Isoform-Selective Physical Coupling of TRPC3 Channels to IP3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Abstract

Many vasoconstrictors bind to phospholipase C (PLC)-coupled receptors on arterial smooth muscle cells, leading to an intracellular inositol 1,4,5-trisphophate (IP3) elevation and vasoconstriction. IP3-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but signaling mechanisms mediating this effect are unknown. Here, we studied the mechanisms by which IP3Rs stimulate TRPC channels in smooth muscle cells of resistance-size cerebral arteries. Immunofluorescence resonance energy transfer (immuno-FRET) microscopy in smooth muscle cells indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels. Endothelin-1 (ET-1), a PLC-coupled receptor agonist, elevated the immuno-FRET signal between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. IP3R1 co-immunoprecipitated with TRPC3, but not with TRPC6. An antibody targeting TRPC3 channels and TRPC3 channel knockdown with short hairpin RNA inhibited IP3-induced nonselective cation current (ICat) activation, whereas an antibody to TRPC6 and TRPC6 channel knockdown had no effect. Biotinylation indicated that ET-1 did not alter total or plasma membrane-localized TRPC3. RT-PCR demonstrated that a calmodulin and IP3R binding (CIRB) domain is present on the C-terminus of both TRPC3 and TRPC6 channels. A CIRB domain peptide attenuated IP3- and ET-1-induced ICat activation. A peptide corresponding to the IP3R region that can interact with TRPC channels activated ICat. A HIV-1 TAT-conjugated CIRB domain peptide reduced IP3- and ET-1-induced vasoconstriction in pressurized arteries. These data indicate that IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial smooth muscle cells, leading to ICat activation and vasoconstriction. Data also indicate that close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction.