Molecular Characterization of Organelle Channels

描述(申请人提供)：两个孔通道(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钙通道和钠通道的成孔亚基关系最密切。每个TPC蛋白包含12个假定的跨膜(TM)1-螺旋，具有两个潜在的孔环。这一结构特征表明，TPC可能代表了从单一结构域6-TM通道到四结构域钙和钠通道的中间进化步骤。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，提示TPC很可能是细胞内细胞器的钙通透通道。我们进一步证明TPC2是烟酸-腺嘌呤二核苷酸磷酸(NAADP)受体的一个可能的候选者。我们假设脊椎动物的TPC是细胞器通道，参与细胞内小泡运输、融合和钙信号的各个方面。我们研究的长期目标是了解哺乳动物细胞中的钙信号机制。长期以来，NAADP从不同于内质网的内部池中释放钙离子。最近的研究表明，NAADP敏感池与溶酶体样酸性隔室有关。因此，对TPC2作为NAADP受体的研究提供了一个独特的机会来解决这一相当独特但也鲜为人知的钙信号通路。本研究的目的是验证上述假说，并为研究细胞器钙通透通道的结构和功能的钙信号新领域奠定基础。我们有三个具体目标。目的1验证TPC2是NAADP受体的假设。我们将研究NAADP与含有TPC2的细胞膜的结合，并研究NAADP在高表达TPC2的细胞以及那些TPC2表达被RNA干扰下调的细胞中引起的钙反应。目的2将研究一种新的细胞内机制，通过膜拉伸刺激NAADP的产生，通过TPC2通道从溶酶体库中释放钙。目的3将研究TPC1-3在亚细胞囊泡运输中的分子动力学，并确定TPC细胞器靶向的分选信号。这些全面的研究将极大地加深我们对这些新的离子通道的了解，并阐明它们在正常人体生理和病理生理学中的潜在作用。公共卫生相关性：两孔通道(TPC)是与电压门控钙通道相关的新型离子通道，但它们表达于细胞内细胞器，如内小体和溶酶体，而不是质膜。其中，TPC2可能参与了第二信使烟酸-腺嘌呤二核苷酸磷酸(NAADP)对溶酶体钙释放的调节。该项目将阐明TPC在细胞内囊泡运输、融合和钙信号转导的各个方面的分子功能，并阐明它们在正常人体生理和病理生理学中的潜在作用。

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.