Regulation of Cx26 and Cx32 Channels by Cytosolic Interdomain Interactions

项目摘要 连接蛋白形成间隙连接通道，介导直接的细胞间分子通讯， 在发育、生理学和对创伤/炎症的反应中。连接蛋白的缺陷导致人类 病理学阐明调节连接蛋白通道功能的分子机制对于 了解它们在人类生理学和病理生理学中的作用，并确定翻译和 基础科学研究。本项目的长期目标是了解胞质结构域间的相互作用 控制连接蛋白通道的门控。这已被Cx43表征，其中相互作用 在细胞质环（CL）和C-末端结构域（CT）之间介导pH和其他通道门控。 因素CL-CT介导的Cx43调节对于其生理功能至关重要，并且是治疗性的。 用于心血管疾病。然而，CL-CT相互作用在调节其他连接蛋白中的作用， 通道，就像生物医学上一样重要，它们的作用机制还没有被探索。 我们已经发现，Cx 26通道的调制CL-CT相互作用。Cx 26和Cx43是代表性的 两个最大的连接蛋白家族的成员。虽然结构相似，但CL-CT的效果 对Cx 26通道功能相互作用似乎与Cx43中的相互作用根本不同，这表明 CL-CT相互作用是连接蛋白中常见的调节机制，但以连接蛋白特异性方式起作用。 我们建议阐明CL-CT控制通道功能和特性的分子机制， Cx 26及其密切相关的同种型Cx 32。Cx 26是唯一的连接蛋白通道， 分辨率结构，使其成为所有连接蛋白通道的结构基础研究的基础。 这些研究探索了CL-CT相互作用的基础和机制，以及它们的通道特性。 调节，使用成功应用于其他通道的策略，包括使用竞争肽， 工程化二硫键、宏观和单通道记录以及突变分析。的 实验利用完整的通道，补充肽NMR。我们建议（a）确定 CL-CT相互作用参与通道门控，（B）鉴定CL-CT相互作用的位点，和（c） 确定CL-CT相互作用及其影响如何被导致人类疾病的突变改变。 Cx 26和Cx 32在体内分布广泛。Cx 26的突变导致了超过一半的遗传性 感音神经性耳聋是世界范围内的常见病，也会导致严重的皮肤病毁容。Cx 32突变导致 外周脱髓鞘在这两种连接蛋白中，许多致病突变定位于影响CL-CT 互动两种连接蛋白都涉及肿瘤进展和多种病理学和免疫学改变。 生理过程。高分辨率结构的组合，大量的疾病引起的 突变和两名PI在研究Cx 26的门控、渗透性和调节方面的丰富经验， Cx 32通道为CL-CT相互作用的有效、全面的研究提供了基础。

Project Summary Connexin proteins form gap junction channels that mediate direct intercellular molecular communication crucial in development, physiology and response to trauma/inflammation. Defects in connexins cause human pathologies. Elucidation of the molecular mechanisms that regulate connexin channel function is essential for understanding their roles in human physiology and pathophysiology, and to identify targets for translational and basic science studies. The long-term goal of this project is to understand the cytosolic interdomain interactions that control the gating of connexin channels. This has been characterized for Cx43, in which interactions between the cytoplasmic loop (CL) and the C-terminal domain (CT) mediate channel gating by pH and other factors. The CL-CT mediated regulation of Cx43 is crucial for its physiological function, and is a therapeutic target for cardiovascular pathologies. However, the role of CL-CT interactions in modulation of other connexin channels, just as likely to be biomedically important, and their mechanism of action have not been explored. We have found that Cx26 channels are modulated by CL-CT interactions. Cx26 and Cx43 are representative members of the two largest families of connexins. Though structurally analogous, the effects of CL-CT interaction on Cx26 channel function seem to be fundamentally different from those in Cx43, suggesting that CL-CT interaction is a common modulatory mechanism in connexins, yet operate in connexin-specific ways. We propose to elucidate the molecular mechanisms of CL-CT control of channel function and properties using Cx26 and its closely related isoform Cx32. Cx26 is the only connexin channel for which there is a high- resolution structure, making it the basis for structure-based studies of all connexin channels. The proposed studies explore the basis and mechanisms of CL-CT interactions and channel properties they modulate, using strategies successfully applied to other channels, including use of competing peptides, engineered disulfide linkages, macroscopic and single channel recordings, and mutational analysis. The experiments utilize intact channels, complemented by peptide NMR. We propose to (a) determine the involvement of CL-CT interactions in channel gating, (b) identify the sites of CL-CT interactions, and (c) determine how CL-CT interaction and its effects are altered by mutations that cause human disease. Cx26 and Cx32 are widely distributed in the body. Mutations of Cx26 are responsible for over half the inherited sensorineural deafness worldwide, and also cause serious disfiguring skin disorders. Mutations of Cx32 cause a peripheral demyelination. In both connexins, many disease-causing mutations are positioned to affect CL-CT interaction. Both connexins are implicated tumor progression and a wide variety of pathological and physiological processes. The combination of a high resolution structure, a large number of disease causing mutations and the extensive experience of both PIs in studying gating, permeability and regulation of Cx26 and Cx32 channels provide a basis for productive, comprehensive investigation of CL-CT interactions.