STRUCTURE-FUNCTION STUDIES OF GAP JUNCTIONS

间隙连接的结构功能研究

• 批准号: 3306386
• 负责人: Thaddeus Andrew Bargiello
• 金额: $ 24.77万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306386
• 关键词: Xenopus oocyte; biophysics; chimeric proteins; electrophysiology; gap junctions; membrane channels; protein structure function; site directed mutagenesis; transfection; voltage gated channel

The mechanisms underlying gating of ion channels are not yet fully understood. The application of molecular genetic and biophysical techniques should lead to the description of the properties, organization and primary sequence of the protein domains that are responsible for the voltage dependence of gap junctions. The regulation of intercellular communication by voltage dependent gap junctions has been postulated to play a role in development, neural signalling and integration, and control of secretion. In vertebrates, gap junction proteins are known to be encoded by a small gene family that shares no extensive sequence homology with other ion channels. Several gap junction proteins (connexins) for which cloned DNA are available have been shown to form channels with different voltage sensitivities and kinetics, but the divergence in their primary protein sequence is sufficient to prevent the identification of regions that are required for the expression of voltage dependence. We will focus our initial investigations on two vertebrate connexins Cx26 and Cx32. Gap junctions formed from homopolymers of Cx26 differ markedly in the form and types of voltage dependence when they are compared to junctions formed by homopolymers of the closely related protein, Cx32. Heterotypic channels resulting from the union of Cx26 hemichannels with Cx32 hemichannels are unique in that they display junctional currents that rectify with a fast time course when transjunctional voltages, Vj, are applied. This fast Vj dependent rectification is similar to that described for some electronic synapses formed by gap junctions in the nervous system. We have developed a new procedure for the formation of gene chimeras that is not dependent on the existence of sequence homology between the two domains. We will use this procedure to determine the protein sequences that are responsible for the differences in voltage dependence of Cx26 and Cx32 and the fast rectification of heterotypic channels by examining the properties of channels formed by the expression these chimeras in pairs of Xenopus oocytes. The role of identified protein domains in the process voltage dependent gating can be inferred from biophysical analyses and tested lines the channel lumen and forms a gate that regulates ion flow. If this hypothesis is verified, the relationship between this domain and other regions of the molecule that function in the expression of voltage dependence will be explored. In the long term these studies should provide an account of the molecular mechanisms that underlie the process of voltage gating of gap junctions. The descriptions of such molecular mechanisms may have applicability to gating of other voltage dependent of ion channels and the should provide information concerning the relationship between protein structure and its function.

离子通道的门控的机制尚未完全 理解。 分子遗传和生物物理的应用 技术应导致属性，组织的描述 以及负责蛋白质结构域的主要序列 间隙连接的电压依赖性。 细胞间的调节 通过电压依赖性间隙连接的通信已被假定为 在开发，神经信号传导和整合以及控制中发挥作用 分泌。 在脊椎动物中，已知间隙连接蛋白是 由一个没有广泛序列同源性的小基因家族编码 与其他离子通道。 几种间隙连接蛋白（连接蛋白） 显示哪些克隆DNA已显示出与 不同的电压敏感性和动力学，但它们的差异 一级蛋白质序列足以防止鉴定 表达电压依赖性所需的区域。 我们 我们的初步调查将把两个脊椎动物连接蛋白CX26和 CX32。 由CX26的均聚物形成的间隙连接在 将电压依赖性的形式和类型进行比较 由密切相关蛋白质CX32的均聚物形成的连接。 CX26半通道与 CX32半通道是独一无二的，因为它们显示了连接电流 当跨连接电压VJ为快速的时间过程中 应用。 这种快速的VJ依赖性纠正与所描述的相似 对于神经系统中的间隙连接形成的某些电子突触。 我们已经开发了一种新的程序，用于形成基因嵌合体 不依赖两者之间的序列同源性 域。 我们将使用此过程来确定蛋白质序列 负责CX26和 CX32和通过检查异型通道的快速纠正 这些嵌合体形成的通道的性质成对 爪蟾卵母细胞。 确定的蛋白质结构域在此过程中的作用 可以通过生物物理分析来推断依赖电压的门控和 测试线腔管道并形成调节离子流量的栅极。 如果验证了该假设，则该领域与 在电压表达中起作用的分子的其他区域 依赖将被探讨。 从长远来看，这些研究应提供 构成电压过程基础的分子机制的说明 间隙连接的门控。 这种分子机制的描述可能 具有离子通道其他电压依赖性的门控和 应提供有关蛋白质之间关系的信息 结构及其功能。