STRUCTURE/FUNCTION STUDIES OF GAP JUNCTIONS

间隙连接的结构/功能研究

• 批准号: 2654964
• 负责人: Thaddeus Andrew Bargiello
• 金额: $ 30.52万
• 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 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2654964
• 关键词: Xenopus oocyte; alternatives to animals in research; aminoacid; biophysics; chimeric proteins; computer simulation; electrophysiology; gap junctions; gene mutation; hereditary motor and sensory neuropathy; membrane channels; membrane proteins; protein structure function; site directed mutagenesis; transfection; voltage gated channel

Gap junctions formed by Cx26 and Cx32, although closely related in sequence, display significant differences in sensitivity to transjunctional voltage (vi) and in single channel conductance. These differences provide a means to define the molecular mechanisms that underlie voltage dependence and ion permeation of intercellular channels formed by members of the connexin gene family. The X-linked form of Charcot-Marie-Tooth disease (CMT-X) appears to result from "loss of function" mutations in human Cx32. The structure-function studies proposed will provide insight into the molecular basis of this disease and will provide information that is required to define the biological roles of gap junctions. In the long-term, the integration of the results of the proposed biophysical, molecular genetic and computer modelling studies will permit the construction of atomic resolution models of junctions and will further our understanding of the relationship between the structure of transmembrane proteins and their functional properties. The proposed studies of slow V-j-dependent gating should identify other components of the gap junction voltage sensor. It has been proposed that the inherent structural flexibility of proline kinks commonly found in transmembrane domains of receptors plays an important role in the mechanism of signal transduction. Studies are proposed to examine if a similar mechanism underlies the reported ability of a conserved proline residue to function as a "transduction element" in voltage gating of gap junctions. Studies are proposed to refine structural models of the N-terminus of Cx32 and other Group I gap junctions. These should explain how CMT-X mutations that map to this domain have altered intercellular communication. Studies described in this proposal indicate that the fast electrical rectification of Cx32/Cx26 junctions, which resembles the properties of rectifying electrical synapses found in the central nervous system of vertebrates, results from differences in ion permeation of the two connexins. A permeation barrier model is presented that accounts for the observed rectification. Single channel studies are proposed that will refine this permeation barrier model and establish the role of specific amino acids to the formation of barriers and "selectivity filters". Gene chimeras of Cx26 and Cx32 are identified that should lead to the description of the protein domains that form the ion conduction path. A new CMT-X mutation, humCx32S26L is described that forms functional channels characterized by significant reductions in unitary conductance. Studies are proposed to further examine changes in permeation caused by this and other CMT-X mutations that may form functional gap junction channels. These studies should define the molecular basis of CMT-X disease.

缝隙连接由Cx 26和Cx 32形成，尽管在细胞内紧密相关， 序列，显示对 跨接电压（VI）和单沟道电导。这些 差异提供了一种手段来定义分子机制， 细胞间通道的电压依赖性和离子渗透 由连接蛋白基因家族的成员形成。X连锁形式的 腓骨肌萎缩症（CMT-X）似乎是由于“丧失 人Cx 32中的“功能”突变。结构-功能研究建议 将深入了解这种疾病的分子基础， 提供定义gap的生物学作用所需的信息 交叉点从长远来看，整合的结果 拟议的生物物理、分子遗传和计算机模拟研究 将允许构建结点的原子分辨率模型， 将进一步加深我们对 跨膜蛋白及其功能特性。拟议 慢V-j依赖性门控的研究应确定 差距结电压传感器。有人提出， 在跨膜中常见脯氨酸扭结的结构柔性 受体的结构域在信号转导机制中起着重要作用， 转导建议研究是否有类似的机制 是保守的脯氨酸残基发挥作用的能力的基础 作为间隙连接的电压门控中的“转导元件”。研究 提出了完善结构模型的N-末端的Cx 32和 其他I组间隙连接。这些应该可以解释为什么CMT-X突变， 映射到这个域改变了细胞间的通讯。研究 本提案中所述的快速电整流 的Cx 32/Cx 26结，这类似于整流特性 在脊椎动物的中枢神经系统中发现的电突触， 这是由两种连接蛋白的离子渗透差异引起的。一 渗透屏障模型，占观察到的 整改单通道研究提出，将完善这一点 渗透屏障模型，并建立特定氨基酸的作用， 形成屏障和“选择性过滤器”。Cx 26基因嵌合体 和Cx 32被鉴定，这将导致蛋白质的描述 一种新的CMT-X突变， 描述了humCx 32 S26 L，其形成功能性通道，其特征在于 单位电导的显著降低。建议开展研究， 进一步检查这种和其他CMT-X引起的渗透变化 这些突变可能形成功能性间隙连接通道。这些研究 应该能确定CMT-X疾病的分子基础