A role for Orai in TRPC-mediated Ca2+ entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+ entry

Abstract

TRPC and Orai proteins have both been proposed to form Ca(2+)-selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca(2+) chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLCbeta signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W, inhibits Ca(2+) entry through both SOCE and ROCE channels, implicating Orai participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of IP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca(2+) entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Orai and TRPC proteins form complexes that participate in Ca(2+) entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE.

Fig. 1.

Enhancement of SOCE in HEK293 cells stably expressing TRPC1 (T1–8 cells). Cells were cotransfected in 60 mm dishes with empty pcDNA3 (Clontech) or 60 ng of pOrai1 (pcDNA3 carrying the cDNA of wild-type Orai1 under the control of the CMV promoter) and peYFP as described (17). After 24 h, cells were replated onto coverslips for an additional 24 h. SOCE was monitored in cells loaded with Fura2 by dual wavelength ratiometric fluorescence video microscopy as described (44, 17).

Fig. 2.

Effect of varying the input pOrai1 on SOCE in HEK293 cells stably expressing TRPC7 (T7–2 cells).

Fig. 3.

(A) Orai1[R91W] inhibits Ca²⁺ entry triggered by OAG (100 μg/ml) in HEK cells that stably express TRPC3 (clone T3H1), and (B) in HEK-293 cells that transiently express TRPC3. (A) T3H1 cells were transfected with 0.1 μg peYFP (Control, deep blue), 0.1 μg peYFP plus 60 ng of pOrai1[R91W] (R91W, bluish-green), or with 0.1 μg peYFP plus 60 ng pOrai1 (wild-type Orai1, red) and analyzed for their response to OAG. (B) HEK-293 cells were cotransfected with 1.0 μg pTRPC3 and 0.1 μg peYFP (TRPC3, deep blue) or with 1.0 μg pTRPC3, 60 ng pOrai1[R91W] and 0.1 μg peYFP (TRPC3 + W91R, blue-green) and analyzed for their response to OAG. Transfections were in 60 mm dishes as described under Methods. The OAG responses were tested 48 h after transfection. The OAG activation protocol was as described (17). Similar results were obtained with HEK-293 cells stably expressing TRPC6 (data not shown).

Fig. 4.

Wild-type (wt) Orai1 causes the appearance of Gd³⁺-resistant ROCE in HEK-293 cells and Orai1[R91W] inhibits ROCE both in the absence (Left) and the presence (Right) of 1 μM Gd³⁺. Note: no exogenous TRPC was present.

Fig. 5.

Induction of Gd³⁺-resistant ROCE by varying the amount of input pOrai1 shown in ng per 60 mm dish. All transfections also contained constant amounts of plasmids directing the expression of exogenous V1a vasopressin receptor and peYFP. At the concentrations tested the Gd-resistant ROCE was maximal at 50 ng pOrai1 (trace #5). Ten-fold higher amounts of plasmid were inhibitory (trace #6). Trace #1, (ROCE in the absence of Gd³⁺) was obtained from cells transfected only with peYFP and pV1aR. Note: that no exogenous TRPC was present.

Fig. 6.

Model of TRPC (purple):Orai (red) complexes operating as ROCE channels outside of lipid rafts and as SOCE (CRAC) channels within the confines of lipid rafts to which the complex is recruited by the multifunctional C terminus of activated STIM1 (blue; ref. 26). STIM1 is shown interacting by coiled-coiled domain interaction with the C terminus of Orai1 (yellow; ref. 27) and by ionic interaction of its C-terminal KK dipetide with the DD dipeptide of TRPC (light blue; ref. 31). The IP3 receptor (cyan) is shown in its IP3-activated form that confers to it the ability to interact with a region of TRPCs located in their C-termini (pink; ref. 34).

Fig. 7.

Model of the events triggered by phospholipase Cβ activated by a Gq/Gi-coupled GPCR (receptor signaling shown by red arrow). Nonactivated TRPC channels (1) are depicted as TRPC:Orai complexes based on our finding that spontaneous activities of TRPC3 channels (17) and TRPC6 channels (C. Erxleben and D.L. Armstrong, personal communication) are reduced by expression of low levels of Orai1. The receptor-activated TRPC (2) is shown in association with Orai based on the finding reported here that a dominant negative variant of Orai (Orai1[R91W]) inhibits ROCE as well as DAG activated Ca²⁺ entry. Activated STIM1 (3) is shown as a dimer with a dual TRPC and Orai interacting C terminus (dark green) to reflect multimerizaton and activation of its TRPC and Orai interacting ability induced by loss of Ca²⁺ caused by active store depletion (4) triggered by IP3 formed by PLC activity or passive store depletion without activation of PLC (light blue arrows). The activated CRAC channel (5) is shown in a lipid raft to which it is directed by activated STIM1 molecules. (Modified from figure 6 in ref. 19).