Vasoregulation by IP3 receptor coupling to TRPC channels

动脉直径是全身血压和器官血流的主要调节因子， 动脉肌细胞收缩力的变化。动脉肌细胞的收缩状态由以下决定： 几种局部和整体细胞内钙（[Ca 2 +]i）信号。肌醇对Ca ~（2+）信号和直径的调节 1，4，5-磷酸三酯（IP 3），磷脂酶C产生的第二信使知之甚少。的 传统观点认为IP 3通过刺激肌浆网（SR）Ca 2+释放来收缩动脉 在肌细胞中。我们最近发表的数据表明IP 3诱导的血管收缩的一种新机制， 发生独立于SR Ca 2+释放，并通过IP 3受体（IP 3R）-和经典瞬时受体 电位（TRPC）3依赖性阳离子电流（ICat）激活。然而，IP 3R激活的机制 刺激动脉肌细胞中的TRPC通道以调节动脉直径尚不清楚。初步数据 提示肌细胞IP 3Rs和TRPC 3通道之间的物理耦合调节IP 3诱导的 血管收缩数据还表明，动脉肌细胞小窝促进这种IP 3R-TRPC通道 血管调节机制该建议的中心假设是，在脑动脉肌细胞中， 促进TRPC 3通道与IP 3Rs偶联，介导血管收缩和IP 3诱导的膜 去极化、电压依赖性Ca 2+通道激活、[Ca 2 +]i升高和收缩。这项建议 将研究3个具体目标：目标1将检验以下假设：在脑动脉肌细胞中，IP 3R至TRPC 3 IP 3诱导的ICat激活需要通道物理耦合。目标2将检验IP 3R TRPC 3偶联介导IP 3诱导的脑细胞膜去极化、[Ca 2 +]i升高和收缩 动脉目的3将检验动脉肌细胞小窝介导生理和功能性 TRPC 3通道与IP 3R的偶联。研究这些目标的实验将整合在 分子、细胞和完整组织水平，包括Ca 2+成像、FRET、电生理学、加压动脉 肌造影和基因抑制

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.