Molecular Characterization of Organelle Channels

描述（由申请人提供）：这两个孔通道（tpc）属于电压门控离子通道超族。这三个TPCN基因编码的蛋白质与电压门控Ca2+和Na+通道的孔形成亚基最密切相关。每个TPC蛋白含有12个假定的跨膜（TM） 1-螺旋和两个潜在的孔环。这一结构特征表明，TPCs可能代表了从单畴6-TM通道到四畴Ca2+和Na+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，这表明TPC1很可能是胞内细胞器的Ca2+渗透通道。我们进一步表明，TPC2是烟酸-腺嘌呤二核苷酸磷酸（NAADP）受体的合理候选。我们假设脊椎动物的TPCs是细胞器通道，参与细胞内囊泡运输、融合和Ca2+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Ca2+信号传导机制。人们早就认识到NAADP从一个不同于内质网的内部池中释放Ca2+。最近的研究表明，naadp敏感池与溶酶体样酸性区室有关。因此，TPC2作为NAADP受体的研究提供了一个独特的机会来解决这个相当独特，但也知之甚少的Ca2+信号传导途径。本研究的目的是验证上述假设，并为关注细胞器Ca2+可渗透通道的结构和功能的Ca2+信号传导的新领域奠定基础。我们有三个具体目标。目的1将验证TPC2是NAADP受体的假设。我们将研究NAADP与含TPC2膜的结合，并研究过表达TPC2的细胞以及受RNA干扰抑制TPC2表达的细胞中NAADP诱发的Ca2+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，导致Ca2+通过TPC2通道从溶酶体释放。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TPC1-3的细胞器靶向的分类信号。这些综合研究将极大地增强我们对这些新型离子通道的认识，并揭示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（TPCs）是与电压门控钙通道相关的新型离子通道，但它们在胞内细胞器（如核内体和溶酶体）上表达，而不是在质膜上表达。其中之一，TPC2，可能参与介导钙从溶酶体释放，以响应第二信使，烟酸-腺嘌呤二核苷酸磷酸（NAADP）。该项目将阐明TPCs在细胞内囊泡运输、融合和钙信号传导等方面的分子功能，并揭示其在正常人体生理和病理生理中的潜在作用。

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.