The role of two-pore channels in integrative calcium signaling

双孔通道在整合钙信号传导中的作用

• 批准号: 7863955
• 负责人: MICHAEL X ZHU
• 金额: $ 29.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7863955
• 关键词: Abbreviations; Ablation; Affinity; Animals; Autophagocytosis; Binding; Biochemistry; Biological Assay; Calcium Signaling; Calcium ion; Cations; Cell Line; Cell Signaling Process; Cells; Cellular biology; Chickens; Coupled; Coupling; Cyclic ADP-Ribose; Data; Electrophysiology (science); Endoplasmic Reticulum; Endosomes; Exhibits; Functional disorder; Gated Ion Channel; Genes; Genetic; Goals; Hepatocyte; Human; Image; Inositol; Kidney; Lasers; Ligands; Lipid Bilayers; Liver; Lung; Lysosomal Storage Diseases; Lysosomes; Mammalian Cell; Mediating; Membrane; Molecular; Molecular Biology; Mus; NAADP; Null Lymphocytes; Organelles; Pancreas; Pattern; Pharmacology; Physiological; Physiology; Plants; Play; Population; Potassium Channel; Property; Proteins; Protons; RNA Interference; Radiolabeled; Reporting; Research; Role; Ryanodine; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Second Messenger Systems; Shapes; Signal Pathway; Signal Transduction; Specificity; Testing; Thapsigargin; Tissues; Transmembrane Domain; Vacuole; bafilomycin A1; base; cell type; design; extracellular; flash photolysis; functional outcomes; heparin receptor; interdisciplinary approach; knockout gene; member; novel; overexpression; public health relevance; radiotracer; receptor; research study; response; second messenger; voltage

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.

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Ca 2+信号传导的机制和生理功能。Ca 2+从细胞内储存的动员代表了重要的细胞信号传导过程。在三种已知的Ca 2+动员第二信使中，肌醇1，4，5-三磷酸（IP 3）、环ADP-核糖（cADPR）和烟酸腺嘌呤二核苷酸磷酸（NAADP），NAADP的特征最少。最近的研究表明，NAADP动员钙从溶酶体相关的酸性细胞器，然而，分子身份的钙释放通道和具体的内部存储参与NAADP刺激的钙释放仍然难以捉摸。 这两个孔通道（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Ca 2+和Na+通道的成孔亚基最密切相关。每个TPC蛋白含有12个假定的跨膜1-螺旋，具有两个潜在的孔环。我们最近的研究表明，TPC 1和TPC 3的表达的内体膜和TPC 2的溶酶体膜上，表明TPC最有可能是酸性细胞器的Ca 2+渗透通道。我们发现，TPC 2富集膜结合NAADP具有高亲和力，在低纳摩尔浓度和细胞过度表达TPC 2有增强的响应细胞内应用NAADP。NAADP反应通过破坏溶酶体的质子梯度和RNAi介导的TPC 2表达沉默以及小鼠中Tpc 2基因的遗传消融来消除。此外，NAADP引起的Ca 2+信号耦合到Ca 2+从内质网释放，这表明NAADP和IP 3受体之间的串扰。 当前项目的目标是通过深入表征这些新型Ca 2+释放通道（Aim 1）来验证TPC形成介导不同内溶酶体群体的Ca 2+释放的NAADP受体的假设，并探索NAADP诱导的酸性储存的Ca 2+释放和内质网的Ca 2+动员之间的功能性串扰，以及额外的Ca 2+信号通路（Aim 2）。一个多学科的方法，采用分子生物学，生物化学，药理学，细胞生物学和电生理学将被用来实现这两个特定的目标。这些全面的研究应该大大提高我们对NAADP信号的理解，并揭示了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类生理学和病理生理学中的参与，特别是因为溶酶体Ca 2+处理与自噬和溶酶体贮积病有关。 公共卫生相关性：钙离子对细胞信号传导非常重要。在已知的三种诱导细胞内Ca ~（2+）释放的第二信使中，IP 3、cADPR和NAADP，NAADP诱导的Ca ~（2+）释放的机制和生理功能是最不清楚的。本项目的研究结果表明，双孔通道是表达于核内体和溶酶体上的NAADP受体，它们在调节酸性细胞器的Ca 2+释放和通过与内质网上的Ca 2+释放通道的交叉作用形成Ca 2+信号传导中发挥重要作用。