Regulation of Cx26 and Cx32 Channels by Cytosolic Interdomain Interactions

胞浆域间相互作用对 Cx26 和 Cx32 通道的调节

• 批准号: 9041629
• 负责人: Jorge Enrique Contreras
• 金额: $ 29.7万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9041629
• 关键词: Address; Affect; Basic Science; Biochemical; C-terminal; Cardiovascular Pathology; Cardiovascular system; Cell Proliferation; Communication; Complement; Connexin 43; Connexins; Cysteine; Defect; Demyelinations; Development; Disease; Electrophysiology (science); Engineering; Family; Functional disorder; GJB2 gene; Generations; Goals; Health; Human; Human Pathology; Inflammation; Inherited; Intervention; Investigation; Mammalian Cell; Mediating; Molecular; Mutate; Mutation; Oocytes; Pathologic Processes; Pathology; Peptides; Peripheral; Permeability; Physiological; Physiological Processes; Physiology; Point Mutation; Positioning Attribute; Property; Protein Isoforms; Proteins; Regulation; Resolution; Role; Sensorineural Hearing Loss; Signal Transduction; Site; Structure; Translational Research; Trauma; Work; base; deafness; disease-causing mutation; experience; gap junction channel; human disease; in vivo; insight; member; mutant; nervous system disorder; reconstitution; research study; response; skin disorder; therapeutic target; tumor progression

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.

描述（由申请方提供）：连接蛋白蛋白形成差距连接通道，介导在发育、生理学和创伤/炎症/疾病反应中至关重要的直接细胞间分子通讯。连接蛋白的突变会导致人类疾病。阐明调节连接蛋白通道功能的分子机制对于理解其在人类生理学和病理生理学中的作用以及确定转化和基础科学研究的目标至关重要。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质结构域间的相互作用。在连接蛋白43（Cx43）中，胞质环（CL）和C-末端结构域（CT）之间的相互作用至关重要地涉及通道的生理功能，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，正如可能在生物医学上重要的那样，尚未被探索。我们已经发现，Cx 26通道的调制CL-CT相互作用。Cx 26和Cx43是两个最大的连接蛋白家族的代表成员。虽然结构相似，CL-CT相互作用对Cx 26通道功能的影响似乎是根本不同的Cx43，这表明CL-CT相互作用是一种常见的调节机制，在连接蛋白，但在连接蛋白特异性的方式运作。我们建议使用Cx 26及其密切相关的异构体Cx 32来阐明CL-CT控制通道功能的分子机制。Cx 26是唯一具有高分辨率结构的连接蛋白通道，使其成为所有连接蛋白通道基于结构研究的基石。拟议的研究探索CL-CT相互作用的基础和机制及其调节的通道特性，使用成功应用于其他通道的策略，包括宏观和单通道记录，使用竞争肽，工程Cys连接和突变分析。实验利用完整的通道，补充肽NMR。我们建议（a）确定通道门控中CL-CT相互作用的参与，（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相互作用及其效应。Cx 26和Cx 32在体内分布广泛。Cx 26突变导致全球超过一半的遗传性非综合征感音神经性耳聋，以及严重的毁容皮肤病。Cx 32突变导致外周脱髓鞘。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，因此了解其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。大量致病突变和两名PI在研究Cx 26和Cx 32通道门控和渗透性方面的丰富经验为CL-CT相互作用的生产性、信息性研究、病理突变如何改变它们以及对通道功能的影响提供了基础。