The molecular nature of store-operated Ca 2؉ -selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca 2؉ -selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca 2؉ entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca 2؉ entry and Ca 2؉ -selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca 2؉ -selective store-operated Ca 2؉ entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca 2؉ entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca 2؉ sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.

Store-operated Ca2؉ entry (SOCE) is likely the most common mode of regulated influx of Ca 2؉ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca 2؉ current (I CRAC ). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca 2؉ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I CRAC -like currents in cells coexpressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than ؉50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca 2؉ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.In many cell types, the activation of phospholipase C through G protein-coupled receptors liberates Ca 2ϩ from the lumen of the endoplasmic reticulum (ER current (I CRAC ), first described in mast cells (3) and since recorded in several cell types (4). Until recently, the mechanism by which I CRAC is activated by store depletion, as well as the channels themselves, was unknown. However, the discoveries of both STIM1 (5, 6) and Orai1 (CRACM1) (7-9) have revealed two key molecular components of the I CRAC -signaling pathway.It is now clear that STIM1 functions as the Ca 2ϩ sensor within the ER, whereas members of the family of Orai proteins (including Orai1, -2, and -3) function as pore-forming subunits of CRAC channels in the plasma membrane. When intracellular Ca 2ϩ stores are depleted, STIM1 rearranges from a fibrillar localization that depends on microtubules to discrete punctate structures near the plasma membrane (6, 10 -12). Remarkably, Orai1 channels also rearrange into punctate structures, in response to store depletion, that coincide with those formed by STIM1 (13-15). Thus, highly orchestrated molecular rearrangements underlie I CRAC activation.Overexpression of Orai1 together with STIM1 in HEK293 cells produces unusually large currents with biophysical properties similar to I CRAC (9, 16 -18), suggesting that either these two proteins are sufficien...

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca 2؉ sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca 2؉ for channels composed primarily of Orai1, Orai2, and Orai3, by coexpressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I crac ), but Orai3 failed to produce any detectable Ca 2؉ -selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca 2؉ and Ba 2؉ or Mg 2؉ solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na ؉ currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na ؉ currents in HEK293 cells, were all inhibited by extracellular Ca 2؉ with a half-maximal concentration of ϳ20 M. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca 2؉ , indicating similar affinities for Ca 2؉ within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca 2؉ depotentiation.Store-operated or capacitative calcium entry channels subtend or support important Ca 2ϩ signaling pathways in a wide variety of cell types (1). The best characterized store-operated mechanism involves a current termed calcium release-activated calcium (CRAC) 2 current or I crac . The recent discoveries of Stim1 as an endoplasmic reticulum Ca 2ϩ sensor (2, 3) and Orai1 as a pore-forming subunit of the T-cell CRAC channel (4 -6) provide powerful tools for understanding the mechanisms of activation and regulation of these channels, as well as their specific roles in various physiological pathways. In addition to Orai1, two homologs, Orai2 and -3, have been shown to be capable of forming store-operated channels (7). Knowledge of the properties of these three Orai forms is necessary to understand the specific functions of the subunits and to implicate specific molecular components of native store-operated channels. In this study, we focused on the well characterized modulatory effects of extracellular Ca 2ϩ on CRAC channels (8, 9). Extracellular Ca 2ϩ has two effects on CRAC channels. It reduces conductance, or blocks the channels, by binding to a selectivity filter in the channel pore (10), but it also augments channel function by binding to an undefined extracellular site, a process known as potentiation (11). The latter role of extracellular Ca 2ϩ can be revealed by protocols in which extracellular divalent ...

The process of store-operated Ca2+ entry (SOCE), whereby Ca2+ influx across the plasma membrane is activated in response to depletion of intracellular Ca2+ stores in the endoplasmic reticulum (ER), has been under investigation for greater than 25 years; however, only in the past 5 years have we come to understand this mechanism at the molecular level. A surge of recent experimentation indicates that STIM molecules function as Ca2+ sensors within the ER that, upon Ca2+ store depletion, rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels of the Orai family. The molecular and biophysical data that have led to these findings are discussed in this review, as are several controversies within this rapidly expanding field.

2+-release-activated Ca 2+ (CRAC) current. Reversal by ML-9 resulted in full re-establishment of the tubular EYFP-Stim1 localization. A constitutively active EFhand mutant of EYFP-Stim1 was also reversed by ML-9, regardless of the Ca 2+ store content. Inhibition by ML-9 was not due to MLCK inhibition as other inhibitors of MLCK had no effect. Finally, we provide evidence that EYFP-Stim1 punctae form in specific predetermined cellular loci. We conclude that SOCE is tightly coupled to formation of Stim1 puncta, and both SOCE and puncta formation involve a dynamic, reversible signaling complex that probably consists of components in addition to Stim1 and Orai channels. Supplementary material available online at

Activation of surface membrane receptors coupled to phospholipase C results in the generation of cytoplasmic Ca2+ signals comprised of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. A primary mechanism for this Ca2+ entry process is attributed to store-operated Ca2+ entry, a process that is activated by depletion of Ca2+ ions from an intracellular store by inositol 1,4,5-trisphosphate. Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques. Pharmacological manipulation of this Ca2+ signaling process has been somewhat limited; but recent identification of key molecular players, STIM and Orai family proteins, has provided new approaches. Here we describe practical methods involving fluorescent Ca2+ indicators and electrophysiological approaches for dissecting the observed intracellular Ca2+ signal to reveal characteristics of store-operated Ca2+ entry, highlighting the advantages, and limitations, of these approaches.

Depletion of intracellular Ca2+ stores induces Ca2+ influx across the plasma membrane through store-operated channels (SOCs). This store-operated Ca2+ influx is important for the replenishment of the Ca2+ stores, and is also involved in many signaling processes by virtue of the ability of intracellular Ca2+ to act as a second messenger. For many years, the molecular identities of particular SOCs, as well as the signaling mechanisms by which these channels are activated, have been elusive. Recently, however, the mammalian proteins STIM1 and Orai1 were shown to be necessary for the activation of store-operated Ca2+ entry in a variety of mammalian cells. Here we present molecular, pharmacological, and electrophysiological properties of SOCs, with particular focus on the roles that STIM1 and Orai1 may play in the signaling processes that regulate various pathways of store-operated entry.