Voltage-gated sodium channels in lysosomal physiology

质膜的生物物理性质和调控已经被广泛地研究了几年 几十年。人们已经发现了数百个离子通道。它们本质上调节细胞的各个方面。 生物学和生理功能，从肌肉收缩、神经元信号到激素 分泌和基因表达。相比之下，细胞内细胞器膜的生物物理性质 被调查的要少得多。在这个提议中，我们扩展了我们对溶酶体的初步研究 膜，重点是溶酶体钠通道。溶酶体是体内的消化和再循环中心 哺乳动物细胞。它们在细胞清除、营养循环、能量产生和 发信号。溶酶体功能障碍会导致严重的疾病，如溶酶体储存疾病和 包括帕金森氏症和阿尔茨海默氏症在内的神经退行性疾病。最近的电生理记录， 分子克隆、蛋白质化学和小鼠遗传学研究已经开始定义 溶酶体膜。完整的溶酶体电流钳记录发现溶酶体的一个子集 产生类似动作电位的膜去极化尖峰。产生尖峰的能力是至关重要的 依赖于由两孔重复通道1形成的新型电压门控钠渗透通道 (TPC1)蛋白质。此外，初步研究表明，TPC通道与代谢状态相耦合 细胞的养分利用率，以及细胞器的管腔pH。我们提出了三个具体目标来 扩大我们的研究范围。膜片钳记录将被用来检验溶酶体兴奋性是 广泛表达，在兴奋性和非兴奋性细胞中均有表达。兴奋性是不是 还将测试新陈代谢状态和营养素的可获得性(目标1)。TPC通道是 通过mTOR激酶由胞浆中的ATP浓度控制。我们将使用生化方法来定义 通道对三磷酸腺苷敏感性的潜在机制(目标2)。TPC通道通常是有选择性的 钠，而且有证据表明，这些通道也是钙渗透的。在目标3中，生化和 将进行电生理实验，以确定通道渗透性及其调节 钙(目标3)。鉴于溶酶体的生理重要性，拟议的研究将有助于填补 我们对溶酶体膜的性质及其功能的理解上的知识差距 正常和病理状态，如神经退行性疾病。

The biophysical properties and the regulation of plasma membranes have been extensively studied for several decades. Hundreds of ion channels have been discovered. They regulate essentially every aspect of cell biology and physiological functions, ranging from muscle contraction and neuronal signaling to hormone secretion and gene expression. In contrast, the biophysical properties of intracellular organelle membranes have been much less investigated. In this propose, we extend our preliminary studies of lysosomal membranes, with focus on lysosomal sodium channels. Lysosomes are the digestion and recycling center in mammalian cells. They play central roles in cellular clearance, nutrient recycling, energy generation and signaling. Dysfunction of lysosomes leads to severe diseases such as lysosomal storage diseases and neurodegenerative diseases including Parkinson’s and Alzheimer’s. Recent electrophysiological recordings, molecular cloning, protein chemistry and mouse genetics studies have started to define the properties of lysosomal membranes. Whole-lysosome current-clamp recording has discovered that a subset of lysosomes generate action potential-like membrane depolarization spikes. The ability to generate spikes is critically dependent on a novel voltage-gated sodium-permeable channel formed by the two-pore repeat channel 1 (TPC1) protein. In addition, preliminary studies suggest that TPC channels are coupled to the metabolic state and nutrient availability of the cell, and to the luminal pH of the organelle. We propose three specific aims to expand our studies. Patch clamp recordings will be used to test the hypothesis that lysosomal excitability is widely expressed and can be found in both excitable and non-excitable cells. Whether the excitability is regulated by metabolic state and the availability of nutrients will also be tested (Aim 1). TPC channels are controlled by cytosolic ATP concentration via the mTOR kinase. We will use biochemical approaches to define the mechanisms underlying the channel’s ATP sensitivity (Aim 2). TPC channels are generally selective for sodium, and evidences suggest that the channels are also calcium-permeable. In Aim 3, biochemical and electrophysiological experiments will be performed to define the channel permeation and its regulation by calcium (Aim 3). Given the physiological importance of lysosomes, the proposed studies will help fill the knowledge gap in our understanding of the properties of lysosomal membranes and their functions under normal and pathological states such as neurodegenerative diseases.