Molecular Characterization of Organelle Channels

细胞器通道的分子表征

• 批准号: 8138060
• 负责人: MICHAEL X ZHU
• 金额: $ 3.49万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8138060
• 关键词: Molecular; Characterization; Organelle; Channels

DESCRIPTION (provided by applicant): The two pore channels (TPCs) belong to the voltage-gated ion channel superfamily. The three TPCN 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 (TM) 1-helices with two potential pore loops. This structure feature suggests that TPCs may represent an intermediate evolutionary step from the single domain 6-TM channels to the four-domain Ca2+ and Na+ channels. 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 intracellular organelles. We further show that TPC2 is a plausible candidate of the receptor for nicotinic acid-adenine dinucleotide phosphate (NAADP). We hypothesize that vertebrate TPCs are organelle channels involved in various aspects of intracellular vesicle trafficking, fusion, and Ca2+ signaling. The long-term objective of our research is to understand Ca2+ signaling mechanisms in mammalian cells. It has long been recognized that NAADP releases Ca2+ from an internal pool that is distinct from the endoplasmic reticulum. More recent studies show that the NAADP-sensitive pool is associated with lysosome-like acidic compartments. Thus, the study of TPC2 as an NAADP receptor provides a unique opportunity to address this rather distinct, but also poorly understood, pathway of Ca2+ signaling. The goals of the proposed research are to test the aforementioned hypothesis and to set a foundation for a new area of Ca2+ signaling focusing on the structures and functions of organelle Ca2+ permeable channels. We have three specific aims. Aim 1 will test the hypothesis that TPC2 is an NAADP receptor. We will examine the binding of NAADP to TPC2-containing membranes and study NAADP-evoked Ca2+ response in cells overexpressing TPC2 as well as those that have TPC2 expression knocked down by RNA interference. Aim 2 will examine a novel intracellular mechanism by which membrane stretch stimulates NAADP production, causing Ca2+ release from lysosomal stores via the TPC2 channel. Aim 3 will examine the molecular dynamics of TPC1-3 in subcellular vesicle trafficking and to determine sorting signals for the organelle targeting of TPCs. These comprehensive studies will greatly enhance our understanding on these novel ion channels and shed light on their potential roles in normal human physiology and pathophysiology. PUBLIC HEALTH RELEVANCE: Two pore channels (TPCs) are novel ion channels related to voltage-gated calcium channels but they are expressed on intracellular organelles such as endosomes and lysosomes instead of plasma membrane. One of them, TPC2, is likely involved in mediating calcium release from lysosomes in response to the second messenger, nicotinic acid-adenine dinucleotide phosphate (NAADP). The proposed project will elucidate the molecular functions of TPCs in various aspects of intracellular vesicle trafficking, fusion, and calcium signaling and shed light on their potential roles in normal human physiology and pathophysiology.

描述（由申请人提供）：两种孔通道（TPC）属于电压门控离子通道超家族。这三个TPCN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个推定的跨膜（TM）1-螺旋与两个潜在的孔环。这种结构特征表明，TPC可能代表了从单结构域6-TM通道到四结构域Ca ~（2+）和Na ~+通道的中间进化步骤。我们最近的研究表明TPC 1和TPC 3在内体膜上表达，TPC 2在溶酶体膜上表达，这表明TPC最有可能是细胞内细胞器的Ca 2+渗透通道。我们进一步表明，TPC 2是一个合理的候选人的受体烟酸腺嘌呤二核苷酸磷酸（NAADP）。我们假设脊椎动物TPC是细胞器通道，参与细胞内囊泡运输、融合和Ca 2+信号传导的各个方面。我们研究的长期目标是了解哺乳动物细胞中的Ca 2+信号传导机制。长期以来，人们已经认识到NAADP从不同于内质网的内部池释放Ca 2+。最近的研究表明，NAADP敏感池与溶酶体样酸性区室有关。因此，TPC 2作为NAADP受体的研究提供了一个独特的机会，以解决这个相当独特的，但也知之甚少，Ca 2+信号通路。本研究的目的是验证上述假设，并为研究细胞器Ca 2+渗透通道的结构和功能奠定基础。我们有三个具体目标。目的1将检验TPC 2是NAADP受体的假设。我们将研究NAADP与含TPC 2的膜的结合，并研究NAADP诱导的过表达TPC 2的细胞中的Ca 2+反应以及那些通过RNA干扰敲低TPC 2表达的细胞。目的2将研究一种新的细胞内机制，膜拉伸刺激NAADP的生产，导致钙释放从溶酶体存储通过TPC 2通道。目的3将研究TPC 1 -3在亚细胞囊泡运输中的分子动力学，并确定TPC靶向细胞器的分选信号。这些全面的研究将大大提高我们对这些新的离子通道的理解，并阐明它们在正常人体生理和病理生理中的潜在作用。公共卫生相关性：双孔通道（Two pore channels，TPC）是一种与电压门控性钙通道相关的新型离子通道，但其表达于胞内体和溶酶体等细胞器上，而非质膜上。其中之一，TPC 2，可能参与介导钙释放从溶酶体响应第二信使，烟酸腺嘌呤二核苷酸磷酸（NAADP）。该项目将阐明TPC在细胞内囊泡运输，融合和钙信号传导的各个方面的分子功能，并阐明其在正常人体生理学和病理生理学中的潜在作用。