Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1

Abstract

Store-operated Ca 2؉ entry (SOCE) is activated by redistribution of STIM1 into puncta in discrete ER-plasma membrane junctional regions where it interacts with and activates store-operated channels (SOCs). The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. Here we report that caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland (HSG) cells decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of ⌬ERM-STIM1 or STIM1( 684 EE 685 ) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-( 639 KK 640 ) with STIM1( 684 EE 685 ) restored function and the dissociation of TRPC1 from Cav1 in response to store depletion. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1. S tore-operated calcium entry (SOCE) is activated by depletion of endoplasmic reticulum (ER) Ca 2ϩ stores and regulates a variety of critical cellular functions (1). Ca 2ϩ depletion in the ER lumen is detected by the Ca 2ϩ -binding protein STIM1, which oligomerizes into puncta and relocates to discrete ERplasma membrane (ER-PM) junctional regions (2, 3) where it associates with and activates store-operated channels including Orai1 and TRPC1, which are components of CRAC and SOC channels, respectively (4-13). Therefore, the location of these channels in the plasma membrane is likely to be critical for their interaction with peripheral STIM1 and activation. However, mechanisms involved in the precise targeting and retention of the channels at the domains where STIM1 puncta are located are not well-understood.Distinct regions of STIM1 determine aggregation and targeting of the protein to ER-PM junctional domains as well as its clustering with and gating of Orai1 and TRPC1 at these sites. The SAM and coiled-coiled domains are involved in STIM1 aggregation while the polybasic C-terminal region of STIM1 is suggested to target STIM1 to ER-PM junctional regions, which is the likely site for SOCE in native cells (3,(9)(10)(11)14). Thus, it can be predicted that SOCs are either localized in this...

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“…For adenoviral expression, SH-SY5Y cells were infected with Ad-TRPC1 at an MOI of 5. For transient transfection, SH-SY5Y cells (1.5 × 10 5 cells/ml) were transfected with TRPC1 siRNAs (Ambion) or STIM1 siRNA (58) or scrambled control siRNA (Ambion negative control siRNA #1) using HiPerFect transfection reagent (QIAGEN). Cells were passaged and transfected with siRNA every 3 days when the cells were 80%-90% confluent and in log growth phase.…”

“…Immunoprecipitations were carried out as described earlier (58,59). Following stimulation, cells were lysed with RIPA buffer and used for immunoprecipitation.…”

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