Regulation of Cx26 and Cx32 Channels by Cytosolic Interdomain Interactions

项目摘要 缝隙连接蛋白形成缝隙连接通道，介导细胞间直接的分子通讯 在发育、生理和对创伤/炎症的反应中。连接蛋白的缺陷导致人类 病理学。阐明调节缝隙连接蛋白通道功能的分子机制对于 了解它们在人体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。这个项目的长期目标是了解胞质结构域间的相互作用。 控制缝隙连接蛋白通道的门控。这是Cx43的特征，在Cx43中， 在细胞质环(CL)和C末端结构域(CT)之间，通过pH和其他因素调节通道门控 各种因素。CL-CT介导的Cx43调节对其生理功能至关重要，是一种治疗作用 心血管疾病的靶点。然而，CL-CT相互作用在其他连接蛋白调控中的作用 在生物医学上同样重要的渠道，及其作用机制还没有被探索。 我们发现Cx26通道受CL-CT相互作用的调制。Cx26和Cx43具有代表性 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 Cx26通道功能的相互作用似乎与Cx43通道功能的相互作用有根本的不同，这表明 CL-CT相互作用是连接蛋白的一种常见的调控机制，但其作用方式是连接蛋白特有的。 我们建议阐明CL-CT控制通道功能和特性的分子机制 Cx26及其同源异构体Cx32。Cx26是唯一的连接蛋白通道，它有一个高- 分辨率结构，使其成为基于结构的所有连接蛋白通道研究的基础。 拟议的研究探索了CL-CT相互作用的基础和机制以及它们的通道特性 使用成功应用于其他渠道的策略进行调节，包括使用竞争多肽， 工程二硫键、宏观和单通道记录以及突变分析。这个 实验利用完整的通道，并辅之以多肽核磁共振。我们建议(A)厘定 CL-CT相互作用在通道门控中的参与，(B)确定CL-CT相互作用的位置，以及(C) 确定CL-CT相互作用及其影响如何因导致人类疾病的突变而改变。 Cx26和Cx32在体内分布广泛。Cx26的突变导致了超过一半的遗传 全世界的感觉神经性耳聋，还会导致严重的毁容皮肤疾病。Cx32基因突变导致 外周脱髓鞘。在这两种连接蛋白中，许多致病突变都会影响CL-CT 互动。这两种连接蛋白都与肿瘤的进展有关，并与多种病理和 生理过程。结合了高分辨率结构，大量致病 突变和两种PI在研究Cx26和Cx26的门控、通透性和调控方面的丰富经验 Cx32通道为有效、全面地研究CL-CT相互作用提供了基础。

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.