Structural Biology of Connexin Membrane Channels

连接蛋白膜通道的结构生物学

• 批准号: 10809113
• 负责人: Mark Jay Yeager
• 金额: $ 24.91万
• 依托单位: UNIVERSITY OF MIAMI CORAL GABLES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10809113
• 关键词: Structural; Biology; Connexin; Membrane; Channels

Abstract Connexin (Cx) proteins form hexameric hemichannels (HCs) that dock end-to-end to form gap junction channels (GJCs) across the extracellular gap, allowing intercellular exchange of nutrients, metabolites, ions and signaling molecules. Over the last 3 decades our research program has explored the structure and regulation of two Cx isoforms, Cx43 and Cx26. Each is found in many tissues. The former most notably mediates electrical conduction between cardiac myocytes, enabling the normal heartbeat, but also mediating potentially fatal cardiac arrhythmias. The latter, is most well known for its role in the inner ear; mutations of Cx26 are the predominant cause of inherited deafness. Over the last dozen years we have focused on the regulation of Cx26 channels during tissue injury, associated with Ca2+ overload and acidic pH. We determined X-ray structures of the human Cx26 GJC with and without bound Ca2+. To our surprise, the two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Computational analysis revealed that the binding of Ca2+ ions creates a positive electrostatic barrier that blocks K+ permeation. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion. To examine pH-mediated gating of Cx26 GJCs we used cryoEM and single-particle image analysis coupled with H/D exchange and crosslinking mass spectrometry. The results support a steric “ball-and-chain” mechanism in which association of the acetylated N-termini form a pore-occluding, gating particle. Building on this rigorous structural and biophysical analysis of WT channels, we are now in a position to explore (1) the effects of deafness-causing mutations of Cx26 that involve residues that participate in Ca2+ coordination, (2) the effects of mutations of residues implicated in pH regulation, (3) the structure of undocked hemichannels and (4) structures of other connexins, particularly Cx32, mutations of which cause peripheral neuropathy, and also the cardiac connexins Cx43, in the working myocardium, and Cx40, in the specialized conducting tissue. Our structural studies utilize X-ray crystallography, cryoEM, crosslinking, H/D exchange mass spectrometry (HDX) and EPR spectroscopy in a synergistic manner. Functional studies include electrophysiology and proteoliposome-based transport assays. Our research program is fortified by fruitful collaborations with 3 experts: Drs. Andrew Harris (electrophysiology and functional assays), Patrick Griffin (HDX mass spectrometry) and David Cafiso (EPR spectroscopy). Our proposed research provides an opportunity to understand aspects of GJC and HC channel function that have been long-desired, and to initiate exploration of how those structure-function properties operate in several members of the Cx family. Given the importance of proper Cx channel function in development, pathophysiology and response to disease and trauma, this understanding will have substantial biomedical impact. !

摘要 连接蛋白（Cx）蛋白形成六聚体半通道（HC），端对端对接形成间隙连接 跨细胞间隙的GJC，允许营养物质、代谢物、离子的细胞间交换 和信号分子。在过去的30年里，我们的研究计划探索了结构， 调节两种Cx同种型，Cx43和Cx 26。每一种都存在于许多组织中。前者最明显 介导心肌细胞之间的电传导，使正常的心跳，但也介导 可能致命的心律失常后者，最为人所知的是它在内耳中的作用; Cx 26是遗传性耳聋的主要原因。在过去的十几年里，我们一直专注于 组织损伤过程中Cx 26通道的调节，与Ca 2+超载和酸性pH有关。 具有和不具有结合的Ca 2+的人Cx 26 GJC的X射线结构。令我们惊讶的是，这两个结构是 几乎相同，排除了大规模的结构变化和孔的局部空间收缩。 计算分析表明，Ca 2+离子的结合产生了一个正的静电势垒， K+渗透。我们的研究结果为一种独特的通道调节机制提供了结构证据：离子通道 通过静电屏障而不是空间闭塞来阻断传导。为了检查pH介导的门控， 我们使用cryoEM和单颗粒图像分析结合H/D交换和交联来分析Cx 26 GJC。 质谱分析法来这些结果支持了空间位阻的“球链”机制， 乙酰化的N-末端形成孔闭塞的门控颗粒。基于这种严格的结构和生物物理 WT通道的分析，我们现在可以探索（1）导致遗传突变的影响， Cx 26中涉及参与Ca 2+配位的残基，（2）残基突变的影响 涉及pH调节，（3）未对接半通道的结构和（4）其他连接蛋白的结构， 特别是Cx 32，其突变引起周围神经病变，以及心脏连接蛋白Cx43， 工作心肌，和Cx40，在专门的传导组织。我们的结构研究利用X射线 晶体学，cryoEM，交联，H/D交换质谱（HDX）和EPR光谱， 协同方式。功能研究包括电生理学和基于蛋白脂质体的转运测定。 我们的研究计划得到了与3位专家富有成效的合作的加强：Andrew Harris博士（电生理学 和功能测定）、帕特里克格里芬（HDX质谱）和大卫科菲索（EPR光谱）。我们 拟议的研究提供了一个机会，了解GJC和HC通道功能的各个方面， 长期以来一直渴望，并开始探索这些结构功能特性如何在几个 Cx家族的成员。考虑到适当的Cx通道功能在发育中的重要性， 病理生理学和对疾病和创伤的反应，这种理解将具有实质性的生物医学意义。 冲击 !