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: 8795731
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795731
• 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; Engineering; Family; Functional disorder; 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（由申请人提供）：连接蛋白形成的间隙连接通道介导了直接的细胞间分子通信在发育，生理和对创伤/炎症/疾病的反应至关重要的情况下。连接蛋白的突变引起人类病理。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和病理生理学中的作用以及识别转化和基础科学研究的靶标至关重要。该项目的长期目标是了解控制通道门控的连接蛋白的胞质间相互作用。在connexin43（CX43）中，细胞质环（CL）与C末端结构域（CT）之间的相互作用至关重要，至关重要的是该通道的生理功能，并且是心血管疗法的靶标。但是，尚未探索Cl-CT相互作用在其他连接蛋白通道调节中的作用和机制，在生物医学上很重要。我们发现CX26通道通过CL-CT相互作用调节。 CX26和CX43是两个最大的连接素家族的代表成员。尽管在结构上相似，但Cl-CT相互作用对CX26通道功能的影响似乎与CX43中的函数根本不同，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运行。我们建议使用CX26及其密切相关的同工型CX32阐明通道功能CL-CT控制的分子机制。 CX26是唯一具有高分辨率结构的连接通道，使其成为所有连接蛋白通道基于结构的研究的基石。拟议的研究探索了成功应用于其他通道的策略，包括宏观和单个通道记录，使用竞争性肽，工程CYS链接和突变分析，探索了他们调节Cl-CT相互作用和信道特性的基础和机制。这些实验利用完整的通道，补充了肽NMR。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相互作用的位点，以及（c）确定CL-CT相互作用及其效果如何通过引起人类疾病的突变改变。 CX26和CX32广泛分布在体内。 CX26突变导致一半以上的一半以上的遗传性非综合性感觉性耳聋以及严重毁容的皮肤疾病。 CX32突变引起周围脱髓鞘。值得注意的是，在两个连接素中，许多引起疾病的突变都在CL和CT结构域中。两种连接素都参与了广泛的病理和生理过程，因此了解这些连接蛋白和其他人的失调基础具有广泛的生物医学意义。引起突变的大量疾病以及PI在研究CX26和CX32通道的门控和渗透性方面的广泛经验为CL-CT相互作用的生产性，信息性研究提供了基础，如何通过病理突变改变它们以及对通道功能的影响。