The role of two-pore channels in integrative calcium signaling

描述（由申请人提供）：我们研究的长期目标是了解哺乳动物细胞中Ca2+信号传导的机制和生理功能。Ca2+从细胞内储存的动员代表了一个重要的细胞信号传导过程。在已知的三种Ca2+动员第二信使，肌醇1,4,5-三磷酸（IP3），环ADP-核糖（cADPR）和烟酸腺嘌呤二核苷酸磷酸（NAADP）中，NAADP的特征最少。最近的研究表明，NAADP从溶酶体相关的酸性细胞器中动员Ca2+；然而，Ca2+释放通道的分子身份和参与naadp刺激的Ca2+释放的特定内部储存仍然难以捉摸。这两个孔通道（TPCs）属于电压门控离子通道超族。这三个TPC基因编码的蛋白质与电压门控Ca2+和Na+通道的孔形成亚基最密切相关。每个TPC蛋白含有12个假定的跨膜1-螺旋和两个潜在的孔环。我们最近的研究表明，TPC1和TPC3在内体膜上表达，TPC2在溶酶体膜上表达，这表明TPC1很可能是酸性细胞器的Ca2+渗透通道。我们发现富含TPC2的细胞膜在低纳摩尔浓度下与NAADP具有高亲和力，并且过表达TPC2的细胞对细胞内应用NAADP的反应增强。通过破坏溶酶体的质子梯度和rnai介导的TPC2表达沉默，以及TPC2基因在小鼠中的遗传消融，NAADP反应被消除。此外，NAADP诱导的Ca2+信号与内质网Ca2+释放耦合，表明NAADP和IP3受体之间存在交叉对话。当前项目的目标是测试假设，即TPCs形成NAADP受体，介导Ca2+释放来自不同的内溶酶体群体，深入表征这些新的Ca2+释放通道（Aim 1），并探索NAADP诱导的Ca2+释放从酸性储存和内质网Ca2+动员之间的功能交叉对话，以及额外的Ca2+信号通路（Aim 2）。采用多学科的方法，包括分子生物学、生物化学、药理学、细胞生物学和电生理学，来完成这两个特定的目标。这些全面的研究将极大地增强我们对NAADP信号传导的理解，并揭示这一重要细胞信号传导途径在广泛的细胞类型中的作用，以及它们在正常人体生理和病理生理中的作用，特别是因为溶酶体Ca2+处理与自噬和溶酶体贮病有关。

DESCRIPTION (provided by applicant): The long-term objective of our research is to understand the mechanisms and physiological functions of Ca2+ signaling in mammalian cells. Ca2+ mobilization from intracellular stores represents an important cell signaling process. Of the three known Ca2+ mobilization second messengers, inositol 1,4,5-trisphosphate (IP3), cyclic ADP- ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), NAADP is least characterized. Recent studies indicate that NAADP mobilizes Ca2+ from lysosome-related acidic organelles; however, the molecular identity of the Ca2+ release channels and the specific internal stores involved in NAADP-stimulated Ca2+ release remain elusive. The two pore channels (TPCs) belong to the voltage-gated ion channel superfamily. The three TPC genes encode proteins that are most closely related to the pore-forming subunit of voltage-gated Ca2+ and Na+ channels. Each TPC protein contains 12 putative transmembrane 1-helices with two potential pore loops. Our recent studies show expression of TPC1 and TPC3 on endosomal membranes and that of TPC2 on lysosomal membranes, suggesting that TPCs are most likely Ca2+- permeable channels of acidic organelles. We show that TPC2-enriched membranes bind to NAADP with a high affinity at low nanomolar concentrations and cells overexpressing TPC2 have enhanced response to intracellular application of NAADP. NAADP response is abolished by disrupting proton gradient of lysosomes and RNAi-mediated silencing of TPC2 expression, as well as genetic ablation of the Tpc2 gene in mice. Furthermore, the NAADP-elicited Ca2+ signal is coupled to Ca2+ release from the endoplasmic reticulum, suggesting a cross-talk between NAADP and IP3 receptors. The goals of the current project are to test the hypothesis that TPCs form NAADP receptors that mediate Ca2+ release from different endo-lysosome populations with in-depth characterization of these novel Ca2+ release channels (Aim 1), and to explore the functional cross-talk between NAADP-induced Ca2+ release from acidic stores and Ca2+ mobilization from endoplasmic reticulum, as well as additional Ca2+ signaling pathways (Aim 2). A multidisciplinary approach employing molecular biology, biochemistry, pharmacology, cell biology, and electrophysiology will be used to accomplish the two specific aims. These comprehensive studies should greatly enhance our understanding on NAADP signaling and shed new lights on the roles of this important cell signaling pathway in a broad spectrum of cell types and their involvement in normal human physiology and pathophysiology especially because lysosomal Ca2+ handling has been implicated in autophagy and lysosomal storage diseases. PUBLIC HEALTH RELEVANCE: Calcium ions are very important for cell signaling. Of the three second messengers known to induce Ca2+ release from intracellular Ca2+ storage pools, IP3, cADPR and NAADP, the mechanism and physiological function of NAADP-induced Ca2+ release are the least understood. The proposed project will show that two-pore channels are NAADP receptors expressed on endosomes and lysosomes and they play important functions in regulating Ca2+ release from acidic organelles and shaping Ca2+ signaling via cross-talking to Ca2+ release channels on endoplasmic reticulum.