Regulation of Cx26 and Cx32 Channels by Cytosolic Interdomain Interactions

描述（由申请人提供）：连接蛋白形成间隙连接通道，介导直接的细胞间分子通信，在发育，生理和对创伤/炎症/疾病的反应中至关重要。连接蛋白的突变引起人类疾病。阐明调节连接蛋白通道功能的分子机制对于理解它们在人体生理和病理生理中的作用，以及确定转化和基础科学研究的目标至关重要。该项目的长期目标是了解控制通道门控的连接蛋白的细胞质结构域间相互作用。在connexin43 （Cx43）中，细胞质环（CL）和c端结构域（CT）之间的相互作用对通道的生理功能至关重要，并且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，可能在生物医学上很重要，尚未被探索。我们发现Cx26通道被CL-CT相互作用调制。Cx26和Cx43是两个最大的连接蛋白家族的代表成员。虽然在结构上类似，但CL-CT相互作用对Cx26通道功能的影响似乎与Cx43有根本不同，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的方式起作用。我们建议利用Cx26及其密切相关的异构体Cx32来阐明CL-CT控制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，是所有连接蛋白通道基于结构研究的基石。这些研究探索了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、竞争肽的使用、工程Cys连接和突变分析。实验利用完整的通道，辅以肽核磁共振。我们建议(a)确定CL-CT相互作用参与通道门控，(b)确定CL-CT相互作用的位点，以及(c)确定导致人类疾病的突变如何改变CL-CT相互作用及其影响。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损性皮肤病都是由Cx26突变引起的。Cx32突变引起外周脱髓鞘。值得注意的是，在这两种连接蛋白中，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。大量的致病突变和pi在研究Cx26和Cx32通道的门控和通透性方面的丰富经验为CL-CT相互作用、它们如何被病理突变改变以及对通道功能的影响提供了富有成果的信息性研究基础。

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.