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

项目概要本提案将清晰地描绘一个独特的质子家族的分子功能， 敏感离子通道，称为酸敏感离子通道（ASIC）。主要位于中部， 在外周神经系统中，ASIC发挥着从突触可塑性到疼痛的广泛作用。在酸中毒相关 中枢神经系统病理学如脑缺血、ASIC与引起神经变性有关。这些 细胞外pH值低于7时，通道被酸性变化激活，其中溶液中过量的质子与通道结合， 大的细胞外结构域并允许孔打开。ASIC主要分布在三个州：1。在 在生理或碱性pH下，通道处于非活性静息状态。2.在轻度酸化过程中， 通道恢复到质子结合的非活性构象，称为脱敏状态。3.在初始重 酸化时，通道处于开放状态，但长时间暴露，通道脱敏。而 这些状态的晶体结构有助于阐明这些状态转变之间的沟道中的潜在变化， 需要实验测试以在功能上评估它们是否与通道门控相关。阐明 这个，具体有以下两个目标。目标1将确定构象变化， 必要和充分的激活机制，使用非规范氨基酸掺入诱导 在经历大的构象变化的区域发生光遗传交联和侧链异构化， 测试功能性后果。具体来说，这一目标将决定酸性口袋的贡献， 掌域激活。目的2：探讨β11-12连接子调控脱敏的机制。 首先了解Asp 415的作用，连接子中的一个残基，以及它在 通过使用定点突变和光交联进行脱敏。此外，我们将调查 通过ASIC亚型的系统发育比较，以及β11-12接头的周围环境如何影响ASIC亚型的系统发育， 环境可以控制连接体的运动，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对ASIC门控过程的知识空白， ASIC靶向治疗的发展。此外，本研究将为光遗传学奠定基础。 通过遗传密码扩展来调节ASIC。这些工具将提供更精确的控制， 在体外和体内模型系统中的通道活性。与此同时， 这份建议书将支持我在阅读、写作和沟通技巧方面的成长，使我成为一名成功的科学家。 从技术上讲，训练计划作为一个指导，以进一步提高我的电生理技能，即膜片钳， 并加强我在光遗传学、遗传密码扩展和分子建模方面的知识和技能。的 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，提供必要的设施和指导，以完成我的研究金培训计划。

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.