Molecular Physiology of TRPC Channels

描述（由申请人提供）：本研究计划的长期目标是了解控制激素诱导的Ca2+内流的分子机制。受体操作和存储操作的阳离子通道（ROC和SOC）代表了几乎所有细胞系统中重要的Ca2+内流途径。ROC和SOC在介导包括胞吐和收缩在内的许多细胞功能中起着至关重要的作用。在肾上腺染色质细胞（ACC）中，循环激素激活的ROC和SOC通道与电压门控的Ca2+通道协同作用，调节儿茶酚胺的胞吐，儿茶酚胺是血管张力、心输出量和心率的重要调节因子。对于ACC中激素激活的ROC和SOC的分子性质知之甚少。越来越多的证据表明，典型瞬时受体电位（TRPC）通道可以在各种细胞类型中形成或成为ROC和SOC的组成部分。我们已经表明，TRPC通道在ACC中表达，TRPC的过表达增加了持续的钙内流和儿茶酚胺分泌。一个决定Ca2+内流通过TRPCs的因素是通道通透性。TRPC通道通透性的调控尚不清楚，然而，某些TRPC通道的异构体表现出降低的通透性。我们假设TRPCs介导ACC中激素诱发的胞吐作用，它们的异质化有助于调节这种能力。本项目旨在确定内源性TRPC通道在ACC中的作用和调控。为此，我们将使用分子生物学，生化，膜片钳和荧光成像方法来解决以下具体目标：(1)确定哪些trpc在ACC中内源性表达；(2)确定内源性trpc在ACC中的表达调控机制；(3)确定控制异质TRPC渗透率的分子决定因素。总之，这些研究将阐明循环激素激活的儿茶酚胺胞吐在ACC中的调节机制。儿茶酚胺是心血管功能的重要调节因子，因此，了解ACC中控制儿茶酚胺分泌的机制是一个重要的卫生保健问题。

DESCRIPTION (provided by applicant): The long-term objective of this research proposal is to understand the molecular mechanisms involved in controlling hormone-induced Ca2+ influx. Receptor-operated and store-operated cation channels (ROC and SOC) represent vital Ca2+ influx pathways in almost all cellular systems. ROC and SOC play a crucial role in mediating numerous cellular functions including exocytosis and contraction. In adrenal chromaffin cells (ACC), circulating-hormone-activated ROC and SOC channels act in concert with voltage-gated Ca2+ channels to modulate exocytosis of catecholamines, essential regulators of vascular tone, cardiac output and heart rate. Little is known about the molecular nature of hormone-activated ROC and SOC in ACC. A growing body of evidence suggests that Canonical Transient Receptor Potential (TRPC) channels can form, or be a component of, ROC and SOC in various cell types. We have shown that TRPC channels are expressed in ACC and that over-expression of TRPCs increases sustained Ca influx and catecholamine secretion. A factor that determines Ca2+ influx through TRPCs is channel permeability. Regulation of TRPC channel permeability is not well understood, however, certain heteromers of TRPC channels exhibit reduced permeability. We hypothesize that TRPCs mediate hormone-evoked exocytosis in ACC and their heteromerization serves to regulate this ability. This project is aimed to identify the role and the regulation of endogenous TRPC channels in ACC. To do this we will use molecular biological, biochemical, patch-clamp and fluorescence imaging methods to address the following specific aims: (1) identify which TRPCs are endogenously expressed in ACC; (2) identify mechanisms regulating the expression of endogenous TRPCs in ACC; and (3) identify the molecular determinants governing heteromeric TRPC permeability. Together these studies will shed light on the mechanisms regulating circulating-hormone-activated catecholamine exocytosis in ACC. Catecholamines are crucial regulators of the cardiovascular function, therefore, understanding mechanisms involved in controlling secretion of catecholamines in ACC is an important health care issue.