The Calcium Store Sensor, STIM1, Reciprocally Controls Orai and CaV1.2 Channels (original) (raw)
Related papers
Orai1 and STIM Reconstitute Store-operated Calcium Channel Function
Journal of Biological Chemistry, 2006
The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca 2؉ entry (SOCE) in human embryonic kidney 293 cells and the Ca 2؉ release-activated Ca 2؉ current (I CRAC ) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca 2؉ entry by up to 103-fold. This entry is entirely storedependent since the same coexpression causes no measurable store-independent Ca 2؉ entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (storeindependent) Ca 2؉ entry. STIM proteins are known to mediate Ca 2؉ store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca 2؉ entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.
Biochemical and functional properties of the store-operated Ca2+ channels
Cellular Signalling, 2009
Store-operated calcium entry (SOCE) is a major mechanism for Ca 2+ entry in excitable and non-excitable cells. The best-characterised store-operated current is I CRAC , but other currents activated by Ca 2+ store depletion have also been reported. The recent identification of the proteins stromal interaction molecule 1 (STIM1) and Orai1 has shed new light on the nature and regulation of SOC channels. STIM1 has been presented as the endoplasmic reticulum (ER) Ca 2+ sensor that communicates the content of the Ca 2+ stores to the storeoperated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and coclustering with the Ca 2+ channel subunit, Orai1. Interestingly, TRPC1, which has long been proposed as a SOC channel candidate, associates with Orai1 and STIM1 in a ternary complex that appears to increase the variability of SOC currents available to modulate cell function.
Role of STIM and Orai proteins in the store-operated calcium signaling pathway
Cell Calcium, 2007
Ca 2+ signals are universal among cells in regulating a spectrum of cellular responses. Phospholipase C-coupled receptors activate two components of Ca 2+ signals-rapid Ca 2+ release from ER stores, followed by slower Ca 2+ entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca 2+ entry process remained until recently a molecular mystery. The recent discovery of the necessity for STIM1 and Orai proteins in this process has provided crucial information on the coupling mechanism between stores and PM Ca 2+ entry. STIM1 is a single spanning membrane protein with an unpaired Ca 2+ binding EF-hand and appears to function as the sensor of ER luminal Ca 2+ , and, through redistribution in the ER, transduces information directly to the PM. Orai1 is a tetra-spanning PM protein and functions as the highly Ca 2+ -selective channel in the PM that is gated through interactions with the store-activated ER Ca 2+ sensor. Recent evidence shows the two proteins together are necessary and sufficient for the function of store-operated Ca 2+ entry. However, many questions arise about how and where the interactions of the STIM1 and Orai1 proteins occur within cells. Here we discuss recent information and ideas about the coupling between these proteins that leads to store-operated channel activation.
Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1
Journal of Biological Chemistry, 2009
With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca 2؉ influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D,D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K,D640K) that can be rescued only by the STIM1(K684E,K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1(K684E,K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity. Ca 2ϩ influx across the plasma membrane mediates many of the cellular functions of Ca 2ϩ , and several diseases, such as pancreatitis (1) and severe combined immune deficiency syndrome (2), are the result of aberrant Ca 2ϩ influx. In non-excitable cells, Ca 2ϩ influx is mediated by store-operated Ca 2ϩ channels (SOCs) 4 (3). The molecular identity of the SOCs and their regulation by Ca 2ϩ stored in the endoplasmic reticulum (ER) have been revealed over the last few years with the discoveries of STIM1 (4, 5) and the Orai channels (2, 6, 7). STIM1 is the ER Ca 2ϩ content sensor. It has an N-terminal EF hand, which resides in the ER lumen, as well as cytoplasmic C-terminal Ezrin-Radixin-Moesin (ERM), serine/proline, and lysine-rich domains (8, 9). Ca 2ϩ release from the ER results in the assembly of STIM1 into puncta at ER/plasma membrane interfaces. The STIM1 puncta subsequently interact with and activate the SOCs (9-11). The two Ca 2ϩ influx channel types that are regulated by STIM1 and function as SOCs are the TRPC (12-17) and Orai (2, 6, 7) channels. Deletion of TRPC1 and TRPC4 in mice (18, 19) and silencing of several TRPC channels (TRPCs) by siRNA (9, 20, 21) showed that TRPCs contribute to receptor-stimulated and store-mediated Ca 2ϩ influx in various cell types. More recently, we showed that all TRPCs, except TRPC7, are gated by STIM1 (13) and that this regulation requires the lysine-rich domain at the C terminus of STIM1 (12, 22). Furthermore, native STIM1 is sufficient to fully activate the ectopically expressed TRPCs (12, 13). The Orai family consists of Orai1, Orai2, and Orai3 (2, 6, 7). Orai1 mediates the CRAC current, and co-expression of the Orais with STIM1 is obligatory for the Orais to function as channels (23-26). In fact, expression of Orai1 alone results in inhibition of the native Ca 2ϩ influx (27, 28), possibly as a result of scavenging the native STIM1 (14). The expression and function of the Orais, TRPCs, and STIM1 in the same cells raise the question of the contribution of each channel to the native SOCs. Several recent studies suggest that Orai1 and TRPC channels may interact with and contribute to Ca 2ϩ influx by SOCs. In the human salivary gland cell line HSG, knockdown (KD) of Orai1 alone reduced SOCs-mediated Ca 2ϩ influx by more than 90%, and KD of TRPC1 alone reduced Ca 2ϩ influx by more than 50% (29). Co-expression of Orai1, TRPC1, and STIM1 in HEK cells resulted in a larger SOC-mediated Ca 2ϩ influx than expression of the individual channels with STIM1 (30). Accordingly, a complex of Orai1-TRPC1-STIM1 could be co-immunoprecipitated from HEK cells transfected with Orai1, STIM1, and TRPC1 (29). KD of Orai1 and expression of Orai1 channel mutants reduced the
A Novel Modulator of STIM2-Dependent Store-Operated Ca2+ Channel Activity
Acta Naturae
Store-operated Ca2+ entry is one of the main pathways of calcium influx into non-excitable cells, which entails the initiation of many intracellular processes. The endoplasmic reticulum Ca2+ sensors STIM1 and STIM2 are the key components of store-operated Ca2+ entry in mammalian cells. Under physiological conditions, STIM proteins are responsible for store-operated Ca2+ entry activation. The STIM1 and STIM2 proteins differ in their potency for activating different store-operated channels. At the moment, there are no selective modulators of the STIM protein activity. We screened a library of small molecules and found the 4-MPTC compound, which selectively inhibited STIM2-dependent store-operated Ca2+ entry (IC50 = 1 M) and had almost no effect on the STIM1-dependent activation of store-operated channels.
Journal of Biological Chemistry, 2008
Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca 2؉ selective I CRAC and relatively Ca 2؉ selective I SOC , respectively. STIM1 has been proposed to communicate the Ca 2؉ content of the intracellular Ca 2؉ stores to the plasma membrane storeoperated Ca 2؉ channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca 2؉ stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca 2؉. In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca 2؉ permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca 2؉ entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca 2؉ entry.
STIM1, an essential and conserved component of store-operated Ca2+ channel function
The Journal of Cell Biology, 2005
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conser...
Journal of Biological Chemistry, 2011
Activation of Ca 2؉ release-activated Ca 2؉ channels by depletion of intracellular Ca 2؉ stores involves physical interactions between the endoplasmic reticulum Ca 2؉ sensor, STIM1, and the channels composed of Orai subunits. Recent studies indicate that the Orai3 subtype, in addition to being store-operated, is also activated in a store-independent manner by 2-aminoethyldiphenyl borate (2-APB), a small molecule with complex pharmacology. However, it is unknown whether the store-dependent and -independent activation modes of Orai3 channels operate independently or whether there is cross-talk between these activation states. Here we report that in addition to causing direct activation, 2-APB also regulates storeoperated gating of Orai3 channels, causing potentiation at low doses and inhibition at high doses. Inhibition of store-operated gating by 2-APB was accompanied by the suppression of several modes of Orai3 channel regulation that depend on STIM1, suggesting that high doses of 2-APB interrupt STIM1-Orai3 coupling. Conversely, STIM1-bound Orai3 (and Orai1) channels resisted direct gating by high doses of 2-APB. The rate of direct 2-APB activation of Orai3 channels increased linearly with the degree of STIM1-Orai3 uncoupling, suggesting that 2-APB has to first disengage STIM1 before it can directly gate Orai3 channels. Collectively, our results indicate that the store-dependent and -independent modes of Ca 2؉ release-activated Ca 2؉ channel activation are mutually exclusive: channels bound to STIM1 resist 2-APB gating, whereas 2-APB antagonizes STIM1 gating.
STIM1 has a plasma membrane role in the activation of store-operated Ca2+ channels
Proceedings of the National Academy of Sciences, 2006
Receptor-induced Ca 2؉ signals are key to the function of all cells and involve release of Ca 2؉ from endoplasmic reticulum (ER) stores, triggering Ca 2؉ entry through plasma membrane (PM) ''storeoperated channels'' (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca 2؉ -binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca 2؉ sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca 2؉ sensor, STIM1 functions within the PM to control operation of the Ca 2؉ entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca 2؉ release-activated Ca 2؉ (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current (I CRAC) into a constitutively active, Ca 2؉ store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties.
Journal of Biological Chemistry, 2008
We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca 2؉-selective current with Ca 2؉-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca 2؉ release-activated Ca 2؉ (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca 2؉ influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca 2؉ entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.