The role of two-pore channels in integrative calcium signaling

描述(申请人提供)：我们研究的长期目标是了解哺乳动物细胞中钙信号的机制和生理功能。细胞内钙离子的动员是一个重要的细胞信号传递过程。在已知的3种钙动员第二信使中，1，4，5-三磷酸肌醇(IP3)、环状ADP-核糖(CADPR)和烟酸腺嘌呤二核苷酸磷酸(NAADP)是最不典型的。最近的研究表明，NAADP可以从溶酶体相关的酸性细胞器中动员钙离子；然而，NAADP刺激的钙释放过程中涉及的钙释放通道和特定的内库的分子同一性仍然是未知的。两个孔通道(TPC)属于电压门控离子通道超家族。这三个TPC基因编码的蛋白质与电压门控钙离子和钠离子通道的成孔亚基关系最密切。每个TPC蛋白含有12个可能的跨膜1-螺旋，有两个潜在的孔环。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TPC很可能是酸性细胞器的钙渗透通道。我们发现，在低纳摩尔浓度下，TPC2富集膜与NAADP以高亲和力结合，并且高表达TPC2的细胞对NAADP的细胞内应用的反应增强。NAADP反应通过破坏溶酶体的质子梯度、RNAi介导的TPC2表达沉默以及TPC2基因的遗传消融而被取消。此外，NAADP诱导的钙信号与内质网的钙释放相耦合，提示NAADP和IP3受体之间存在串扰。本项目的目标是通过对这些新的钙释放通道的深入研究，验证TPC形成NAADP受体介导不同内切酶群体钙释放的假说(目标1)，并探索NAADP诱导的酸性钙释放和内质网钙动员之间的功能串扰，以及其他钙信号通路(目标2)。将使用分子生物学、生物化学、药理学、细胞生物学和电生理学等多学科方法来实现这两个特定目标。这些全面的研究将极大地提高我们对NAADP信号的理解，并为这一重要的细胞信号通路在广泛的细胞类型中的作用及其在正常人体生理和病理生理学中的参与提供新的线索，特别是因为溶酶体钙处理已涉及自噬和溶酶体储存疾病。 与公共健康相关：钙离子对细胞信号非常重要。在已知的诱导细胞内钙离子释放的三种第二信使IP3、cADPR和NAADP中，NAADP诱导的钙释放的机制和生理功能最不清楚。该项目将表明，双孔通道是表达在内质体和溶酶体上的NAADP受体，它们通过与内质网上的钙释放通道交叉对话，在调节酸性细胞器的钙释放和塑造钙信号方面发挥重要作用。

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.