Structural Biology of Connexin Membrane Channels

摘要 连接蛋白(Cx)形成六聚体半通道(Hcs)，端对端对接形成缝隙连接 穿过细胞外间隙的通道(GJCs)，允许细胞间营养物质、代谢物、离子的交换 和信号分子。在过去的30年里，我们的研究计划探索了 Cx43和Cx26两种Cx亚型的调控。每一种都存在于许多组织中。前者最引人注目的是 介导心肌细胞之间的电传导，使正常的心跳，但也调解 潜在致命的心律失常。后者最为人所知的是它在内耳中的作用； Cx26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 Cx26通道在组织损伤过程中的调节，与钙超载和酸性pH有关。我们决定 结合钙和不结合钙的人Cx26 GJC的X射线结构令我们惊讶的是，这两座建筑 几乎相同，既排除了大规模的结构变化，也排除了毛孔的局部立体收缩。 计算分析表明，钙离子的结合产生了一个正的静电屏障，阻止了 钾渗透。我们的结果为离子通道调节的一种独特机制提供了结构性证据 通过静电屏障而不是立体闭塞的传导阻断。检查pH介导的门控 Cx26 GJCs我们使用了低温电子显微镜和单粒子图像分析，并结合H/D交换和交联 质谱学。研究结果支持一种立体的“球和链”机制，在这种机制中， 乙酰化的N-末端形成封闭气孔的门控颗粒。建立在这种严谨的结构和生物物理基础上 分析WT通道，我们现在能够探索(1)导致耳聋的突变的影响 涉及参与钙配位的残基的Cx26，(2)残基突变的影响 与pH调节有关，(3)非对接半突的结构和(4)其他连接蛋白的结构， 特别是Cx32，它的突变导致周围神经病变，以及心脏连接蛋白Cx43，在 工作心肌和Cx40，在专门的传导组织中。我们的结构研究利用X射线 结晶学、低温EM、交联、H/D交换质谱学和电子顺磁共振波谱 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分析。 我们的研究计划通过与三位专家富有成效的合作而得到加强：安德鲁·哈里斯博士(电生理学 和功能分析)、Patrick Griffin(HDX质谱学)和David Cafiso(EPR光谱学)。我们的 拟议的研究为了解GJC和HC通道功能的各个方面提供了机会 ，并开始探索这些结构-功能属性如何在几个 CX家族的成员。鉴于适当的CX通道功能在开发中的重要性， 对于疾病和创伤的病理生理学和反应，这一认识将具有实质性的生物医学意义 冲击力。 好了！

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. !