Voltage-gated sodium channels in lysosomal physiology

溶酶体生理学中的电压门控钠通道

• 批准号: 9753478
• 负责人: Dejian Ren
• 金额: $ 49.83万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753478
• 关键词: Action Potentials; Aging; Alzheimer' s Disease; Autophagocytosis; Binding Proteins; Biochemical; Biophysics; Calcium; Cardiac; Cardiac Myocytes; Cell membrane; Cell physiology; Cells; Cellular biology; Closure by clamp; Complex; Coupled; Digestion; Disease; Dominant-Negative Mutation; EF Hand Motifs; Electrophysiology (science); Environment; Exocytosis; FRAP1 gene; Face; Feedback; Fibroblasts; Functional disorder; Gene Expression; Generations; Genetic study; Heart; Hormone secretion; Ion Channel; Ion Channel Protein; Kidney; Kinetics; Knock-out; Knowledge; Lysosomal Storage Diseases; Lysosomes; Mammalian Cell; Mediating; Membrane; Membrane Potentials; Metabolic; Molecular Cloning; Mus; Muscle Contraction; Neurodegenerative Disorders; Neuroglia; Neurons; Nutrient; Organelles; Parkinson Disease; Pathologic; Permeability; Phosphotransferases; Physiological; Physiology; Play; Property; Protein Chemistry; Proteins; Recycling; Regulation; Reporting; Research; Role; Shapes; Signal Transduction; Sodium; Sodium Channel; Structure; Surface; System; Testing; Tissues; biophysical analysis; biophysical properties; experimental study; extracellular; heart cell; improved; lysosome membrane; mTOR protein; macrophage; neurotransmission; novel; patch clamp; protein degradation; recruit; repaired; sensor; voltage; voltage clamp

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.

质膜的生物物理特性和调节已被主要研究了几个 几十年来，人们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传导到激素 相反，细胞内细胞器膜的生物物理特性。 在这项提案中，我们扩展了对溶酶​​体的初步研究。 膜，重点关注溶酶体钠通道 溶酶体是消化和回收中心。 它们在细胞清除、营养循环、能量产生和 溶酶体信号功能障碍会导致严重的疾病，例如溶酶体贮积病和 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定义 溶酶体膜。全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 此外，初步研究表明 TPC 通道与代谢状态相关。 和细胞的营养可用性，以及细胞器的腔内 pH 值，我们提出了三个具体目标。 扩大我们的研究将使用膜片钳记录来检验溶酶体兴奋性的假设。 广泛表达，可在兴奋性和非兴奋性细胞中发现。 代谢状态的调节和营养物质的可用性也将受到测试（目标 1）。 通过 mTOR 激酶受胞质 ATP 浓度控制。我们将使用生化方法来定义。 通道 ATP 敏感性的潜在机制（目标 2）通常具有选择性。 钠，并且有证据表明这些通道也是钙可渗透的。 In Aim 3、生化和 将进行电生理学实验来定义通道渗透及其调节 鉴于溶酶体的生理重要性，拟议的研究将有助于填补钙（目标 3）。 我们对溶酶体膜的特性及其功能的理解存在知识差距 正常和病理状态，例如神经退行性疾病。