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.

CRACM1 (Orai1) constitutes the pore subunit of CRAC channels that are crucial for many physiological processes 1-6 . A point mutation in CRACM1 has been associated with SCID disease in humans 2 . We have generated CRACM1 deficient mice using gene trap, where β-galactosidase (LacZ) activity identifies CRACM1 expression in tissues. We show here that the homozygous CRACM1 deficient mice are considerably smaller in size and are grossly defective in mast cell degranulation and cytokine secretion. FcεRI-mediated in vivo allergic reactions were also inhibited in CRACM1-/-mice. Other tissues expressing truncated CRACM1-LacZ fusion protein include skeletal muscles, kidney and regions in the brain and heart. Surprisingly, no CRACM1 expression was seen in the lymphoid regions of thymus. Accordingly, we found no defect in T cell development. Thus, our data reveal novel crucial roles for CRAC channels including a putative role in excitable cells.

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...