ABSTRACT: KEY POINTS:In vascular smooth muscle cells (VSMCs), activation of Ca2+ -permeable store-operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease. Activation of TRPC1-based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). We investigated the identity of the PKC isoform involved in activating TRPC1-based SOCs in rat mesenteric artery VSMCs. TRPC1-based SOCs were reduced by PKC? inhibitors and knockdown of PKC? expression. Store depletion induced interactions between TRPC1 and PKC? and PKC?-dependent phosphorylation of TRPC1. Furthermore, generation of store-operated interactions between PIP2 and TRPC1 and activation of TRPC1-based SOCs by PIP2 required PKC?. These findings reveal that PKC? activity has an obligatory role in activating TRPC1-based SOCs, through regulating PIP2 -mediated channel opening. ABSTRACT:In vascular smooth muscle cells (VMSCs), stimulation of Ca2+ -permeable canonical transient receptor potential channel 1 (TRPC1)-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1-based SOCs are activated. Stimulation of TRPC1-based SOCs requires protein kinase C (PKC) activity, with store-operated PKC-dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Experimental protocols used to activate TRPC1-based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+ -insensitive, which are characteristics of the novel group of PKC isoforms (?, ?, ?, ?). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1-based SOCs in contractile rat mesenteric artery VSMCs. Store-operated whole-cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKC? was expressed in VSMCs, and selective PKC? inhibitory peptides and knockdown of PKC? expression with morpholinos oligomers inhibited TRPC1-based SOCs. TRPC1 and PKC? interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKC? knockdown. In addition, store-operated PIP2 and TRPC1 interactions were blocked by PKC? inhibition, and PKC? was required for PIP2 -mediated activation of TRPC1 currents. These results identify the involvement of PKC? in stimulation of TRPC1-based SOCs and highlight that store-operated PKC? activity is obligatory for channel opening by PIP2 , the probable activating ligand.
