Isoform-Selective Physical Coupling of TRPC3 Channels to IP 3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Abstract

Rationale : Inositol 1,4,5-trisphosphate (IP 3 )-induced vasoconstriction can occur independently of intracellular Ca 2+ release and via IP 3 receptor (IP 3 R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Objectives : Study mechanisms by which IP 3 Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. Methods and Results : Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP 3 Rs (IP 3 R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C–coupled receptor agonist, elevated immuno-FRET between IP 3 R1 and TRPC3, but not between IP 3 R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP 3 R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP 3 -induced nonselective cation current ( I Cat ) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP 3 R1 and TRPC3, IP 3 -induced I Cat activation, and ET-1 and IP 3 -induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP 3 R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP 3 R N-terminal region that can interact with TRPC channels activated I Cat . A TRPC3 CIRB domain peptide attenuated IP 3 - and ET-1–induced I Cat activation and vasoconstriction. Conclusions : IP 3 stimulates direct coupling between IP 3 R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to I Cat activation and vasoconstriction. Close spatial proximity between IP 3 R1 and TRPC3 establishes this isoform-selective functional interaction.