Molecular physiology of TRPML channels

项目摘要 内溶酶体系统是细胞信号和生理所必需的。内吞囊泡的功能 受多种离子通道的调节，包括瞬时受体电位的粘脂亚家族 (TRPML)通道，主要定位于内小体和溶酶体。这些通道传导钙离子 和Na+电流从囊泡腔到细胞质，并在膜运输中起关键作用。 胞吐和自噬。TRPML1基因突变导致一种严重的溶酶体--IV型粘脂沉积症 储存障碍和TRPML3突变导致小鼠耳聋和色素缺陷，强调 这些通道的关键生理重要性。TRPML通道的活跃性很强 受PIP2、pH、Na+、Ca~(2+)等内源因子调控。复杂的监管反过来又控制着 这些通道的生理功能。这个项目的目标是阐明分子 这些生理因素对TRPML3的调节机制。TRPML3由共同的 和独特的机制。与其他TRPML一样，TRPML3被PI(3，5)P2激活并被PI(4， 5)P2。然而，低pH值和低Na+对其有独特的抑制作用。这种抑制作用可能使溶酶体 TRPML3在生理条件下处于失活状态。溶酶体的中和或损伤可能会缓解这种情况 抑制并激活TRPML3。我们最近已经解决了全长人TRPML3的冷冻-EM结构 闭合、开放和低pH抑制状态。这些结构显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的“抑制记忆” 依赖于Na+和氨基酸H283。我们将在这些令人兴奋的发现的基础上，确定 低pH，Na+，PI(3，5)P2和低pH，Na+，PI(3，5)P2和 PI(4，5)P2.我们将进行结构导向的突变研究，以检验腔内毛孔- 环和H283是pH传感器，而跨膜片段S1和S2充当变构换能器， 将低pH、Na+和PIP2诱导的局部构象变化转化为 增强或抑制通道活性。我们将获得WT和H283A突变通道的冷冻-EM结构 不同pH、不同碱性离子与膜脂的络合物及络合物中WT通道的变化 PI(3，5)P2或PI(4，5)P2在不同的pH和Na+浓度下。这些研究将产生丰富而深入的成果 对TRPML3渠道调控的机械性见解，为 ML IV和其他内细胞性囊泡相关疾病的治疗策略。

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.