Molecular Physiology of TRPC Channels

描述（由申请人提供）：本研究提案的长期目标是了解控制钙离子诱导的Ca 2+内流的分子机制。受体操纵和储存操纵的阳离子通道（ROC和SOC）代表了几乎所有细胞系统中重要的Ca 2+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关键作用。在肾上腺嗜铬细胞（ACC）中，循环激素激活的ROC和SOC通道与电压门控Ca 2+通道协同作用，调节血管张力、心输出量和心率的重要调节因子儿茶酚胺的胞吐作用。ACC中经典瞬时受体电位（Canonical Transient Receptor Potential，TRPC）通道的分子机制尚不清楚，但越来越多的证据表明TRPC通道可以在多种细胞中形成或参与ROC和SOC。我们已经表明，TRPC通道在ACC中表达，并且TRPC的过度表达增加了持续的Ca内流和儿茶酚胺分泌。决定Ca 2+通过TRPC内流的因素是通道渗透性。TRPC通道渗透性的调节还不清楚，然而，TRPC通道的某些异聚体表现出降低的渗透性。我们推测TRPC介导ACC中的细胞外分泌，它们的异聚化起到调节这种能力的作用。本课题旨在研究内源性TRPC通道在ACC中的作用及其调控，主要利用分子生物学、生物化学、膜片钳和荧光成像等方法，研究以下几个方面：（1）ACC中内源性TRPC的表达;（2）ACC中内源性TRPC表达的调控机制;和（3）鉴定控制异聚体TRPC渗透性的分子决定因素。这些研究将揭示ACC中调节循环激素激活的儿茶酚胺胞吐的机制。儿茶酚胺是心血管功能的重要调节剂，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.