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: 8711872
• 负责人: Jorge Enrique Contreras
• 金额: $ 28.85万
• 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 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8711872
• 关键词: Affect; Basic Science; Binding; C-terminal; Cardiovascular Pathology; Cell Proliferation; Cells; Communication; Complement; Connexin 43; Connexins; Cysteine; Defect; Demyelinations; Development; Disease; Disulfide Linkage; Engineering; Family; Functional disorder; Generations; Goals; Health; Human; Human Pathology; Inflammation; Inherited; Intervention; Investigation; Mammalian Cell; Mass Spectrum Analysis; Mediating; Molecular; Mutate; Mutation; Oocytes; Pathologic Processes; Pathology; Peptides; Peripheral; Permeability; Physiological; Physiological Processes; Physiology; Point Mutation; Positioning Attribute; Post-Translational Protein Processing; Property; Protein Isoforms; Proteins; Public Health; Regulation; Resolution; Role; Sensorineural Hearing Loss; Signal Transduction; Site; Structure; Translational Research; Trauma; base; deafness; disease-causing mutation; experience; gap junction channel; human disease; in vivo; member; mutant; nervous system disorder; research study; response; skin disorder; therapeutic target; tumor progression

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.

项目概要 连接蛋白形成间隙连接通道，介导直接的细胞间分子通讯至关重要 发育、生理学和对创伤/炎症的反应。连接蛋白的缺陷会导致人类 病理学。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，并确定转化和病理生理学的目标 基础科学研究。该项目的长期目标是了解胞质域间相互作用 控制连接蛋白通道的门控。这是 Cx43 的特征，其中相互作用 细胞质环 (CL) 和 C 端结构域 (CT) 之间通过 pH 和其他因素介导通道门控 因素。 CL-CT 介导的 Cx43 调节对其生理功能至关重要，并且是一种治疗方法 心血管病理的目标。然而，CL-CT 相互作用在调节其他连接蛋白中的作用 通道，可能在生物医学上很重要，但其作用机制尚未被探索。 我们发现 Cx26 通道受到 CL-CT 相互作用的调节。 Cx26和Cx43为代表 连接蛋白两个最大家族的成员。尽管结构相似，但 CL-CT 的效果 Cx26 通道功能上的相互作用似乎与 Cx43 中的相互作用有根本不同，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运作。 我们建议利用 CL-CT 控制通道功能和特性的分子机制 Cx26 及其密切相关的异构体 Cx32。 Cx26 是唯一具有高连接蛋白通道 分辨率结构，使其成为所有连接蛋白通道基于结构的研究的基础。 所提出的研究探讨了 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，包括使用竞争肽， 工程二硫键、宏观和单通道记录以及突变分析。这 实验利用完整的通道，并辅以肽 NMR。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在体内广泛分布。 Cx26 突变导致一半以上的遗传 全世界范围内的感音神经性耳聋，还会导致严重的毁容性皮肤病。 Cx32突变导致 周围性脱髓鞘。在这两种连接蛋白中，许多致病突变都会影响 CL-CT 相互作用。两种连接蛋白都与肿瘤进展和多种病理学和 生理过程。高分辨率结构、大量致病因素的组合 突变以及两位 PI 在研究 Cx26 和 Cx26 的门控、通透性和调节方面的丰富经验 Cx32 通道为 CL-CT 相互作用的高效、全面的研究提供了基础。