Molecular domains determining gap junction channel properties

决定间隙连接通道特性的分子域

• 批准号: RGPIN-2020-05194
• 负责人: Bai, Donglin
• 金额: $ 2.62万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740442
• 关键词: Molecular; domains; determining; gap; junction

Background Gap junction (GJ) channels are specialized membrane pore structures between cells, allowing ions and small signalling/metabolic molecules in one cell to pass on to the neighbour cells. The building blocks for GJ channels are connexins. 20 different connexin genes are identified in the rodent genome. Six identical or different connexins oligomerize to form a hemichannel. Back-to-back docking of two hemichannels forms a whole GJ channel. Intercellular communication through GJ channels is essential for synchronized, coordinated cellular activities in many tissues and organs. The GJ channel pore size, the switch control for opening/closing, and the modulations by ions and chemicals are different depending on the connexin subtypes that organize the channel. The molecular domains and residues responsible for these channel properties are largely unknown. Long-term Objectives To reveal how neuronal and lens connexin (Cx36, Cx46, and Cx50) domains/residues and their interactions with chemicals regulate GJ channel properties. Short-term Objectives In this proposal we aim to identify the roles of pore-lining domains/residues of Cx36, Cx46, and Cx50 on their GJ channel properties, including single channel conductance, transjunctional voltage (Vj) and pH dependent gating. Approaches To evaluate the role of the critical domains and individual residues, we will use molecular biology techniques to engineer cDNA constructs with a candidate domain switched among Cx36, Cx46, and Cx50. Mutagenesis will be used to generate point variants to further evaluate the roles of individual amino acid residues. These cDNA construct of chimeras and variants will be transfected into connexin deficient N2A cells for functional tests. Dual patch clamp will be used to study voltage- and chemical-dependent gating as well as single channel conductance. Immunolabelling with connexin-specific antibodies or tagging fluorescent protein (GFP or RFP) at the carboxyl terminus will be used to study the localization of these connexin chimeras/variants. We have successfully used these approaches to identify the roles of amino terminus (NT) and the first extracellular domain (E1) in single channel conductance and transjunctional voltage-dependent gating of Cx50. Significance GJ channels are ubiquitous in various tissues and play important roles in development, growth, differentiation and many physiological processes. How each connexin domain or residue controls to its GJ channel properties are not clear. Recent studies with cryo-electron microscopy (CryoEM) revealed high resolution Cx46/Cx50 GJ structures with many novel interactions among domains, subunits, and local water/lipids. Yet the functional implications are not identified. The knowledge on structure-function relationship is crucial in understanding of the physiological functions of these channels in synchronized neuronal activities and the lens physiology.

背景间隙连接通道（GJ channels）是细胞间的一种特殊的膜孔结构，允许一个细胞中的离子和小的信号/代谢分子传递到相邻的细胞。GJ通道的结构单元是连接蛋白。在啮齿动物基因组中鉴定了20种不同的连接蛋白基因。六个相同或不同的连接蛋白寡聚形成半通道。两个半通道背靠背对接形成一个完整的GJ通道。通过GJ通道的细胞间通讯对于许多组织和器官中同步、协调的细胞活动是必不可少的。GJ通道的孔径大小、打开/关闭的开关控制以及离子和化学物质的调节是不同的，这取决于组织通道的连接蛋白亚型。负责这些通道特性的分子结构域和残基在很大程度上是未知的。长期目标揭示神经元和透镜连接蛋白（Cx 36，Cx46和Cx 50）结构域/残基及其与化学物质的相互作用如何调节GJ通道特性。短期目标在这个提议中，我们的目标是确定孔衬结构域/Cx 36，Cx46和Cx 50的残基对它们的GJ通道特性的作用，包括单通道电导，跨接电压（Vj）和pH依赖性门控。为了评估关键结构域和单个残基的作用，我们将使用分子生物学技术来工程化具有在Cx 36、Cx46和Cx 50之间切换的候选结构域的cDNA构建体。诱变将用于产生点变体，以进一步评价单个氨基酸残基的作用。将嵌合体和变体的这些cDNA构建体转染到连接蛋白缺陷型N2 A细胞中用于功能测试。双膜片钳将用于研究电压和化学依赖性门控以及单通道电导。在羧基末端用连接蛋白特异性抗体或标记荧光蛋白（GFP或RFP）的免疫标记将用于研究这些连接蛋白嵌合体/变体的定位。我们已经成功地使用这些方法来确定的作用氨基末端（NT）和第一个胞外结构域（E1）的单通道电导和跨连接的电压依赖性门控的Cx 50。 意义GJ通道广泛存在于各种组织中，在发育、生长、分化和许多生理过程中发挥重要作用。每个连接蛋白结构域或残基如何控制其GJ通道特性尚不清楚。最近的研究与冷冻电子显微镜（CryoEM）揭示了高分辨率的Cx46/Cx 50 GJ结构域，亚基，和当地的水/脂质之间的许多新的相互作用。然而，功能的影响没有确定。结构-功能关系的知识对于理解这些通道在同步神经元活动和透镜生理学中的生理功能至关重要。