An ADP-ribose dependent cation channel encoded by LTRPC2

描述（由申请人提供）：在许多细胞中， 对启动和维持特定细胞反应至关重要。 除了广泛存在的钙池操纵的钙内流机制外， 分子本质仍然难以捉摸，其他假定的钙内流途径 通过鉴定越来越多的编码 钙渗透性阳离子通道。我们的初步数据描述了 ADP-核糖调控的新型钙内流途径的研究 （ADPR）。这一特征包括确定一个新的高度 特异性ADP-核糖水解酶NUDT 9，其与 先前鉴定的基因LTRPC 2的C-末端区域，以及功能性 证明LTRPC 2编码的蛋白产物是ADPR门控的， 钙离子通道我们还发现了 胰腺β细胞和人单核细胞中的ADPR依赖性电导。基于 基于这些数据，我们建议探索调节ADPR介导的 钙进入重组和生理系统。在具体目标1中， 将分析LTRPC 2离子通道的生物物理和分子方面 通过使用钙成像和电生理学的组合， 分析表达重组LTRPC 2的细胞。为了识别结构 ADPR依赖性门控LTRPC 2的基础上，我们将表达和表征 针对C-末端核苷酸的截短的或嵌合的LTRPC 2构建体 结构域，推定的ADPR结合区。我们还建议系统地 改变该结构域中的氨基酸以评估结构-功能关系 其赋予ADPR门控的选择性。在具体目标2中，我们将讨论 ADPR门控通道在调节 β细胞的钙稳态。我们会调查 的天然ADPR门控通道，并将它们与重组ADPR门控通道进行比较。 LTRPC 2.我们将评估它们在细胞反应中的功能作用， 通过比较ADPR门控的Ca 2+信号的相对贡献， 钙池操纵的钙内流和电压依赖性钙通道的钙通道。 最后，我们将寻求确定负责ADPR的机制 通过研究主要的酶和参与其生产的途径， 新陈代谢.

DESCRIPTION (provided by applicant): Sustained calcium entry, in many cells, is fundamental to the initiation and maintenance of specific cellular responses. In addition to the widespread store-operated calcium influx mechanism, whose molecular nature remains elusive, other putative calcium influx pathways have emerged through identification of a growing number of genes coding for calcium-permeable cation channels. Our preliminary data describe the molecular characterization of a novel calcium-entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific ADP-ribose hydrolase, NUDT9, which shares high homology with the C-terminal region of a previously identified gene, LTRPC2, and the functional demonstration that LTRPC2 encodes a protein product that is an ADPR-gated calcium-permeable cation channel. We also identified natively expressed ADPR-dependent conductances in pancreatic beta cells and human monocytes. Based on these data, we propose to explore the mechanisms that regulate ADPRmediated calcium entry in recombinant and physiological systems. In Specific Aim 1, we will analyze the biophysical and molecular aspects of LTRPC2 ion channel function by using a combination of calcium imaging and electrophysiological analysis of cells that express recombinant LTRPC2. To identify the structural basis for ADPR-dependent gating of LTRPC2, we will express and characterize truncated or chimeric LTRPC2 constructs directed towards the C-terminal nudix domain, the putative ADPR binding region. We also propose to systematically alter amino acids within this domain to assess structure-function relationships that confer selectivity for ADPR gating. In Specific Aim 2, we will address the functional and physiological role of ADPR-gated channels in the regulation of calcium homeostasis of beta cells. We will investigate the specific properties of native ADPR-gated channels and compare them with those of recombinant LTRPC2. We will assess their functional role in the cellular responses of the above cells by comparing the relative contributions of ADPR-gated Ca2+ signals to those of store-operated Ca2+ influx and voltage-dependent Ca2+ channels. Finally, we will seek to identify the mechanisms responsible for ADPR production by investigating the major enzymes and pathways involved in its metabolism.