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.

项目摘要 连接蛋白形成介导直接细胞间分子通信至关重要的间隙连接通道至关重要 在发育，生理和对创伤/炎症的反应中。连接蛋白的缺陷引起人类 病理。阐明调节连接蛋白通道功能的分子机制对于 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 基础科学研究。该项目的长期目标是了解胞质间相互作用 控制连接蛋白通道的门控。这是CX43的特征，其中相互作用 在细胞质环（Cl）和C末端结构域（CT）之间介导通道门控之间，pH和其他 因素。 CL-CT介导的CX43调节对于其生理功能至关重要，并且是一种治疗性 心血管病理的靶标。但是，CL-CT相互作用在其他连接素的调节中的作用 渠道在生物医学上很重要，并且尚未探索它们的作用机理。 我们发现CX26通道通过CL-CT相互作用调节。 CX26和CX43是代表性的 两个最大家族的连接素家族的成员。尽管在结构上类似，但Cl-CT的影响 CX26通道功能的相互作用似乎与CX43中的相互作用根本不同，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行。 我们建议阐明使用使用 CX26及其密切相关的同工型CX32。 CX26是唯一具有高度的连接通道 分辨率结构，使其成为所有连接蛋白通道基于结构研究的基础。 拟议的研究探讨了Cl-CT相互作用和通道特性的基础和机制 使用成功应用于其他渠道的策略调节，包括使用竞争肽， 设计的二硫键，宏观和单个通道记录以及突变分析。这 实验利用完整的通道，补充肽NMR。我们建议（a）确定 Cl-CT相互作用参与通道门控，（b）识别Cl-CT相互作用的位点，（C） 确定CL-CT相互作用及其影响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的突变负责遗传的一半以上 全世界的感觉性耳聋，也引起严重毁容皮肤疾病。 CX32原因的突变 外围脱髓鞘。在这两种连接素中，许多引起疾病的突变都定位为影响CL-CT 相互作用。两种连接蛋白都涉及肿瘤的进展和多种病理学， 生理过程。高分辨率结构的结合，大量疾病导致 PI在研究门控，CX26和 CX32通道为CL-CT相互作用的生产，全面研究提供了基础。