Dynamics of Acid-sensing ion channels

酸敏感离子通道的动力学

• 批准号: 10218221
• 负责人: John Bankston
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10218221
• 关键词: ASIC channel; Binding; Binding Sites; Cells; Complex; Data; Distal; Drug Targeting; Electrophysiology (science); Event; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Homo; Ion Channel; Ions; Length; Macromolecular Complexes; Measures; Modeling; Molecular; Molecular Conformation; Neurodegenerative Disorders; Neurons; Pain; Pain management; Peripheral Nervous System; Prevalence; Property; Protein Family; Protein Isoforms; Proteins; Protons; Regulation; Research; Sodium; Stroke; Structure; System; Techniques; Transition Elements; Transmembrane Domain; Work; desensitization; extracellular; fluorophore; insight; interest; member; patch clamp; receptor; sensor; stoichiometry; targeted treatment; therapy design

Project Summary Acid-sensing ion channels (ASICs) are critical sensors of extracellular pH that contribute to excitability in cells in both the central and peripheral nervous system. ASICs couple the binding of extracellular protons to the opening of a sodium selective pore. Preliminary research has suggested that ASICs may be viable targets in the treatment of pain as well as ischemic events such as stroke. There are 5 ASIC isoforms that give rise to at least 7 different channel subunits. ASICs form both homo- and heterotrimers and their precise properties are governed by the channel composition. Research in my lab focuses on the molecular mechanisms underlying ASIC function and how these channels are fine tuned in neurons. Our first focus is on using cutting-edge techniques to measure conformational changes in ASICs. Using a FRET approach that replaces the donor fluorophore with a transition metal ion, we can measure channel dynamics in full-length ASICs in real cells. With these data, we can build mechanistic models for how ASICs open, close, and desensitize. Despite the prevalence of heteromeric ASIC complexes in neurons, little is known about the stoichiometry of ASIC heteromers or the mechanism of heteromer formation. Using a new fluorescence approach called spatial intensity distribution analysis (SpIDA), we will be able to look at how different ASIC isoforms heteromerize. Previous work has looked at the stoichiometry of ASIC1a/ASIC2a heteromers, but no other combination has been studied. Our work will provide the first look at heteromerization between these other ASIC combinations. In principle, this approach is also compatible with looking at stoichiometry of endogenous receptors in neurons. We will begin to build our system in that direction. Lastly, we are interested in the macromolecular complexes that ion channels form. ASICs are known to associate with the Stomatin (STOM) family of proteins. We have demonstrated that STOM binds to ASIC3 and reduces the current by almost 200-fold. In addition, we have localized the binding site for STOM on ASIC3 to two critical regions. The first is the distal C-terminus and the second in the first transmembrane domain (TM1). Extending this work, we plan to use patch clamp electrophysiology to determine the mechanism of STOM-dependent regulation of ASIC3. In addition, we hope to extend this work to include other members of the STOM family including Stomatin-like protein 3 (STOML3). Overall, these studies will provide new insights in two how ASICs function both at the structural and cellular levels.

项目总结