Obligatory role for PKCδ in PIP₂-mediated activation of store-operated TRPC1 channels in vascular smooth muscle cells

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 PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ-dependent phosphorylation of TRPC1. Furthermore, generation of store-operated interactions between PIP₂ and TRPC1 and activation of TRPC1-based SOCs by PIP₂ required PKCδ. These findings reveal that PKCδ 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 (δ, ε, η, θ). 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. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1-based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store-operated PIP₂ and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP₂-mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1-based SOCs and highlight that store-operated PKCδ activity is obligatory for channel opening by PIP₂, the probable activating ligand.