Structural basis of the gating mechanism of connexin 26 and related hearing loss

连接蛋白26门控机制及相关听力损失的结构基础

• 批准号: 10303738
• 负责人: Weihua Qiu
• 金额: $ 22.9万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-09 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303738
• 关键词: Amino Acids; Biochemical; Biological Assay; Calcium; Calcium ion; Calmodulin; Cell membrane; Cell physiology; Cells; Chemicals; Clinical; Complex; Connexins; Cryoelectron Microscopy; Data; Detergents; Dyes; Environment; Family; Functional disorder; GJB2 gene; Glean; Hearing problem; Hela Cells; Human; Human body; Ion Exchange; Lead; Lipids; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Mutation; Outcome; Pathology; Physiological; Physiological Processes; Play; Point Mutation; Polymers; Proteins; Protocols documentation; Resolution; Role; Sampling; Second Messenger Systems; Senter syndrome; Solid; Structure; System; Testing; Transmembrane Domain; base; deaf; density; gap junction channel; hearing impairment; improved; insight; intercellular communication; interest; member; mutant; nanoparticle; novel strategies; particle; protein complex; small molecule; therapeutic protein

SUMMARY Connexin 26, as a gap junction channel, directly regulates the cell-to-cell exchange of ion, metabolites, and second messengers. Mutations of connexin 26 can cause severe hearing loss. However, the molecular gating mechanism on the structural basis of connexin 26 is still unclear, especially the closed state and malfunction of connexin 26 mutations. We have developed the native cell membrane nanoparticle (NCMN) system for high-resolution single-particle cryo-EM structure determination and functional studies of membrane proteins within their physiological conditions. We recently solved the cryo-EM structures of connexin 26 in the absence or presence of calcium ions in an open state at 2.3 Å and 2.9 Å associated with about 36 lipid molecules via the NCMN system. However, the close state of connexin 26 with high resolution is still uncovered. We hypothesize that our NCMN system's application to structure determination of Cx26 and Cx26-R75W in the presence and absence of calcium and calmodulin can provide unique structural information. Aim 1: To investigate the CLOSED conformation of the gating mechanism of Cx26. Aim 2: To elucidate the molecular basis of how the single point mutation R75W in Cx26 leads to profound hearing loss. The proposed experimental results will provide insights into detailed structural information and biochemical analysis in understanding the gating mechanism, also provide mechanistic details on abnormal channel gating function detected in this hearing disorder. The structural information may lead to some novel strategy to modulate the malfunction CX26-R75W for hearing loss.

概括 连接蛋白26作为间隙连接通道，直接调节离子的细胞间交换， 代谢物和第二使者。连接蛋白26的突变会导致严重的听力损失。 但是，连接蛋白26的结构基础上的分子门控机制尚不清楚， 特别是连接蛋白26突变的封闭状态和故障。我们已经开发了 用于高分辨率单粒子冷冻EM的天然细胞膜纳米颗粒（NCMN）系统 膜蛋白在其生理中的结构确定和功能研究 状况。我们最近在不存在或存在的情况下解决了连接蛋白26的冷冻EM结构 在2.3Å处的开放状态下的钙离子与约36个脂质分子通过 NCMN系统。但是，仍然发现具有高分辨率的连接蛋白26的近距离状态。 我们假设我们的NCMN系统在结构CX26和 CX26-R75W在存在和不存在钙和Calmofluin的情况下可以提供独特的 结构信息。目标1：研究门控机制的闭合构象 CX26。目标2：阐明单点突变R75W中的分子基础 导致大量听力损失。提出的实验结果将为您提供见解 详细的结构信息和生化分析在理解门控机制时， 还提供有关此听证会中检测到的异常通道门控函数的机械详细信息 紊乱。结构信息可能会导致一些新的策略来调节故障 CX26-R75W用于听力损失。