Molecular physiology of TRPML channels

项目摘要 内溶酶体系统对于细胞信号传导和生理学是必不可少的。内吞囊泡的功能 由多种离子通道调节，包括瞬时受体电位的粘脂亚家族 （TRPML）通道，其主要位于内体和溶酶体中。这些通道传导Ca 2 + 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。TRPML 1突变导致IV型粘脂沉积症（ML IV），一种严重的溶酶体 储存障碍和TRPML 3突变导致小鼠耳聋和色素沉着缺陷，强调了 这些通道至关重要的生理意义。TRPML通道的活动强烈 受内源性因子如PIP 2、pH、Na+和Ca 2+的调节。复杂的调节反过来控制着 这些通道的生理功能。本项目的目标是阐明 这些生理因素对TRPML 3的调节机制。TRPML 3受两种常见的 独特的机制。与其他TRPML一样，TRPML 3被PI（3，5）P2激活并被PI（4， 5）P2。然而，它是唯一的抑制鲁米那低pH值和Na+。这种抑制作用可能使溶酶体 TRPML 3在生理条件下无活性。溶酶体的中和或损伤可能缓解这种情况 抑制并激活TRPML 3。我们最近已经解决了全长人TRPML 3的冷冻-EM结构， 封闭、开放和低pH抑制状态。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， 低pH、Na+、PI（3，5）P2和PI（3，5）P3对TRPML 3调控的潜在因素和构象变化 PI（4，5）P2.我们将进行结构导向诱变研究，以检验管腔孔- 环和H283是pH传感器，跨膜区段S1和S2作为变构转换器， 将低pH、Na+和PIP 2诱导的局部构象变化转化为全局构象变化， 增强或抑制通道活性。我们将获得WT和H283 A突变通道的冷冻电镜结构 在不同pH和不同碱离子下与膜脂质复合，以及复合物中WT通道 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刻的 对TRPML 3渠道监管的机械见解，并为发展提供新知识 ML IV和其他内吞囊泡相关疾病的治疗策略。

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.