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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=763332
• 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.

背景缝隙连接通道是细胞间特殊的膜孔结构，允许一个细胞中的离子和小的信号/代谢分子传递给邻近的细胞。GJ通道的构建块是连接蛋白。在啮齿动物基因组中发现了20个不同的连接蛋白基因。六个相同或不同的连接蛋白齐聚形成半通道。两个半通道的背靠背对接形成了一个完整的GJ通道。通过GJ通道的细胞间通信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通道的孔大小、打开/关闭的开关控制以及离子和化学物质的调制根据组织通道的连接蛋白亚型而不同。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。长期目标是揭示神经元和晶状体连接蛋白(Cx36、Cx46和CX50)结构域/残基及其与化学物质的相互作用如何调节GJ通道的特性。短期目标在这项建议中，我们的目标是确定Cx36、Cx46和CX50的孔衬结构域/残基对其GJ通道特性的作用，包括单通道电导、跨结电压(VJ)和pH依赖门控。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设计候选结构域在Cx36、Cx46和CX50之间切换的cDNA构建。突变将被用来产生点变异，以进一步评估单个氨基酸残基的作用。这些构建的嵌合体和变异体将被导入连接蛋白缺失的N2A细胞进行功能测试。双膜片钳将被用来研究电压和化学依赖的门控以及单通道电导。用连接蛋白特异性抗体或在羧基末端标记荧光蛋白(GFP或RFP)的免疫标记将被用来研究这些连接蛋白嵌合体/变体的定位。我们已经成功地利用这些方法确定了氨基末端(NT)和第一胞外域(E1)在CX50的单通道电导和跨连接电压依赖门控中的作用。意义GJ通道广泛存在于各种组织中，在发育、生长、分化等多种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。最近的低温电子显微镜研究揭示了高分辨率的CX46/CX50 GJ结构，它们具有许多新的结构域、亚单位和局部水/脂之间的相互作用。然而，功能上的影响还没有确定。对结构-功能关系的了解对于理解这些通道在神经元同步活动和晶状体生理学中的生理功能至关重要。