STRUCTURE/FUNCTION STUDIES OF GAP JUNCTIONS

由Cx26和Cx32形成的缝隙结，尽管在 序列，显示出显著的敏感性差异 跨结电压(Vi)和单通道电导。这些 差异提供了一种方法来定义分子机制， 细胞间通道的离子渗透与衬底电压的关系 由连接蛋白基因家族的成员组成。的X-链接形式 Charcot-Marie-Tooth病(CMT-X)似乎是由于 人类Cx32“功能”突变。结构-功能研究 将提供对这种疾病的分子基础的洞察并将 提供定义GAP生物学作用所需的信息 交汇点。从长远来看，一体化的成果 拟议的生物物理学、分子遗传学和计算机模拟研究 将允许构建结的原子分辨率模型和 将进一步加深我们对结构之间关系的理解 跨膜蛋白及其功能特性的研究。建议数 慢V-j依赖门控的研究应确定 缝隙结电压传感器。已经有人提出，固有的 跨膜中常见的脯氨酸扭结的结构柔韧性 受体结构域在信号机制中起着重要作用 转导。有人建议进行研究，以检验是否存在类似的机制 强调了已报道的保守的Pro残基发挥作用的能力 作为间隙结电压门控中的“转换元件”。研究 提出了完善Cx32和Cx32 N末端结构模型的建议 其他I类缝隙连接。这些应该解释了CMT-X突变是如何 映射到这个区域改变了细胞间的通讯。研究 本方案中所述的快速电气整流 Cx32/Cx26结，类似于整流的性质 脊椎动物中枢神经系统中发现的电突触， 这是由于这两种连接蛋白的离子渗透性不同所致。一个 提出了考虑观测数据的渗透势垒模型。 整改。建议进行单渠道研究，以完善这一点 渗透屏障模型和建立特定氨基酸对 形成屏障和“选择性过滤器”。Cx26基因嵌合体 和Cx32被鉴定出来，这应该导致对蛋白质的描述 形成离子传导路径的磁区。一种新的CMT-X突变， 描述了HumCx32S26L，其形成功能通道，其特征在于 单位电导显著降低。建议进行研究以 进一步检查此CMT-X和其他CMT-X引起的渗透变化 可能形成功能性缝隙连接通道的突变。这些研究 应明确CMT-X病的分子基础。

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.