Optical interrogation of acid-sensing ion channel activation and desensitization through genetic code expansion

通过遗传密码扩展对酸敏感离子通道激活和脱敏进行光学询问

• 批准号: 10312523
• 负责人: Matthew L Rook
• 金额: $ 4.59万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312523
• 关键词: ASIC channel; Acidosis; Amino Acids; Area; Automobile Driving; Binding; Biological Models; Biophysics; Brain; Cells; Cerebral Ischemia; Cerebrum; Communication; Crystallization; Development; Disease; Drug Design; Electrophysiology (science); Environment; Extracellular Domain; Family; Fellowship; Fright; Funding; Future; Genetic Code; Goals; Growth; In Vitro; Individual; Ion Channel; Ion Channel Gating; Kinetics; Knowledge; Lead; Learning; Link; Mentors; Methods; Modeling; Molecular; Molecular Conformation; Movement; Mutagenesis; Nerve Degeneration; Nervous system structure; Neurons; Optics; Outcome; Pain; Paint; Parkinson Disease; Pathology; Peptides; Peripheral Nervous System; Permeability; Pharmacology; Phylogenetic Analysis; Physiological; Physiology; Play; Positioning Attribute; Process; Protein Isoforms; Protons; Pruritus; Reading; Regulation; Reporting; Research; Rest; Role; Rotation; Scientist; Sensory Process; Side; Site; Site-Directed Mutagenesis; Sodium; Sodium Channel; Stretching; Structure; Synaptic plasticity; Techniques; Testing; Time; Toxin; Training; Universities; Writing; alkalinity; base; beta pleated sheet; brain tissue; crosslink; desensitization; extracellular; in vivo Model; molecular modeling; optogenetics; patch clamp; sensor; skills; targeted treatment; therapeutic target; tool; virtual

Project Summary This proposal will paint a clear picture of the molecular function of a unique family of proton- sensitive ion channels, known as Acid-sensing ion channels (ASICs). Located primarily in the central and peripheral nervous system, ASICs play a wide range of roles from synaptic plasticity to pain. In acidosis-related CNS pathologies like cerebral ischemia, ASICs have been implicated in causing neurodegeneration. These channels are activated by acidic shifts in extracellular pH below 7, where excess protons in solution bind the large extracellular domain and allow for the pore to open. ASICs primarily reside in three states: 1. At physiological or alkaline pH, the channel resides in an inactive resting state. 2. During mild acidification, the channel reverts to a proton-bound, inactive conformation known as the desensitized state. 3. Upon initial heavy acidification, the channel is in an open state, but over prolonged exposures, the channel desensitizes. While the crystal structures of these states help illuminate potential changes in the channel between these state transitions, experimental testing is required to functionally assess whether they are pertinent to channel gating. To elucidate this, the two following specific aims will be employed. Aim 1 will determine conformational changes that are necessary and sufficient for the mechanisms of activation using noncanonical amino acid incorporation to induce optogenetic crosslinking and sidechain isomerization at regions that undergo large conformational changes and test the functional consequences. Specifically, this aim will determine the contributions of the acidic pocket and palm domain to activation. Aim 2 will evaluate the mechanism of β11-12 linker regulation of desensitization by first understanding the role of Asp415, a residue within the linker, and its conformational consequences in desensitization through the use site-directed mutagenesis and photocrosslinking. Further, we will investigate the surrounding environment of the β11-12 linker by a phylogenetic comparison of ASIC isoforms and how this environment can control the movement of the linker, which we have previously shown to control desensitization. The outcomes of these two aims will fill the gaps in our knowledge for ASIC gating processes to allow for the development of ASIC-targeted therapeutics. Furthermore, this study will lay the groundwork for optogenetic modulation of ASICs through genetic code expansion. These tools will provide more precise control over the channels activity in in vitro and in vivo model systems. The fellowship training plan that accompanies this proposal will support my growth in reading, writing and communication skills to become a successful scientist. Technically, the training plan acts as a guide to further enhance my electrophysiological skills, i.e. patch clamp, and bolster my knowledge and skill in optogenetics, genetic code expansion and molecular modelling. The University of Rochester and the Department of Pharmacology and Physiology is renowned for its ion channel research, which provides the necessary facilities and mentoring required to complete my fellowship training plan.

项目概述这项提案将清楚地描绘出一种独特的质子家族的分子功能-- 敏感离子通道，称为酸敏离子通道(ASIC)。主要位于中部和 在周围神经系统中，ASICs发挥着从突触可塑性到疼痛的广泛作用。与酸中毒相关 中枢神经系统的病理，如脑缺血，ASICs被认为与导致神经退行性变有关。这些 细胞外pH低于7的酸性变化激活了通道，其中溶液中过量的质子结合了 较大的胞外区域，并允许毛孔打开。ASIC主要驻留在三个状态：1.在 生理或碱性pH时，通道处于不活跃的休眠状态。2.在轻度酸化过程中， 通道回复到质子结合的、不活跃的构象，称为脱敏状态。3.在最初沉重的情况下 酸化时，通道处于打开状态，但长时间暴露后，通道会变得不敏感。而当 这些状态的晶体结构有助于照亮这些状态转变之间的通道中的潜在变化， 需要进行实验测试，以从功能上评估它们是否与通道选通有关。为了澄清 对此，将采用以下两个具体目标。目标1将确定以下构象变化 非规范氨基酸掺入诱导激活机制的充要条件 发生较大构象变化的区域的光生交联和侧链异构化 测试功能后果。具体地说，这一目标将确定酸性口袋和 掌上域激活。目的2评价β11-12接头调节脱敏的机制。 首先了解Asp415的作用，连接子中的残基，以及它在 通过使用定点诱变和光交联法脱敏。此外，我们将调查 通过ASIC11-12连接子亚型的系统发育比较研究β11-12连接子的周围环境 环境可以控制连接子的运动，我们之前已经证明了它可以控制脱敏。 这两个目标的结果将填补我们在ASIC门控工艺方面的知识空白，以允许 ASIC靶向疗法的发展。此外，本研究还将为光遗传学研究奠定基础。 通过遗传密码扩展对ASIC进行调制。这些工具将提供对 在体外和体内模型系统中的通道活性。随之而来的团契培训计划 提案将支持我在阅读、写作和沟通能力方面的增长，以成为一名成功的科学家。 从技术上讲，训练计划起到了指导作用，进一步提高了我的电生理技能，即膜片钳， 并增强了我在光遗传学、遗传密码扩展和分子建模方面的知识和技能。这个 罗切斯特大学药理学和生理学系以其离子通道而闻名 研究，它提供了完成我的团契培训计划所需的必要设施和指导。