Ca2+ channels in non-excitable cells

描述(申请人提供)：该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传导的关键组成部分，它编码着一个普遍存在的钙离子通透性阳离子通道家族，似乎在细胞信号转导、细胞生长和细胞死亡中发挥着重要作用。Trp通道分为三大亚类：TRPC、TRPV和TRPM。哺乳动物中有7种TRPC蛋白，命名为TRPC1-TRPC7。色氨酸通道被认为是四聚体，但实际的亚基组成仍然未知。果蝇色氨酸通道被含有PDZ的支架蛋白INAD控制在一个大型的多聚体“信号丛”中。我们以前报道过免疫亲和素FKBP59，是果蝇色氨酸信号复合体的一员。最近的研究表明，TRPC1、C4和C5也可能存在于包括FKBP52的信号复合体中，并由哺乳动物的INAD样蛋白拴住，而TRPC3、C6和C7可能形成非拴系的异构体，受FKBP12调控。该项目的具体目标是1)确定天然的TRPC通道亚单位组成，2)鉴定存在于TRPC通道信号复合体中的辅助蛋白并对其进行功能鉴定，3)评估免疫亲和素在调节TRPC通道中的作用。为了达到这些目的，将产生针对每个TRPC蛋白的多种特异性抗体。这些抗体将被用作免疫组织化学工具，利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伙伴，即蛋白质组行走。在双向凝胶电泳之后，新的相互作用的蛋白质将通过质谱学来鉴定。通道功能将使用膜片钳和钙成像技术进行评估，并将使用定点突变在异源表达系统中确定结构-功能关系。高通量分析将被用来评估蛋白质-蛋白质的相互作用，有效地研究磷脂酶C、蛋白激酶和钙调蛋白对TRPC通道的调节，并寻找影响通道功能和改变细胞信号的新配体。最终，这些研究将为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)它们作为新的治疗药物的分子靶标的潜在用途提供更多的洞察。

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.