STIM1 Activation Research Articles

The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function

Ca(2+) influx by store-operated Ca(2+) channels (SOCs) mediates all Ca(2+)-dependent cell functions, but excess Ca(2+) influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca(2+) sensor STIM1. Slow Ca(2+)-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448-530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448-490) and STIM1(490-530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448-490) lobe restricted, whereas the STIM1(490-530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.

TRPC1 and Orai1 interact with STIM1 and Mediate Capacitative Calcium Entry Activated by Acute Hypoxia in Mouse Pulmonary Arterial Smooth Muscle Cells

The present study aimed to determine if TRPC1 and Orai1 interact with STIM1 and mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura‐2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). Acute hypoxia also increased the rate of Mn2+ quench of fura‐2 fluorescence that was inhibited by SKF 96365, Ni2+, La3+ and Gd3, exhibiting pharmacological properties characteristic of CCE. The nifedipine‐insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells treated with TRPC1 antibody, in cells transfected with STIM1 siRNA and cells transfected with Orai1 siRNA. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca2+]i and Mn2+ quench rate caused by acute hypoxia, and they were reduced in cells treated with TRPC1 antibody and cells treated with Orai1 siRNA. Furthermore, TRPC1 and Orai1 co‐immunoprecipitate with STIM1 and the precipitation levels of TRPC1 and Orai1 were increased in cells exposed to acute hypoxia. Immunostaining showed co‐localizations of TRPC1‐STIM1 and Orai1‐STIM1, and the co‐localizations of these proteins were more apparent in acute hypoxia. These data provide direct evidence that TRPC1 and Orai1 channels mediate CCE through activation of STIM1 in mouse PASMCs in acute hypoxia. [Supported by HL49254 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

Mitochondrial dynamics and their impact on T cell function

Energy supply is the most prominent function of mitochondria, but in addition, mitochondria are indispensable for a multitude of other important cellular functions including calcium (Ca2+) signaling and buffering, the supply of metabolites and the sequestration of apoptotic factors. The efficiency of those functions highly depends on the proper positioning of mitochondria within the cytosol. In lymphocytes, mitochondria preferentially localize into the vicinity (∼200nm) of the immune synapse (IS). This localization is regulated by motor-based cytoskeleton-mediated transport, the fusion/fission dynamics of mitochondria, and probably also through tethering with the ER. IS formation also induces the accumulation of CRAC/ORAI1 Ca2+ channels, the CRAC/ORAI channel activator STIM1, K+ channels and plasma membrane Ca2+ ATPase (PMCA) within the IS. Such a large agglomeration of Ca2+ binding organelles and proteins highlights the IS as a critical cellular compartment for Ca2+ dependent lymphocyte activation. At the IS, Ca2+ microdomains generated beneath open CRAC/ORAI channels provide a rapid, robust and reliable mechanism for driving cellular responses in mast cells and T cells. Here, we discuss the relevance of motor-based mitochondrial transport, fusion, fission and tethering for mitochondrial localization in T cells and the importance of subplasmalemmal mitochondria to control local CRAC/ORAI1-dependent Ca2+ microdomains at the IS for efficient T lymphocyte activation.

STIM1/Orai1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai1/STIM1 (stromal interaction molecule 1). In addition, we investigated whether ovariectomy in females affects the activation of the Orai1/STIM1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar-Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM1 and Orai1, as shown by the blockade of STIM1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM1 and Orai1. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM1 expression. These findings suggest that augmented activation of STIM1/Orai1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai1 pathway, contributing to vascular protection observed in female rats.

Dendritic EGFP‐STIM1 activation after type I metabotropic glutamate and muscarinic acetylcholine receptor stimulation in hippocampal neuron

Several signaling pathways in neurons engage the endoplasmic reticulum (ER) calcium store by triggering calcium release. After release, ER calcium levels must be restored. In many non-neuronal cell types, this is mediated by store-operated calcium entry (SOCE), a cellular homeostatic mechanism that activates specialized store-operated calcium channels (SOC). Although much evidence supports the existence of SOCE in neurons, its importance has been difficult to determine because of the abundance of calcium channels expressed and the lack of SOC-specific pharmacological agents. We have explored the function of the SOCE-inducing protein STIM1 in neurons. In EGFP-STIM1-expressing hippocampal neurons, the sarco- and endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin caused rapid aggregation (i.e., activation) of STIM1 in soma and dendrites. Upon STIM1 activation by thapsigargin, a dramatic reduction in STIM1 mobility was detected by fluorescence recovery after photobleaching (FRAP). By triggering release of ER calcium with 3,5-dihydroxyphenylglycine (DHPG) or carbachol (Cch), agonists of type I metabotropic glutamate receptors (mGluR) and muscarinic acetylcholine receptors (mAChR), respectively, STIM1 was activated, and calcium entry (likely to represent SOCE) occurred in dendrites. It is therefore possible that neuronal SOCE is activated by physiological stimuli, some of which may alter the postsynaptic calcium signaling properties.

Activation of STIM1‐Orai1 involves an intramolecular switching mechanism

The cytosolic level of Ca2+ has to be under tight control since Ca2+ is a potent regulator of many pivotal cellular functions. Store operated calcium entry (SOCE) is a major calcium entry mechanism present in virtually all cell types. Its activity is controlled by the luminal Ca2+ concentration of the ER. The stromal interaction molecule (STIM1) located in the ER, which is the Ca2+ sensor, and the highly Ca2+ selective Orai1 channels in the plasma membrane are the two main component of this machinery. Decreased ER luminal Ca2+ concentrations trigger oligomerization and clustering of STIM1, which, in turn, becomes capable of activating Orai1 channels. The Orai1 activating region within the STIM1 cytoplasmic part has been recently identified (CAD/SOAR) Here, we identified an acidic motif in STIM1 that keeps CAD/SOAR domain in an inactive state. Intriguingly, the sequence of the STIM1 acidic motif shows substantial similarity to that of the C‐terminal coiled‐coil segment of Orai1, which is the postulated site of interaction with STIM1. Mutations within this acidic region make STIM1 constitutively active presumably by preventing the intramolecular interaction with a short basic segment within the CAD/SOAR domain that is also essential for Orai1 activation. We propose that the CAD/SOAR domain is released from an intramolecular clamp during STIM1 activation, allowing activation of Orai1 channels.

Role of the STIM1 C-terminal Domain in STIM1 Clustering

Store-operated Ca(2+) entry (SOCE) represents a ubiquitous Ca(2+) influx pathway activated by the filling state of intracellular Ca(2+) stores. SOCE is mediated by coupling of STIM1, the endoplasmic reticulum Ca(2+) sensor, to the Orai1 channel. SOCE inactivates during meiosis, partly because of the inability of STIM1 to cluster in response to store depletion. STIM1 has several functional domains, including the Orai1 interaction domain (STIM1 Orai Activating Region (SOAR) or CRAC Activation Domain (CAD)) and STIM1 homomerization domain. When Ca(2+) stores are full, these domains are inactive to prevent constitutive Ca(2+) entry. Here we show, using the Xenopus oocyte as an expression system, that the C-terminal 200 residues of STIM1 are important to maintain STIM1 in an inactive state when Ca(2+) stores are full, through predicted intramolecular shielding of the active STIM1 domains (SOAR/CAD and STIM1 homomerization domain). Interestingly, our data argue that the C-terminal 200 residues accomplish this through a steric hindrance mechanism because they can be substituted by GFP or mCherry while maintaining all aspects of STIM1 function. We further show that STIM1 clustering inhibition during meiosis is independent of the C-terminal 200 residues.

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced store-operated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1(high)/STIM2(low) tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women.

Orai1 and STIM1 Mediate Capacitative Ca2+ Entry in Mouse Pulmonary Arterial Smooth Muscle Cells

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that TRPC1 and STIM1 mediates the sustained component of capacitative Ca2+ entry (CCE) but the molecular candidate(s) that mediate the transient component of CCE remains unknown. The aim of the present study was to further examine if Orai1 mediates the transient component of CCE through activation of STIM1 protein in mouse PASMCs.In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i).The transient but not the sustained rise in [Ca2+]i was partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 siRNA. Moreover, over-expression of STIM1 enhanced the rise in [Ca2+]i and the increase in Mn2+ quench of fura-2 fluorescence caused by CPA and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to co-immunoprecipitate with STIM1 and immunostaining showed co-localization of Orai1 and STIM1 proteins. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

STIM1 Is a Calcium Sensor Specialized for Digital Signaling

When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry, which is initiated by calcium sensor proteins, STIM1 and STIM2, in the endoplasmic reticulum [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, whereas a threshold of calcium depletion is required for STIM1 activation [4]. Here we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium and that this drop is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

TRPC1 and STIM1 mediate capacitative Ca2+ entry in mouse pulmonary arterial smooth muscle cells

Previous studies in pulmonary arterial smooth muscle cells (PASMCs) showed that the TRPC1 channel mediates capacitative Ca(2+) entry (CCE), but the molecular signal(s) that activate TRPC1 in PASMCs remains unknown. The aim of the present study was to determine if TRPC1 mediates CCE through activation of STIM1 protein in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca(2+) concentration ([Ca(2+)](i)). The transient but not the sustained rise in [Ca(2+)](i) was partially inhibited by nifedipine. In addition, CPA increased the rate of Mn(2+) quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni(2+), La(3+) and Gd(3+), exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive sustained rise in [Ca(2+)](i) and the increase in Mn(2+) quench of fura-2 fluorescence caused by CPA were both inhibited in cells pretreated with antibody raised against an extracellular epitope of TRPC1. Moreover, STIM1 siRNA reduced the rise in [Ca(2+)](i) and Mn(2+) quench of fura-2 fluorescence caused by CPA, whereas overexpression of STIM1 resulted in a marked increase in these responses. RT-PCR revealed TRPC1 and STIM1 mRNAs, and Western blot analysis identified TRPC1 and STIM1 proteins in mouse PASMCs. Furthermore, TRPC1 was found to co-immunoprecipitate with STIM1, and the precipitation level of TRPC1 was increased in cells subjected to store depletion. Taken together, store depletion causes activation of voltage-operated Ca(2+) entry and CCE. These data provide direct evidence that CCE is mediated by TRPC1 channel through activation of STIM1 in mouse PASMCs.

TRPC1 Mediates Capacitative Calcium Entry through Activation of STIM1 in Mouse Pulmonary Arterial Smooth Muscle Cells

The present study aimed to determine if TRPC1 and STIM1 mediate capacitative Ca2+ entry (CCE) in mouse pulmonary arterial smooth muscle cells (PASMCs). In primary cultured PASMCs loaded with fura‐2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). The transient but not sustained rise in [Ca2+]i was partially inhibited by nifedipine but they were both inhibited by SKF 96365, Ni2+, La3+ and Gd3+. In addition, CPA increased the rate of Mn2+ quench of fura‐2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. The nifedipine‐insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells pretreated with antibodies raised against extracellular epitopes of TRPC1 and STIM1. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca2+]i and Mn2+ quench rate caused by CPA, and they were reduced by TRPC1 antibody. RT‐PCR and Western blot analysis revealed TRPC1 and STIM1 mRNAs and proteins. Furthermore, TRPC1 was found to co‐immunoprecipitate with STIM1. Taken together, store‐depletion causes activation of voltage‐operated Ca2+ entry and CCE. These data provide direct evidence that TRPC1 channel mediates CCE through activation of STIM1 in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats

Disturbances in the regulation of cytosolic calcium (Ca 2+ ) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca 2+ entry. Although STIMs act as Ca 2+ sensors for the intracellular Ca 2+ stores, Orai is the putative pore-forming component of Ca 2+ release–activated Ca 2+ channels at the plasma membrane. We hypothesized that augmented activation of Ca 2+ release–activated Ca 2+ /Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca 2+ influx. Depletion of intracellular Ca 2+ stores, which induces Ca 2+ release–activated Ca 2+ activation, was performed by placing arteries in Ca 2+ free-EGTA buffer. The addition of the Ca 2+ regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 μmol/L), an inhibitor of the sarcoplasmic reticulum Ca 2+ ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca 2+ release–activated Ca 2+ channel inhibitors 2-aminoethoxydiphenyl borate (100 μmol/L) or gadolinium (100 μmol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca 2+ levels in hypertension.

Functional Stoichiometry Of The Unitary Calcium-release-activated Calcium Channel Revealed By Single-molecule Imaging

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca2+ releaseactivated Ca2+ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca2+ stores were depleted, the channels clustered in aggregates containing ∼1,300 channels, amplifying the local Ca2+ entry.

A novel Stim1-dependent, non-capacitative Ca2+ entry pathway is activated by B cell receptor stimulation and depletion of Ca2+ stores

In most non-excitable cells, the depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE), which is a Ca(2+)-selective and La(3+)-sensitive entry pathway. Here, we report a novel mechanism of La(3+)-resistant Ca(2+) entry that is synergistically regulated by B cell receptor (BCR) stimulation and Ca(2+) store depletion (B-SOC). In the wild-type (WT) DT40 cells, BCR stimulation with anti-IgM antibodies induced Ca(2+) release and subsequent Ca(2+) entry in the presence of 0.3 microM La(3+) which blocks CCE completely. In the inositol 1,4,5-trisphosphate receptor-deficient (IP(3)R-KO) cells, BCR stimulation elicited neither Ca(2+) release nor Ca(2+) entry. However, under pretreatment of thapsigargin (ThG), BCR stimulation induced La(3+)-resistant Ca(2+) entry into both WT and IP(3)R-KO cells. These results indicate that BCR stimulation and Ca(2+) store depletion work in concert to activate the La(3+)-resistant Ca(2+) entry pathway. B-SOC was inhibited by tyrosine kinase inhibitor, genistein. In addition, B-SOC was completely abolished in Stim1-deficient cells and was restored by overexpression of yellow fluorescent protein (YFP)-tagged Stim1, but was unaffected by double knockdown of Orai1/Orai2. These results demonstrate a unique non-CCE pathway, in which Ca(2+) entry depends on Stim1 and tyrosine kinase activation. It is likely that similar regulation of Ca(2+) entry occurs in other cell types including salivary gland cells.

Functional stoichiometry of the unitary calcium-release-activated calcium channel

Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca(2+) stores were depleted, the channels clustered in aggregates containing approximately 1,300 channels, amplifying the local Ca(2+) entry.

STIM1 carboxyl-terminus activates native SOC, Icrac and TRPC1 channels

Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.