Molecular Physiology of TRPC Channels

描述(由申请人提供)：这项研究计划的长期目标是了解控制激素诱导的钙内流的分子机制。受体操作和存储操作的阳离子通道(ROC和SOC)代表着几乎所有细胞系统中重要的钙内流途径。ROC和SOC在调节包括胞吐和收缩在内的多种细胞功能中起着至关重要的作用。在肾上腺嗜铬细胞(ACC)中，循环激素激活的ROC和SOC通道与电压门控钙通道协同作用，调节儿茶酚胺的排泄，儿茶酚胺是血管张力、心输出量和心率的基本调节器。激素激活的ROC和SOC在ACC中的分子本质知之甚少。越来越多的证据表明，典型的瞬时受体潜力(TRPC)通道可以在不同类型的细胞中形成ROC和SOC，或者是ROC和SOC的组成部分。我们已经证明，TRPC通道在ACC中表达，TRPC的过度表达增加了持续的钙内流和儿茶酚胺的分泌。决定钙离子通过TRPC内流的一个因素是通道通透性。对TRPC通道通透性的调节还不是很清楚，然而，某些TRPC通道的异构体表现出通透性降低。我们假设TRPC在ACC中介导激素诱导的胞吐作用，它们的异构化作用调节这一能力。本项目的目的是确定内源性TRPC通道在ACC中的作用和调节。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特定目标：(1)确定哪些TRPC在ACC中内源性表达；(2)确定调控内源性TRPC在ACC中表达的机制；(3)确定控制异构体TRPC通透性的分子决定因素。总而言之，这些研究将阐明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中儿茶酚胺分泌的调控机制是一个重要的保健问题。

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.