Endothelin‐1 constricts cerebral arteries by stimulating physical coupling between type 1 IP 3 receptors and TRPC3 channels in myocytes

Vasoconstrictors elevate inositol 1,4,5‐trisphophate (IP 3 ), but signaling mechanisms activated by this second messenger in arterial myocytes are poorly understood. Recently, we described that IP 3 ‐induced vasoconstriction can occur independently of intracellular Ca 2+ release and via IP 3 receptor (IP 3 R) and TRPC3 channel activation (Xi et al., Circ Res. 2008; 102:1118‐26). Here, we tested the hypothesis that IP 3 Rs stimulate TRPC channels due to physical coupling between these proteins in cerebral artery myocytes. An IP 3 R1 antibody co‐immunoprecipitated both IP 3 R1 and TRPC3 channels from cerebral artery lysate. IP 3 R1 and TRPC3‐bound fluorescent (Cy2, Cy3) secondary antibodies generated significant immunofluorescence resonance energy transfer (N‐FRET) in myocytes. In contrast, antibodies bound to IP 3 R1 and TRPM4 generated weak N‐FRET. Endothelin‐1 (ET‐1), a PLC‐coupled receptor agonist, increased N‐FRET between IP 3 R1 and TRPC3‐bound fluorescent antibodies. A peptide corresponding to the TRPC channel C‐terminus calmodulin and IP 3 R binding domain (CIRB) reduced ET‐1 and IP 3 ‐induced I Cat 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 IP 3 R1 and TRPC3, leading to cation current activation and vasoconstriction. R01 HL67061.