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Acid-Sensing Ion Channel gating: Conformations and Consequences

酸敏感离子通道门控：构象和后果

基本信息

批准号：
10027391
负责人：
David Malcom MacLean
金额：
$ 38.5万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10027391
关键词：
ASIC channel Ablation Acids Address Amino Acids Attenuated Baroreflex Basic Science Biophysics Brain Cardiac Cell Death Chickens Coupling Cryoelectron Microscopy Crystallization Drug Design Electrophysiology (science)Event Exhibits Extracellular Domain Fluorescence Resonance Energy Transfer Fright Genetic Impairment Investigation Ion Channel Gating Ion Channel Protein Ischemic Stroke Knowledge Label Lead Learning Link Maps Mediating Membrane Proteins Methods Molecular Molecular Conformation Motion Movement Nervous system structure Neurons Optical Methods Pain Pharmacology Phenotype Proteins Regulation Reporting Rest Role Signal Transduction Site Structure Testing Toxin cancer cell cell type desensitization drug development epithelial to mesenchymal transition experimental study extracellular fluorescence lifetime imaging innovation insight operation patch clamp protein protein interaction protonation response scaffold structured data therapeutic development

项目摘要

PROJECT SUMMARY/ABSTRACT Extracellular pH is a highly dynamic and ubiquitous signal and many cell types exhibit robust electrophysiological responses upon extracellular acidification, particularly in the nervous system. Acid-sensing ion channels (ASICs) are thought to mediate the majority of these responses since various ASIC subunits are expressed at high levels in many neuronal types and genetic ablation of ASIC subunits dramatically reduces acid-evoked responses. Consequently, ASICs are vital players in cell death following ischemic stroke. However, ASICs are more than simply an `extracellular acid alarm'. Genetic deletion, or pharmacological manipulation, of specific ASIC subunits can produce a wide array of phenotypes ranging from attenuated fear learning, deficits in pain and mechanosensation, problems in cardiac autonomic regulation and the baroreflex and impairment of the epithelial to mesenchymal transition in various cancer cells. These myriad roles of ASICs have motivated structural and biophysical investigation, leading to crystal or cryo-EM structures of chicken ASIC1 in the resting, toxin-stabilized open and desensitized states. This structural data, combined with functional experiments, have led to a general outline of how ASICs function. Briefly, protonation of key acidic residues in the extracellular domain, in regions known as the palm domain and the acidic pocket, leads to global rearrangements of the extracellular domain as well as channel gating. Yet we lack a clear picture of the molecular events linking protonation with these observed conformational changes and activation or desensitization. To address this gap in our knowledge, we will combine electrophysiology with the power of photo-responsive non-canonical amino acids to test specific molecular hypotheses of protonation-gating coupling. In addition, there has been no structural data for the ASIC intracellular domains. To address this gap, we will employ a combination of fluorescence lifetime imaging, as well as patch clamp FRET to map the overall topology and motions of the intracellular domain using innovative methods of site specific labelling. Finally, despite some reports of ASIC protein-protein interactions and signal transduction capacity, we lack a clear picture of how ASICs scaffold with other proteins. We will address this final knowledge gap using targeted protein labeling and downstream analysis of interactions. Taken together, these proposed experiments will provide new insights into the operation of these important signaling entities, and may help guide drug development.

项目总结/文摘