Structural Biology of Connexin Membrane Channels

连接蛋白膜通道的结构生物学

• 批准号: 10033332
• 负责人: Mark Jay Yeager
• 金额: $ 52.29万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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/10033332
• 关键词: 3-Dimensional; Arrhythmia; Atomic Force Microscopy; Behavior; Binding; Biological Assay; Caliber; Cardiac; Cardiac Myocytes; Cells; Charcot-Marie-Tooth Disease; Charge; Classification; Collaborations; Computer Analysis; Connexin 43; Connexins; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Deuterium; Development; Disease; Disulfides; Docking; Electron Spin Resonance Spectroscopy; Electrophysiology (science); Electrostatics; Event; Family; Florida; Fruit; Functional disorder; GJB2 gene; Gap Junctions; Hearing; Heart; Homology Modeling; Human; Hydrogen; Image; Image Analysis; Ion Channel; Ions; Iris; Labyrinth; Maps; Mass Spectrum Analysis; Mediating; Membrane; Metabolic; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mutation; Myocardium; N-terminal; Nutrient; Peripheral Nerves; Peripheral Nervous System Diseases; Physiological; Physiological Processes; Positioning Attribute; Property; Protein Isoforms; Proteins; Regulation; Research; Resolution; Roentgen Rays; Role; Signaling Molecule; Spectrum Analysis; Structure; Surface; Techniques; Testing; Tissues; Trauma; X-Ray Crystallography; alpha helix; base; biophysical analysis; constriction; crosslink; deafness; experimental study; extracellular; flexibility; gap junction channel; genetic deafness; insight; interest; member; mutant; nanodisk; particle; polypeptide; programs; proteoliposomes; response; small molecule; structural biology; success; sudden cardiac death; tissue injury

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. !

抽象的 连接蛋白（CX）蛋白质形成的六聚体半通道（HCS），端到端靠座以形成间隙连接 横跨细胞外间隙的通道（GJC），允许营养，代谢物，离子的细胞间交换 和信号分子。在过去的三十年中，我们的研究计划探索了结构和 调节两个CX同工型CX43和CX26。每个组织都在许多组织中发现。前者最著名的 介导心肌细胞之间的电传导，使得正常的心跳，但也介导 潜在的致命心律不齐。后者以其在内耳中的作用而闻名。突变 CX26是遗传耳聋的主要原因。在过去的十几年中，我们专注于 在组织损伤过程中调节CX26通道，与Ca2+过载和酸性pH有关。我们确定 具有和没有结合Ca2+的人CX26 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，Ca2+离子的结合产生了一个阳性静电屏障，可以阻塞 K+渗透。我们的结果为通道调节的独特机制提供了结构证据：离子 通过静电屏障而不是空间阻塞传导阻滞。检查pH介导的门控 CX26 GJC我们使用了冷冻和单粒子图像分析，并与H/D交换和交联 质谱法。结果支持一个空间的“球链”机制 乙酰化的N末端形成了孔隙闭合的门控颗粒。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）引起死亡的突变的影响 CX26涉及参与Ca2+协调的残留物，（2）残留物突变的影响 在pH调节中实施，（3）未循环的半通道和（4）其他连接素的结构， 特别是CX32，其突变引起周围神经病，也引起心脏连接素CX43 在专门的导电组织中工作的心肌和CX40。我们的结构研究利用X射线 晶体学，冷冻，交联，H/D交换质谱法（HDX）和EPR光谱法 协同方式。功能研究包括电生理学和基于蛋白质体的运输测定法。 我们的研究计划与3位专家：DRS的富有成果的合作加强了。安德鲁·哈里斯（Andrew Harris）（电生理学 和功能分析），帕特里克·格里芬（HDX质谱法）和大卫·卡菲索（EPR光谱法）。我们的 拟议的研究提供了了解具有GJC和HC渠道功能方面的机会 长期以来一直是探索这些结构功能在几个中如何运作的探索 CX家族的成员。考虑到适当的CX通道功能在开发中的重要性， 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 影响。 呢