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-2015-04110
• 负责人: Bai, Donglin
• 金额: $ 2.77万
• 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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=612778
• 关键词: Molecular; domains; determining; gap; junction

Background Gap junction 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 gap junction channels are a group of transmembrane proteins called connexins (Cxs). 20 different types of connexins are identified in the rodent genome. Six connexins oligomerize to form a cylinder-shaped half channel known as a hemichannel. Docking of two hemichannels forms a complete gap junction channel. Communication through gap junction channels is essential for synchronized, coordinated cellular activities and exchanging of small molecules between cells in tissues. The gap junction channel pore size, the switch control for opening/closing, and the modulations by chemicals are different depending on the connexin subtypes that organize the channel. The molecular domains responsible for these channel properties are largely unknown. Objectives This research program aims to reveal the critical domains in connexins responsible for the gap junction channel pore size and gating controls, by voltage and/or chemicals, in the gap junctions of the neuronal connexin (Cx36), the eye lens connexin (Cx50) and the inner ear connexin (Cx26). Approaches To evaluate the role of the critical domains, we will use molecular techniques to engineer cDNA constructs with the candidate domains of Cx36 swapping the corresponding domains of Cx50 (or Cx26). Mutagenesis will be used to generate point mutations to evaluate the roles of individual amino acid residues in the channel properties. These chimeras and mutants will be introduced into gap junction deficient neuroblastoma (N2A) cells for functional tests. Dual patch clamp will be used to study voltage- and chemical-dependent gating and single channel conductance. Fluorescent proteins and immunolabeling with antibodies will also be used to study the localization of these chimera/mutant connexins. We will also study the structure model of Cx26 and homology structure models of Cx36 and Cx50 to reveal the structural basis of the functional changes of the chimeras/mutants. We have successfully used these approaches to identify the roles of the amino terminus on single channel conductance and transjunctional voltage-dependent gating of Cx50 and Cx36. Significance Gap junction channels are ubiquitous in various tissues and play important roles in development, growth, differentiation, and many physiological processes, including synchronized neuronal activities. How each connexin domain contributes to the whole gap junction, and the structure-function relationships for gap junction channels are not clear for Cx36, Cx50, Cx26 and closely related connexins. Yet the knowledge is crucial in understanding physiological functions of these channels in synchronized neuronal activities and possibly other synchronized physiological processes.

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。缝隙连接通道的构建块是一组称为连接蛋白(CXS)的跨膜蛋白。在啮齿动物基因组中发现了20种不同类型的连接蛋白。六个连接蛋白齐聚形成一个圆柱形的半通道，称为半通道。两个半通道的对接形成了一个完整的缝隙连接通道。通过缝隙连接通道的通讯对于同步、协调的细胞活动和组织中细胞间的小分子交换是必不可少的。缝隙连接通道的孔大小、打开/闭合的开关控制以及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在很大程度上是未知的。 目标 本研究旨在揭示在神经元连接蛋白(Cx36)、眼晶状体连接蛋白(CX50)和内耳连接蛋白(Cx26)的缝隙连接中，连接蛋白中负责缝隙连接通道孔径大小和受电压和/或化学物质控制的门控的关键结构域。 方法 为了评估关键结构域的作用，我们将使用分子技术来设计Cx36的候选结构域与CX50(或Cx26)的相应结构域。突变将被用来产生点突变，以评估单个氨基酸残基在通道特性中的作用。这些嵌合体和突变体将被引入缝隙连接缺陷神经母细胞瘤(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和单通道电导。荧光蛋白和抗体免疫标记也将被用来研究这些嵌合体/突变连接蛋白的定位。我们还将研究Cx26的结构模型以及Cx36和CX50的同源结构模型，以揭示嵌合体/突变体功能变化的结构基础。我们已经成功地使用这些方法来确定氨基末端在CX50和Cx36的单通道电导和跨连接电压依赖门控中的作用。 意义 缝隙连接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着重要作用，包括神经元的同步活动。对于Cx36、CX50、Cx26和密切相关的连接蛋白，每个连接蛋白结构域如何对整个缝隙连接起作用，以及缝隙连接通道的结构与功能关系尚不清楚。然而，这一知识对于理解这些通道在同步神经元活动中的生理功能以及可能的其他同步生理过程至关重要。