Vasoregulation by IP3 receptor coupling to TRPC channels

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Arterial diameter, a principal modulator of systemic blood pressure and organ blood flow is regulated by changes in the contractility of arterial myocytes. The contractile status of arterial myocytes is determined by several local and global intracellular calcium ([Ca2+]i) signals. Ca2+ signal and diameter regulation by inositol 1,4,5-trisphophate (IP3), a phospholipase C-generated second messenger is poorly understood. The conventional view has been that IP3 constricts arteries by stimulating sarcoplasmic reticulum (SR) Ca2+ release in myocytes. Our recently published data indicated a novel mechanism of IP3-induced vasoconstriction that occurred independently of SR Ca2+ release, and via IP3 receptor (IP3R)- and canonical transient receptor potential (TRPC) 3-dependent cation current (ICat) activation. However, mechanisms by which IP3R activation stimulates TRPC channels in arterial myocytes to regulate arterial diameter are unclear. Preliminary data suggest that physical coupling between myocyte IP3Rs and TRPC3 channels regulates IP3-induced vasoconstriction. Data also indicate that arterial myocyte caveolae facilitate this IP3R-TRPC channel vasoregulatory mechanism. The central hypothesis of this proposal is that in cerebral artery myocytes, caveolae facilitate TRPC3 channel coupling with IP3Rs to mediate vasoconstrictor and IP3-induced membrane depolarization, voltage-dependent Ca2+ channel activation, [Ca2+]i elevation, and contraction. This proposal will investigate 3 specific aims: Aim 1 will test the hypothesis that in cerebral artery myocytes, IP3R to TRPC3 channel physical coupling is required for IP3-induced ICat activation. Aim 2 will test the hypothesis that IP3R to TRPC3 coupling mediates IP3-induced membrane depolarization, [Ca2+]i elevation, and constriction in cerebral arteries. Aim 3 will examine the hypothesis that arterial myocyte caveolae mediate physical and functional coupling of TRPC3 channels to IP3Rs. Experiments to study these aims will integrate techniques performed at molecular, cellular, and intact tissue levels, including Ca2+ imaging, FRET, electrophysiology, pressurized artery myography, and gene suppression.