Molecular physiology of TRPML channels

TRPML 通道的分子生理学

基本信息

批准号：
10006011
负责人：
Jian Yang
金额：
$ 36.26万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10006011
关键词：
Alkali Metals Alkalies Amino Acids Animals Autophagocytosis Binding Sites Biological Process Cell physiology Complex Cryoelectron Microscopy Cytoplasm Defect Development Disease Electrophysiology (science)Elements Endocytic Vesicle Endogenous Factors Endosomes Event Exocytosis Foundations Functional disorder Ganglioside Sialidase Deficiency Disease Head Histidine Homeostasis Human Incubated Ion Channel Ions Knowledge Length Link Lipid Bilayers Lipids Lumen of the Lysosome Lysosomes Maps Membrane Membrane Lipids Memory Modification Molecular Molecular Conformation Mus Mutagenesis Mutate Mutation Pathogenesis Phenotype Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Physiological Physiology Pigmentation physiologic function Play Property Regulation Rod Role Side Signal Transduction Structure System Testing Therapeutic Transducers Transmembrane Domain Vesicle Work deafness experimental study extracellular gain of function mutation human disease insight loss of function mutation mutant novel protonation receptor response sensor therapeutic development trafficking treatment strategy

项目摘要

Project Summary The endolysosomal system is essential for cell signaling and physiology. The functions of endocytic vesicles are regulated by a variety of ion channels, including the mucolipin subfamily of transient receptor potential (TRPML) channels, which are localized primarily in endosomes and lysosomes. These channels conduct Ca2+ and Na+ currents from the vesicle lumen to the cytoplasm and are critically involved in membrane trafficking, exocytosis and autophagy. Mutations in TRPML1 cause mucolipidosis type IV (ML IV), a severe lysosomal storage disorder, and mutations in TRPML3 cause deafness and pigmentation defects in mice, underscoring the crucial physiological importance of these channels. The activities of TRPML channels are strongly regulated by endogenous factors such as PIP2, pH, Na+ and Ca2+. The complex regulation in turn controls the physiological functions of these channels. The objective of this project is to elucidate the molecular mechanisms of regulation of TRPML3 by these physiological factors. TRPML3 is regulated by both common and unique mechanisms. Like other TRPMLs, TRPML3 is activated by PI(3, 5)P2 and suppressed by PI(4, 5)P2. However, it is uniquely inhibited by luminal low pH and Na+. This inhibition presumably keeps lysosomal TRPML3 inactive under physiological conditions. Neutralization or damage of lysosomes likely relieves this inhibition and activates TRPML3. We have recently solved cryo-EM structures of full length human TRPML3 in the closed, open and low-pH-inhibited states. These structures reveal a number of unique structural features and suggest new allosteric regulatory mechanisms. We have also uncovered a novel ‘Inhibition Memory’ that depends on Na+ and amino acid H283. We will build on these exciting findings and determine the structural elements and conformational changes underlying the regulation of TRPML3 by low pH, Na+, PI(3, 5)P2 and PI(4, 5)P2. We will carry out structure-guided mutagenesis studies to test the hypothesis that a luminal pore- loop and H283 are pH sensors and that transmembrane segments S1 and S2 act as allosteric transducers that convert low pH-, Na+-, and PIP2-induced local conformational changes to global conformational changes that either enhance or inhibit channel activity. We will obtain cryo-EM structures of WT and H283A mutant channels in complex with membrane lipids at different pH and with different alkali ions and of WT channels in complex with PI(3, 5)P2 or PI(4, 5)P2 at different pH and Na+ concentrations. These studies will yield rich and deep mechanistic insights into TRPML3 channel regulation and provide new knowledge for the development of therapeutic strategies for ML IV and other endocytic vesicle-related diseases.

项目概要内溶酶体系统对于细胞信号传导和生理学至关重要。内吞囊泡的功能。受多种离子通道调节，包括瞬时受体电位的粘磷脂亚家族 (TRPML) 通道，主要位于内体和溶酶体中，这些通道传导 Ca2+。和 Na+ 电流从囊泡腔到细胞质，并且关键参与膜运输， TRPML1 突变导致粘脂沉积症 IV 型 (ML IV)，这是一种严重的溶酶体疾病。储存障碍和 TRPML3 突变会导致小鼠耳聋和色素沉着缺陷，强调这些通道的至关重要的生理重要性 TRPML 通道的活性很强。受 PIP2、pH、Na+ 和 Ca2+ 等内源因素的调节，复杂的调节反过来控制着。该项目的目的是阐明这些通道的分子功能。 TRPML3 受这些生理因素的调节机制 TRPML3 受这两种共同的调节。与其他 TRPML 一样，TRPML3 被 PI(3, 5)P2 激活并被 PI(4, 5)P2。然而，它受到管腔低 pH 值和 Na+ 的独特抑制。这种抑制可能会保持溶酶体。 TRPML3 在生理条件下失活可能会缓解这种情况。我们最近解决了全长人类 TRPML3 的冷冻电镜结构。这些结构揭示了许多独特的结构特征。并提出了新的变构调节机制，我们还发现了一种新颖的“抑制记忆”。取决于 Na+ 和氨基酸 H283 我们将基于这些令人兴奋的发现并确定其结构。低 pH、Na+、PI(3, 5)P2 和 TRPML3 调节的元素和构象变化 PI(4, 5)P2。我们将进行结构引导诱变研究来检验管腔孔的假设。 Loop 和 H283 是 pH 传感器，跨膜片段 S1 和 S2 充当变构传感器，将低 pH-、Na+- 和 PIP2 诱导的局部构象变化转化为全局构象变化我们将获得 WT 和 H283A 突变通道的冷冻电镜结构。与不同 pH 值和不同碱离子的膜脂形成的复合物以及复合物中 WT 通道的复合物 PI(3, 5)P2 或 PI(4, 5)P2 在不同 pH 和 Na+ 浓度下的研究将产生丰富而深入的成果。对 TRPML3 通道调控的机制见解，为 TRPML3 通道调控的发展提供新知识 ML IV 和其他内吞囊泡相关疾病的治疗策略。