Canonical transient receptor potential 1 (TRPC1) proteins form store-operated channels in vascular smooth muscle cells (VSMCs), which are involved in regulating vascular tone, and cell proliferation and migration (1). As such TRPC1 channels are potential therapeutic targets for cardiovascular disease. Previous studies indicate that TRPC1 channels are activated by distinct pools of phosphatidylinositol 4,5-bisphosphate (PIP2) bound to myristoylated alanine-rich protein kinase C substrate (MARCKS) in a process that requires protein kinase C (PKC)-dependent phosphorylation of channel proteins (2). The present study investigates the unresolved question of how depletion of internal Ca2+ stores is coupled to these TRPC1 channel activation mechanisms. New Zealand white rabbits (2-3 kg) were killed with i.v. injection of sodium pentabarbitone (120mgkg-1) according to the UK Scientific Procedures Act of 1986. Portal veins were dissected, cleaned of fat and endothelium, and enzymatically dispersed into single VSMCs for electrophysiology, confocal imaging, or experiments requiring primary cell culture (2). Veins were also used as tissue lysates for co-immunoprecipitation and Western blotting methods (2). All data is from at least n=6 VSMCs or n=3 tissue lysates, with level of significance of p<0.05. Mean peak amplitudes of previously characterised store-operated whole-cell TRPC1 currents evoked using intracellular dialysis with high BAPTA concentrations (1) were attenuated by over 85% by inclusion of the G-protein inhibitor GDP-β-S (500µM) in the patch pipette solution, external application of the phospholipase C inhibitor U73122 (2µM), and the PKC inhibitor GF109203X (3µM). In addition, transfection of VSMCs with short-hairpin RNA targeted against stromal interaction molecule 1 (STIM1) or application of an externally-acting anti-STIM1 antibody (1:100) also reduced store-operated TRPC1 whole-cell currents by over 80%. Interestingly, similar store-operated whole-cell and single TRPC1 channel currents (1,2) were recorded in VSMCs from both wild-type and Orai1-/- mice, which indicated that Orai1 is not involved in TRPC1 channel activation.Pre-treatment of tissue lysates with BAPTA-AM (50µM) induced co-associations between TRPC1, and Gq-protein, PLCβ1 (but not PLCγ1) and STIM1. BAPTA-AM did not induce interactions between STIM1 and TRPC6 (a non-store-operated TRPC channel subtype). Moreover, BAPTA-AM evoked U73122-sensitive depletion of plasma membrane PIP2 levels following the introduction of the PIP2 sensor PLCδ1-PH-GFP into VSMCs by electroporation.These findings offer the intriguing possibility that STIM1 gates TRPC1 channels in VSMCs through activating a Gq-protein- and PLC-mediated pathway, perhaps by acting as a scaffold protein or as a Gq-protein activator.