TRPC1 is required for functional store-operated Ca2+ channels -: Role of acidic amino acid residues in the S5-S6 region

The exact role of TRPC1 in store-operated calcium influx channel (SOCC) function is not known. We have examined the effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557-620) was deleted or modified by site-directed mutagenesis on thapsigargin- and carbachol-stimulated SOCC activity in HSG. cells. TRPC1 overexpression induced channel activity that was indistinguishable from the endogenous SOCC activity. Transfection with antisense hTRPC1 decreased SOCC activity although characteristics of SOCC-mediated current, I-SOC, were not altered. Expression of TRPC1Delta567-793, but not TRPC1Delta664-793, induced a similar decrease in SOCC activity. Furthermore, TRPC1Delta567-793 was coimmunoprecipitated with endogenous TRPC1. Simultaneous substitutions of seven acidic as in the S5-S6 region (Asp --> Asn and Glu --> Gln) decreased SOCC-mediated Ca2+, but not Na+, current and induced a left shift in E-rev. Similar effects were induced by E576K or D581K, but not D581N or E615K, substitution. Furthermore, expressed TRPC1 proteins interacted with each other. Together, these data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form SOCC and that specific acidic as residues in the proposed pore region of TRPC1 contribute to Ca2+ influx.