TRPC Channels Function Independently Of STIM1 And Orai1

Abstract

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor, and calcium channel, respectively, for CRAC channels, channels underlying store-operated Ca2+ entry (SOCE). However, the roles of these proteins in signaling and constructing other channels with biophysical properties distinct from CRAC channels are not known. We examined the hypothesis that STIM1 or Orai1 can interact with and regulate a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signaling of TRPC channels. Specifically, Ca2+ entry seen after carbachol treatment in cells expressing TRPC1, 3, 5, or 6 were not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to published reports. Disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of ICRAC. This suggests that TRPC signaling and STIM1/Orai1 signaling occur in distinct plasma membrane domains. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents, and single channel events recorded in cell-attached configuration from these cells detected a current with a slope conductance of 33.65 pS, similar to that published for TRPC6. Further, RT-PCR analysis of TRPC transcripts in A10 cells revealed the predominant expression of TRPC1 and TRPC6 mRNA. Using a membrane potential-sensitive dye as an assay, we determined that knockdown of either STIM1 or Orai1 had no effect on the function of this AVP-activated current, while store-operated entry was substantially reduced. Thus, both STIM1 and Orai1 appear to be specific molecular components of the ICRAC pathway and in our studies did not influence the function of exogenously or endogenously expressed TRPC channels.