STRUCTURE/FUNCTION STUDIES OF GAP JUNCTIONS

间隙连接的结构/功能研究

• 批准号: 6229707
• 负责人: Thaddeus Andrew Bargiello
• 金额: $ 10.3万
• 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 2000-12-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6229707
• 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.

CX26和CX32形成的差距连接，尽管在 序列，表现出对 跨连接电压（VI）和单个通道电导率。这些 差异提供了定义分子机制的手段 电压依赖性和离子间通道的渗透 由连接蛋白基因家族成员组成。 X连锁形式的 Charcot-Marie Tooth病（CMT-X）似乎是由于“失去 人类CX32中的功能”突变。结构功能研究提出了 将提供有关该疾病分子基础的见解，将 提供定义间隙生物学作用所需的信息 连接。从长远来看，结果的整合 提出的生物物理，分子遗传和计算机建模研究 将允许建立结合的原子分辨率模型和 将进一步理解结构之间的关系 跨膜蛋白及其功能特性。提议 慢速V-J依赖性门的研究应确定其他组成部分 间隙连接电压传感器。有人提出了固有的 在跨膜中常见的脯氨酸扭结的结构柔韧性 受体领域在信号机理中起重要作用 转导。提出了研究以检查是否具有类似的机制 基础是保守的脯氨酸残留物功能的能力 作为间隙连接电压门控的“转导元件”。研究 提议优化CX32 N端的结构模型和 我的其他差距连接。这些应该解释CMT-X突变如何 该域的地图改变了细胞间通信。研究 该提案中描述的是快速电气纠正 CX32/CX26连接，类似于整流的特性 在脊椎动物的中枢神经系统中发现的电突触， 来自两个连接素的离子渗透的差异结果。一个 提出了渗透性屏障模型，该模型解释了观察到的 纠正。提出了单个通道研究，以完善这一点 渗透屏障模型并确定特定氨基酸的作用 障碍物和“选择性过滤器”的形成。 CX26的基因嵌合体 鉴定出应导致蛋白质描述的CX32 形成离子传导路径的域。新的CMT-X突变， 描述了HUMCX32S26L，形成了以功能通道为特征的功能通道 统一电导的大幅降低。提出了研究 进一步研究由此和其他CMT-X引起的渗透变化 可能形成功能间隙连接通道的突变。这些研究 应定义CMT-X疾病的分子基础。