An ADP-ribose dependent cation channel encoded by LTRPC2

描述(由申请人提供)：在许多细胞中，持续的钙离子进入是 是启动和维持特定细胞反应的基础。 除了广泛存在的商店运营的钙内流机制外，其 分子性质仍然难以捉摸，其他可能的钙内流途径已经 通过鉴定出越来越多的编码基因 钙离子通透性阳离子通道。我们的初步数据描述了分子 ADP-核糖调控的一种新的钙内流途径的特性 (ADPR)。这一特征包括对一部小说的高度鉴定 特异性ADP-核糖水解酶NUDT9，与ADP-核糖水解酶 先前发现的基因LTRPC2的C-末端区域和功能 证明LTRPC2编码一种ADPR门控的蛋白质产物 钙离子通透性阳离子通道。我们还发现了天然表达的 胰岛β细胞和人单核细胞中依赖adpr的电导。基座 在这些数据的基础上，我们建议探索ADPR介导的调节机制 重组系统和生理系统中的钙离子进入。在具体目标1中，我们 将分析LTRPC2离子通道的生物物理和分子方面 结合使用钙成像和电生理的功能 表达重组LTRPC2的细胞分析。要标识结构 LTRPC2依赖于ADPR的门控的基础，我们将表达和表征 指向C-末端小节的截短或嵌合的LTRPC2构建物 结构域，推测为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 这为ADPR门控提供了选择性。在具体目标2中，我们将解决 ADPR门控通道在细胞周期调控中的作用 β细胞的钙稳态。我们将调查具体的性质 并与重组人的ADPR门控通道进行比较 LTRPC2.我们将评估它们在细胞反应中的功能作用 通过比较ADPR门控钙信号的相对贡献 与商店操作的钙内流和电压依赖的钙通道相比。 最后，我们将设法确定负责ADPR的机制 通过研究参与ITS的主要酶和途径进行生产 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.