Ca2+ channels in non-excitable cells

描述（由申请人提供）：本项目的长期目标是了解哺乳动物TRPC通道的结构，功能和调节。TRP基因最初被认为是果蝇光转导的关键组成部分，编码一个普遍存在的Ca 2+渗透性阳离子通道家族，该家族似乎在细胞信号传导、细胞生长和细胞死亡中起着重要作用。TRP通道分为3个主要亚组：TRPC、TRPV和TRPM。有7种哺乳动物TRPC蛋白，命名为TRPC 1-TRPC 7。TRP通道被认为是四聚体，但实际的亚基组成仍然未知。果蝇TRP通道由含PDZ的支架蛋白INAD保持在一个大的多聚体“信号复合物”中。我们先前报道了亲免素FKBP 59是果蝇TRP信号复合体的成员。最近的研究表明，TRPC 1 C4和C5也可能存在于包括FKBP 52的信号复合物中，并由哺乳动物INAD样蛋白质束缚，而TRPC 3，C6和C7可能形成非束缚的异源多聚体，由FKBP 12调节。本项目的具体目标是：1）定义天然TRPC通道亚基组成，2）鉴定和功能表征TRPC通道信号复合物中存在的辅助蛋白，3）评估免疫亲素在TRPC通道调节中的作用。为了这些目的，将产生针对每种TRPC蛋白的多种特异性抗体。抗体将用作免疫组织化学工具，以使用共聚焦成像技术评估组织分布和亚细胞定位，并用于免疫沉淀实验以分离新的体内结合配偶体，即，“蛋白质组行走”。 二维凝胶电泳后，新的相互作用的蛋白质将通过质谱鉴定。 将使用膜片钳和Ca 2+成像技术评估通道功能，并使用定点诱变在异源表达系统中定义结构-功能关系。高通量分析将用于评估蛋白质-蛋白质相互作用，并有效地研究TRPC通道的磷脂酶C，蛋白激酶和钙调蛋白的调节，并确定影响通道功能和修饰细胞信号的新型配体。最终，这些研究将提供更深入的了解1）TRPC通道在信号转导中的作用，2）它们可能参与疾病状态的发展和/或进展，以及3）它们作为新型治疗剂的分子靶点的潜在用途。

DESCRIPTION (provided by applicant): The long-range goal of this project is to understand the structure, function, and regulation of mammalian TRPC channels. TRP genes, originally identified as critical components of Drosophila phototransduction, encode a ubiquitous family of Ca2+-permeable cation channels that appear to play a fundamental role in cell signaling, cell growth, and cell death. TRP channels are divided into 3 major subgroups, TRPC, TRPV and TRPM. There are 7 mammalian TRPC proteins designated TRPC1-TRPC7. TRP channels are thought to be tetrameric, but the actual subunit composition remains unknown. Drosophila TRP channels are held in a large multimeric "signalplex" by the PDZ-containing scaffolding protein, INAD. We previously reported that immunophilin FKBP59, is a member of the Drosophila TRP signalplex. Recent studies suggest that TRPC1 C4, and C5 may also exist in a signalplex that includes FKBP52 and is tethered by a mammalian INAD-like protein, whereas TRPC3, C6, and C7 may form non-tethered heteromultimers, regulated by FKBP12. The specific aims of this project are to 1) define the native TRPC channel subunit composition, 2) identify and functionally characterize accessory proteins present in the TRPC channel signalplex, and 3) evaluate the role of immunophilins in regulation of TRPC channels. Towards these ends, multiple specific antibodies for each TRPC protein will be generated. The antibodies will be employed as immunohistochemical tools to evaluate tissue distribution and subcellular localization using confocal imaging techniques, and for immunoprecipitation experiments to isolate novel in vivo binding partners, i.e., "proteome walking". Following 2-D gel electrophoresis, novel interacting proteins will be identified by mass spectrometry. Channel function will be evaluated using patch-clamp and Ca 2+ imaging techniques, and structure-function relationships will be defined in heterologous expression systems using site-directed mutagenesis. High-throughput assays will be employed to evaluate protein-protein interactions, and to efficiently study regulation of TRPC channels by phospholipase C, protein kinases, and calmodulin, and to identify novel ligands that affect channel function and modify cellular signaling. Ultimately, these studies will provide greater insight into 1) the role TRPC channels in signal transduction, 2) their possible involvement in the development and/or progression of disease states, and 3) their potential use as molecular targets for novel therapeutic agents.