Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells

Abstract

Depletion of sarcoplasmic reticulum (SR) Ca2+ stores activates store-operated channels (SOCs) composed of canonical transient receptor potential (TRPC) 1 proteins in vascular smooth muscle cells (VSMCs), which contribute to important cellular functions. We have previously shown that PKC is obligatory for activation of TRPC1 SOCs in VSMCs, and the present study investigates if the classic phosphoinositol signaling pathway involving Gaq-mediated PLC activity is responsible for driving PKCdependent channel gating. The G-protein inhibitor GDPb-S, anti-Gaq antibodies, the PLC inhibitor U73122, and the PKC inhibitor GF109203X all inhibited activation of TRPC1 SOCs, and U73122 and GF109203X also reduced storeoperated PKC-dependent phosphorylation of TRPC1 proteins. Three distinct SR Ca2+ store-depleting agents, 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid acetoxymethyl ester, cyclopiazonic acid, and N,N,N9,N9- tetrakis(2-pyridylmethyl)ethane-1,2-diamineed,induced translocations of the fluorescent biosensor GFP-PLCd1-PH from the cell membrane to the cytosol, which were inhibited by U73122. Knockdown of PLCb1 with small hairpin RNA reduced both store-operated PLC activity and stimulation of TRPC1 SOCs. Immunoprecipitation studies and proximity ligation assays revealed that store depletion induced interactions between TRPC1 and Gaq, and TRPC1 and PLCb1. We propose a novel activationmechanism forTRPC1 SOCs in VSMCs, in which store depletion induces formation of TRPC1-Gaq-PLCb1 complexes that lead to PKC stimulation and channel gating.—Shi, J., Miralles, F., Birnbaumer, L., Large, W. A., Albert, A. P. Store depletion induces Gaqmediated PLCb1 activity to stimulate TRPC1 channels in vascular smooth muscle cells