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-2014-03650
• 负责人: Bai, Donglin
• 金额: $ 2.55万
• 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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566004
• 关键词: Molecular; domains; determining; gap; junction

Molecular domains determining gap junction channel properties. 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 identical or different connexins oligomerize to form a cylinder-shaped half channel known as a hemichannel. Back-to-back docking of two hemichannels forms a complete gap junction channel. Communication through gap junction channels is essential for synchronized, coordinated cellular activities and exchanging 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 proposal aims to reveal the critical domains in connexin molecule responsible for the gap junction channel pore size and the gating controls by voltage and/or chemicals in the gap junctions of the neuronal connexin (Cx36) and the eye lens connexin (Cx50). 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. Mutagenesis will be used to generate point mutations to further evaluate the roles of individual amino acid residues in the channel properties. These chimeras and mutants will be transfected into gap junction deficient neuroblastoma (N2A) cells for functional tests. Functional investigation with dual patch clamp will be used to study voltage- and chemical-dependent gating and single channel conductance of the gap junction channels. Fluorescent proteins (GFP and RFP) and immunolabelling with antibodies will also be used to study the localization of these chimera/mutant connexins. We have successfully used these approaches to identify the roles of amino terminus in single channel conductance and transjunctional voltage-dependent gating of these two connexins (Xin et al, 2010; 2012). Significance Gap junction channels are ubiquitous in various tissues and play important roles in development, growth, differentiation and many physiological processes, including synchronized heart beat and 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 and many other connexins. Yet the knowledge is crucial in understanding of the physiological functions of these channels in synchronized neuronal activities and possibly other synchronized physiological processes.

决定间隙连接通道特性的分子结构域。背景间隙连接通道是细胞间的特殊膜孔结构，允许一个细胞中的离子和小信号/代谢分子传递到相邻细胞。间隙连接通道的结构单元是一组称为连接蛋白（Cxs）的跨膜蛋白。在啮齿动物基因组中鉴定了20种不同类型的连接蛋白。六个相同或不同的连接蛋白寡聚形成一个圆柱形的半通道，称为半通道。两个半通道的背靠背对接形成完整的间隙连接通道。通过缝隙连接通道进行的通讯对于同步、协调的细胞活动和组织中细胞之间的小分子交换是必不可少的。根据组织通道的连接蛋白亚型，差距连接通道的孔径、打开/关闭的开关控制以及化学物质的调节是不同的。负责这些通道特性的分子结构域在很大程度上是未知的。目的揭示神经元连接蛋白（Cx 36）和眼透镜连接蛋白（Cx 50）的差距连接通道的关键结构域，以及电压和/或化学物质对差距连接通道的门控。为了评估关键结构域的作用，我们将使用分子技术来工程化具有Cx 36的候选结构域的cDNA构建体，其交换Cx 50的相应结构域。诱变将用于产生点突变，以进一步评价单个氨基酸残基在通道特性中的作用。将这些嵌合体和突变体转染到间隙连接缺陷型神经母细胞瘤（N2 A）细胞中进行功能测试。双膜片钳功能研究将用于研究差距连接通道的电压和化学依赖性门控和单通道电导。荧光蛋白（GFP和RFP）和抗体免疫标记也将用于研究这些嵌合体/突变连接蛋白的定位。我们已经成功地使用这些方法来鉴定氨基末端在这两种连接蛋白的单通道电导和跨连接电压依赖性门控中的作用（Xin et al，2010; 2012）。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重要作用，包括同步心跳和神经元活动。对于Cx 36、Cx 50和许多其他连接蛋白，每个连接蛋白结构域如何贡献于整个间隙连接以及间隙连接通道的结构-功能关系尚不清楚。然而，这些知识对于理解这些通道在同步神经元活动和可能的其他同步生理过程中的生理功能至关重要。