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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要本提案将清楚地描绘出独特的质子家族的分子功能敏感的离子通道，称为酸性离子通道（ASIC）。主要位于中央和周围神经系统，ASIC扮演着从合成可塑性到疼痛的各种角色。在与酸中毒有关的中诸如脑缺血之类的中枢神经系统病理，在引起神经退行性方面暗示了ASIC。这些通道通过低于7的细胞外pH中的酸性移动激活，其中溶液中的质子超过质子结合大的细胞外域，并允许孔打开。 ASIC主要居住在三个州：1。生理pH值或碱性pH，该通道处于不活跃的静止状态。 2。在温和酸化期间，通道恢复为质子结合的无活性构象，称为脱敏状态。 3。最初的重量酸化，该通道处于开放状态，但是延长的暴露率会降敏。而这些状态的晶体结构有助于阐明这些状态过渡之间的通道的潜在变化，需要实验测试才能在功能上评估它们是否与通道门控有关。阐明这是，将雇用以下两个特定目标。 AIM 1将确定构象变化是使用非规范氨基酸诱导的必要和足够的激活机制在发生巨大构象变化和测试功能后果。具体而言，此目标将确定酸性口袋和棕榈域激活。 AIM 2将评估β11-12连接器脱敏的机理首先了解Asp415的作用，ASP415是链接器内的住宅及其构象后果通过使用站点定向的诱变和光叠链接链接脱敏。此外，我们将调查通过ASIC同工型的系统发育比较β11-12接头的环境以及如何环境可以控制接头的运动，我们先前已证明它可以控制脱敏。这两个目标的结果将填补我们在ASIC门控过程中的知识中的空白，以允许开发ASIC靶向疗法。此外，这项研究将为光遗传学奠定基础通过遗传代码扩展对ASIC进行调节。这些工具将提供更精确的控制通道在体外和体内模型系统中活动。涉及此的奖学金培训计划提案将支持我在阅读，写作和沟通技巧中的增长，以成为一名成功的科学家。从技术上讲，培训计划是进一步提高我的电生理技能的指南，即贴片夹，并增强我在光遗传学，遗传代码扩展和分子建模方面的知识和技能。这罗切斯特大学和药理学与生理学系以其离子频道而闻名研究提供了完成我的奖学金培训计划所需的必要设施和心理。