Endothelin‐1 constricts cerebral arteries by stimulating physical coupling between type 1 IP3 receptors and TRPC3 channels in myocytes

Abstract

Vasoconstrictors elevate inositol 1,4,5‐trisphophate (IP3), but signaling mechanisms activated by this second messenger in arterial myocytes are poorly understood. Recently, we described that IP3‐induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and TRPC3 channel activation (Xi et al., Circ Res. 2008; 102:1118‐26). Here, we tested the hypothesis that IP3Rs stimulate TRPC channels due to physical coupling between these proteins in cerebral artery myocytes. An IP3R1 antibody co‐immunoprecipitated both IP3R1 and TRPC3 channels from cerebral artery lysate. IP3R1 and TRPC3‐bound fluorescent (Cy2, Cy3) secondary antibodies generated significant immunofluorescence resonance energy transfer (N‐FRET) in myocytes. In contrast, antibodies bound to IP3R1 and TRPM4 generated weak N‐FRET. Endothelin‐1 (ET‐1), a PLC‐coupled receptor agonist, increased N‐FRET between IP3R1 and TRPC3‐bound fluorescent antibodies. A peptide corresponding to the TRPC channel C‐terminus calmodulin and IP3R binding domain (CIRB) reduced ET‐1 and IP3‐induced ICat activation in myocytes. Similarly, a membrane‐permeant (HIV‐1 TAT) CIRB peptide reduced ET‐1‐induced constriction in pressurized arteries. Data suggest that ET‐1 enhances physical coupling between the CIRB domain of IP3R1 and TRPC3, leading to cation current activation and vasoconstriction. R01 HL67061.