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: 8319479
• 负责人: MICHAEL X ZHU
• 金额: $ 28.96万
• 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/8319479
• 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 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; 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 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Ca2+信号传导的机制和生理功能。来自细胞内存储的CA2+动员代表了重要的细胞信号传导过程。在三个已知的CA2+动员中，第二信使：肌醇1,4,5-三磷酸（IP3），环状ADP-核糖（CADPR）和烟酸腺苷二核苷酸磷酸（NAADP），NAADP的特征最少。最近的研究表明，NAADP从与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+释放的特定内部存储仍然难以捉摸。 两个孔通道（TPC）属于电压门控离子通道超家族。三个TPC基因编码与电源门控Ca2+和Na+通道的孔形成亚基最密切相关的蛋白质。每种TPC蛋白都包含12个带有两个潜在孔环的推定跨膜1螺旋。我们最近的研究表明，TPC1和TPC3在内体膜上的表达以及TPC2在溶酶体膜上的表达，这表明TPC很可能是Ca2+ - 可渗透的酸性细胞器通道。我们表明，富含TPC2的膜与NAADP结合，在低纳摩尔浓度下具有高亲和力，过表达TPC2的细胞对NAADP的细胞内应用具有增强的响应。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，可以消除NAADP反应。此外，NAADP引用的Ca2+信号与内质网的Ca2+释放耦合，这表明NAADP和IP3受体之间的串扰。 当前项目的目标是测试以下假说：TPC形成NAADP受体，该假说介导了从不同的内部散热体种群中释放的Ca2+释放，这些新型Ca2+释放渠道的深入表征（AIM 1）（AIM 1）（AIM 1），并探索NAADP诱导的Ca2+ sirdig ca2+ Mobilic and Endoplig and endoplyoplix and eNDopliC and eNDOPLAST及其在酸中的释放之间的功能性交叉序列，以及（目标2）。采用分子生物学，生物化学，药理学，细胞生物学和电生理学的多学科方法将用于实现这两个特定目标。这些全面的研究应大大增强我们对NAADP信号传导的理解，并为这种重要的细胞信号通路在广泛的细胞类型中的作用及其参与正常的人类生理学和病理生理学的参与而开发新的灯光，尤其是因为溶酶体CA2+处理尤其是因为自噬和溶酶体储存疾病涉及。 公共卫生相关性：钙离子对于细胞信号非常重要。在已知诱导细胞内Ca2+存储库中释放Ca2+的三个信使中，在NAADP诱导的Ca2+释放的机理和生理功能中，最不了解。拟议的项目将表明，两个孔通道是在内体和溶酶体上表达的NAADP受体，它们在调节酸性细胞器中释放的Ca2+释放方面起着重要功能，并通过交叉词性通过交叉词性向CA2+释放通道塑造CA2+信号传导。