The TRPC (C-type transient receptor potential) class of ion channels has been hypothesized to participate in store-operated Ca 2؉ entry (SOCE). Recently, however, STIM1 and Orai1 proteins have been proposed to form SOCE channels. Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored. Here we show that Orai1 physically interacts with the N and C termini of TRPC3 and TRPC6, and that in cells overexpressing either TRPC3 or TRPC6 in a store-depletion insensitive manner, these TRPCs become sensitive to store depletion upon expression of an exogenous Orai. Thus, Orai-1, -2, and -3 enhanced thapsigargin-induced calcium entry by 50 -150% in cells stably overexpressing either TRPC3 or TRPC6. Orai1 expression had no significant effect on endogenous, thapsigargin-induced calcium entry in wildtype cells (HEK-293, COS1), in HEK cells expressing a thapsigarginsensitive variant of TRPC3 (TRPC3a), or in HEK cells overexpressing another membrane protein, V1aR. Single-channel cation currents present in membrane patches of TRPC3-overexpressing cells were suppressed by expression of Orai1. We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to these channels.capacitative calcium entry ͉ ion channels ͉ thapsigargin ͉ transmembrane signaling

Receptor-operated Ca 2؉ entry (ROCE) and store-operated Ca 2؉ entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Orai proteins. Another proposes SOCE channels to be composed of both Orai and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the Ca 2؉ stores that activates Ca 2؉ entry when stores are depleted. The role of Orai in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Orai proteins.capacitative calcium entry ͉ signal transduction ͉ store depletion R eported independently by three laboratories at the beginning of 2006, Orai (also known as CRACM) proteins, especially Orai1, have emerged as the molecular candidates for the underlying structure of store-operated Ca 2ϩ entry (SOCE) channels responsible for the store-depletion activated Ca 2ϩ current, Icrac, without apparent role(s) for transient receptor potential ion channels (TRPCs) (reviewed in refs. 1-3). Before the discovery of Orai genes and their gene products, the only candidates presumed to underlie SOCE had been the members of the TRPC class of ion channels, of which there are seven. This proposition was based on primary research reports from many laboratories showing that expression of the cloned TRPCs enhances store-depletion activated Ca 2ϩ entry (cf. table 1.3 in ref. 4) and/or that injection of specific anti-TRPC antibodies or genetic ablation of TRPC genes reduces SOCE and/or Icrac. Moreover, Zagranichnaya et al. (5) showed partial reduction of SOCE in response to siRNAs that targeted TRPC1, TRPC3, or TRPC7, which, when combined, were partially additive. The idea promoted in the reviews listed above that channels responsible for SOCE and Icrac form without involvement of TRPC proteins is especially difficult to reconcile with the total loss of Icrac in vascular endothelial cells seen in TRPC4 knock-out mice (6) and the 80% loss of SOCE found in submaxillary acinar cells of TRPC1 knockout mice (7).Based on the foregoing data, and recognizing that Orai/ CRACMs are part of SOCE/Icrac channels, we proposed that SOCE/Icrac channels may be heteromeric complexes of TRPC and Orai proteins (8). If this were the case, we reasoned, expression of Orai1 in cells expressing excess ...

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e ⩽ [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.

Calcium channels in the plasma membrane rarely remain open for much more than a millisecond at any one time, which avoids raising intracellular calcium to toxic levels. However, the dihydropyridinesensitive calcium channels of the CaV1 family, which selectively couple electrical excitation to endocrine secretion, cardiovascular contractility, and neuronal transcription, have a unique second mode of gating, ''mode 2,'' that involves frequent openings of much longer duration. Here we report that two human conditions, cyclosporin neurotoxicity and Timothy syndrome, increase mode 2 gating of the recombinant rabbit CaV1.2 channel. In each case, mode 2 gating depends on a Ser residue at the cytoplasmic end of the S6 helix in domain I (Ser-439, Timothy syndrome) or domain IV (Ser-1517, cyclosporin). Both Ser reside in consensus sequences for type II calmodulin-dependent protein kinase. Pharmacologically inhibiting type II calmodulin-dependent protein kinase or mutating the Ser residues to Ala prevents the increase in mode 2 gating. We propose that aberrant phosphorylation, or ''phosphorylopathy,'' of the CaV1.2 channel protein contributes to the excitotoxicity associated with Timothy syndrome and with chronic cyclosporin treatment of transplant patients.calcium͞calmodulin-dependent protein kinase type II ͉ calcineurin ͉ dihydropyridine ͉ excitotoxicity