Voltage-gated sodium channels in lysosomal physiology

生物物理性质和质膜的调节已经被广泛研究了几个 几十年已经发现了数百种离子通道。它们基本上调节细胞的各个方面 生物学和生理功能，从肌肉收缩和神经信号到激素 分泌和基因表达。相反，细胞内细胞器膜的生物物理特性 很少被调查。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、能量产生和代谢中起着重要作用。 发信号。溶酶体的功能障碍导致严重的疾病，例如溶酶体贮积病， 神经退行性疾病，包括帕金森氏症和阿尔茨海默氏症。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确定 溶酶体膜全溶酶体电流钳记录发现， 产生动作电位样的膜去极化尖峰。产生尖峰的能力至关重要 依赖于由双孔重复通道1形成的新型电压门控钠渗透通道 （TPC 1）蛋白。此外，初步研究表明，TPC通道与代谢状态耦合 和细胞的营养物质利用率，以及细胞器的腔pH。我们提出三个具体目标， 扩大我们的研究。将使用膜片钳记录来检验溶酶体兴奋性降低的假设。 广泛表达，并且可以在可兴奋和不可兴奋的细胞中发现。无论兴奋性是 还将测试受代谢状态调节的营养素和营养素的可用性（目标1）。TPC信道是 通过mTOR激酶由胞质ATP浓度控制。我们将使用生物化学方法来定义 通道ATP敏感性的机制（目的2）。TPC信道通常是选择性的， 钠，并且证据表明通道也是钙可渗透的。在目标3中，生物化学和 将进行电生理学实验以确定通道渗透及其调节， 钙（目标3）。考虑到溶酶体的生理重要性，拟议的研究将有助于填补 我们在理解溶酶体膜的性质及其功能方面的知识差距， 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.