Regulation of Cx26 and Cx32 Channels by Cytosolic Interdomain Interactions

描述(申请人提供)：连接蛋白形成缝隙连接通道，在发育、生理和对创伤/炎症/疾病的反应中至关重要的直接细胞间分子通信。连接蛋白的突变会导致人类的病理。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以及确定翻译和基础科学研究的靶点是至关重要的。这个项目的长期目标是了解控制通道门控的连接蛋白的胞内结构域之间的相互作用。在连接蛋白43(Cx43)中，细胞质环(CL)和C末端结构域(CT)之间的相互作用是该通道的重要生理功能，也是心血管治疗的靶点。然而，CL-CT相互作用在调节其他连接蛋白通道中的作用和机制还没有被探索，这在生物医学上可能是重要的。我们发现Cx26通道受CL-CT相互作用的调制。Cx26和Cx43是两个最大的连接蛋白家族的代表性成员。虽然在结构上相似，但CL-CT相互作用对Cx26通道功能的影响似乎与Cx43完全不同，这表明CL-CT相互作用是连接蛋白中常见的调控机制，但以连接蛋白特有的方式发挥作用。我们建议利用Cx26及其密切相关的亚型Cx32来阐明CL-CT调控通道功能的分子机制。Cx26是唯一具有高分辨率结构的连接蛋白通道，使其成为基于结构研究所有连接蛋白通道的基石。拟议的研究探索了CL-CT相互作用的基础和机制，以及它们调节的通道特性，使用了成功应用于其他通道的策略，包括宏观和单通道记录、竞争多肽的使用、工程半胱氨酸连接和突变分析。这些实验利用完整的通道，并辅之以多肽核磁共振。我们建议(A)确定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。全世界一半以上的遗传性非综合征性感音神经性耳聋和严重的毁容皮肤疾病都是由Cx26突变引起的。Cx32突变导致外周脱髓鞘。值得注意的是，在这两个连接蛋白中，许多致病突变位于CL和CT结构域。这两种连接蛋白都参与了广泛的病理和生理过程，因此了解它们失调的基础对这些连接蛋白和其他连接蛋白具有广泛的生物医学意义。大量的致病突变以及两位PI在研究Cx26和Cx32通道的门控和通透性方面的丰富经验，为研究CL-CT相互作用、病理突变如何改变它们以及对通道功能的影响提供了富有成效的信息基础。

DESCRIPTION (provided by applicant): Connexin proteins form the gap junction channels that mediate direct intercellular molecular communication crucial in development, physiology and response to trauma/inflammation/disease. Mutations 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 inter-domain interactions of connexin proteins that control channel gating. In connexin43 (Cx43), interactions between the cytoplasmic loop (CL) and the C-terminal domain (CT) is crucially involved physiological function of the channel and is a target for cardiovascular therapies. However, the roles and mechanisms of CL-CT interactions in modulation of other connexin channels, just as likely to be biomedically important, 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 operates in connexin-specific ways. We propose to elucidate the molecular mechanisms of CL-CT control of channel function 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 cornerstone 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 macroscopic and single channel recordings, use of competing peptides, engineered Cys linkages 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. Cx26 mutations cause over half the inherited non-syndromic sensorineural deafness worldwide, and serious disfiguring skin disorders. Cx32 mutations cause a peripheral demyelination. Significantly, in both connexins many disease-causing mutations are in the CL and CT domains. Both connexins are involved in a wide range of pathological and physiological processes, so understanding the basis for their dysregulation has broad biomedical implications, for these connexins and others. The large number of disease causing mutations and the extensive experience of both PIs in studying gating and permeability of Cx26 and Cx32 channels provide a basis for productive, informative investigation of CL-CT interactions, how they are altered by pathological mutations and the effects on channel function.