM1 and devoid of chaperones that appear to be specialized ER compartments dedicated to Ca2+ signalling.     

Evolutionary origins of STIM1 and STIM2 within ancient Ca2+ signaling systems

Human stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca(2+) signaling systems that include numerous plasma membrane (PM), endoplasmic reticulum (ER), and mitochondrial Ca(2+) transporters, channels and regulators. STIM2 and STIM1 function as Ca(2+) sensors with different sensitivities for ER Ca(2+). They translocate to ER-PM junctions and open PM Orai Ca(2+) influx channels when receptor-mediated Ca(2+) release lowers ER Ca(2+) levels. The resulting increase in cytosolic Ca(2+) leads to the activation of numerous Ca(2+) effector proteins that in turn regulate differentiation, cell contraction, secretion and other cell functions. In this review, we use an evolutionary perspective to survey molecular activation mechanisms in the Ca(2+) signaling system, with a particular focus on regulatory motifs and functions of the two STIM proteins. We discuss the presence and absence of STIM genes in different species, the order of appearance of STIM versus Orai, and the evolutionary addition of new signaling domains to STIM proteins.     

cAMP induces stromal interaction molecule 1 (STIM1) puncta but neither Orai1 protein clustering nor store-operated Ca2+ entry (SOCE) in islet cells

The events leading to the activation of store-operated Ca(2+) entry (SOCE) involve Ca(2+) depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca(2+) sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane where it binds to the Ca(2+) channel protein Orai1 to activate Ca(2+) influx. Using confocal and total internal reflection fluorescence microscopy, we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing β-cells and glucagon-secreting α-cells within intact mouse and human pancreatic islets. ER Ca(2+) depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the plasma membrane and activated SOCE. Glucose, which promotes Ca(2+) store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in α- than in β-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in α-cells and retranslocation in β-cells involving raising and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and exchange protein directly activated by cAMP. However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1, and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.     

Store-operated Ca2+ entry: A STIMulating stOrai

Store-operated Ca2+ entry (SOCE) mediates much of the Ca2+ entry evoked by receptors that stimulate phospholipase C. However, for 20 years, the plasma membrane Ca2+ channel and the signal linking its activation to loss of Ca2+ from the endoplasmic reticulum (ER) have eluded detection. But the search might now be over. Two proteins, STIM1 (the ER Ca2+ sensor) and Orai1 (the Ca2+ channel), have recently been identified as the missing links in SOCE.     

Location and function of STIM1 in the activation of Ca2+ entry signals

Store-operated channels (SOCs) mediate Ca(2+) entry signals in response to endoplasmic reticulum (ER) Ca(2+) depletion in most cells. STIM1 senses decreased ER luminal Ca(2+) through its EF-hand Ca(2+)-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca(2+) binding. EF20 cells were viable despite constitutive Ca(2+) entry, allowing study of SOC activation without depleting ER Ca(2+). STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIM(EF)-expressing cells had normal ER Ca(2+) levels but substantially reduced ER Ca(2+) leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca(2+) leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca(2+) binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1(WT), is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIM(EF)-mediated Ca(2+) entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIM(WT). This suggests that PM-STIM1 may play a regulatory role in SOC activation.     

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.     

Increased Confinement and Polydispersity of STIM1 and Orai1 after Ca2+ Store Depletion

STIM1 (a Ca²⁺ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca²⁺-release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca²⁺ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca²⁺ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca²⁺ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca²⁺ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility—measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient—decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca²⁺ influx, which can facilitate the rapid activation of local Ca²⁺ signaling pathways and the efficient replenishing of Ca²⁺ store in the ER in store-operated Ca²⁺ entry.     

Mitofusin 2 regulates STIM1 migration from the Ca2+ store to the plasma membrane in cells with depolarized mitochondria

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.     

Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry

Stromal interaction molecule-1 (STIM1) activates store-operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. Here, we present the atomic structure of the Ca2+-sensing region of STIM1 consisting of the EF-hand and sterile alpha motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+-sensing region of STIM is crucial to SOCE regulation.     

Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

Ca²⁺ (calcium) homoeostasis and signalling rely on physical contacts between Ca²⁺ sensors in the ER (endoplasmic reticulum) and Ca²⁺ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca²⁺ sensors oligomerize upon Ca²⁺ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca²⁺ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca²⁺ have been studied but responses towards elevated cytosolic Ca²⁺ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P₂-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P₂, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca²⁺ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca²⁺ and PI(4,5)P₂ concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca²⁺ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.     

Voltage gating at the selectivity filter of the Ca2+ release-activated Ca2+ channel induced by mutation of the Orai1 protein

The Ca(2+) release-activated Ca(2+) (CRAC) channel is a plasma membrane (PM) channel that is uniquely activated when free Ca(2+) level in the endoplasmic reticulum (ER) is substantially reduced. Several small interfering RNA screens identified two membrane proteins, Orai1 and STIM1, to be essential for the CRAC channel function. STIM1 appears to function in the PM and as the Ca(2+) sensor in the ER. Orai1 is forming the pore of the CRAC channel. Despite the recent breakthroughs, a mechanistic understanding of the CRAC channel gating is still lacking. Here we reveal new insights on the structure-function relationship of STIM1 and Orai1. Our data suggest that the cytoplasmic coiled-coil region of STIM1 provides structural means for coupling of the ER membrane to the PM to activate the CRAC channel. We mutated two hydrophobic residues in this region to proline (L286P/L292P) to introduce a kink in the first alpha-helix of the coiled-coil domain. This STIM1 mutant caused a dramatic inhibition of the CRAC channel gating compared with the wild type. Structure-function analysis of the Orai1 protein revealed the presence of intrinsic voltage gating of the CRAC channel. A mutation of Orai1 (V102I) close to the selectivity filter modified CRAC channel voltage sensitivity. Expression of the Orai1(V102I) mutant resulted in slow voltage gating of the CRAC channel by negative potentials. The results revealed that the alteration of Val(102) develops voltage gating in the CRAC channel. Our data strongly suggest the presence of a novel voltage gating mechanism at the selectivity filter of the CRAC channel.     

Activation of the endoplasmic reticulum Ca2+ pump of pancreatic acini by Ca2+ mobilizing hormones

Stimulation of the pancreatic acinar cells with Ca2+ mobilizing hormones increased the ATP-dependent Ca2+ uptake into the ER of permeabilized cells. Activation of the ER Ca2+ pump resulted in increased apparent affinity for Ca2+ from 0.26 to 0.09 uM and Vmax from 2.68 to 5.74 nmoles/mg prot./min. The apparent affinity of the pump for VO4 = was dependent on [Ca2+]. Activation of the pump also decreased apparent affinity for VO4 = from 12 to 32 uM at [Ca2+] of 0.138 uM. These findings suggest that pump activation is due to acceleration of the rate of the conformational transition between the VO4 = (E2) and Ca2+ (E1) sensitive forms of the pump.