Obligatory Role For Pkc Delta In Pip2-Mediated Activation Of Store-Operated Trpc1 Channels In Vascular Smooth Muscle Cells

Key pointsIn vascular smooth muscle cells (VSMCs), activation of Ca2+-permeable store-operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca(2+)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 delta inhibitors and knockdown of PKC delta expression. Store depletion induced interactions between TRPC1 and PKC delta and PKC delta-dependent phosphorylation of TRPC1. Furthermore, generation of store-operated interactions between PIP(2)and TRPC1 and activation of TRPC1-based SOCs by PIP(2)required PKC delta. These findings reveal that PKC delta activity has an obligatory role in activating TRPC1-based SOCs, through regulating PIP2-mediated channel opening. In vascular smooth muscle cells (VMSCs), stimulation of Ca2+-permeable canonical transient receptor potential channel 1 (TRPC1)-based store-operated channels (SOCs) mediates Ca(2+)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 (delta, epsilon, eta, theta). 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 delta was expressed in VSMCs, and selective PKC delta inhibitory peptides and knockdown of PKC delta expression with morpholinos oligomers inhibited TRPC1-based SOCs. TRPC1 and PKC delta interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKC delta knockdown. In addition, store-operated PIP(2)and TRPC1 interactions were blocked by PKC delta inhibition, and PKC delta was required for PIP2-mediated activation of TRPC1 currents. These results identify the involvement of PKC delta in stimulation of TRPC1-based SOCs and highlight that store-operated PKC delta activity is obligatory for channel opening by PIP2, the probable activating ligand.