STRUCTURE/FUNCTION STUDIES OF GAP JUNCTIONS

离子通道门控的机制还不完全 明白 分子遗传学和生物物理学的应用 技术应该导致描述的属性，组织 和蛋白质结构域的一级序列， 间隙连接的电压依赖性。 细胞间的调节 已经假设通过电压依赖性间隙连接的通信 在发育、神经信号传导和整合以及控制中发挥作用 分泌物。 在脊椎动物中，已知间隙连接蛋白是 由一个小的基因家族编码，没有广泛的序列同源性 其他离子通道。 几种间隙连接蛋白（连接蛋白）， 克隆的DNA已经被证明可以与 不同的电压敏感性和动力学，但在他们的分歧 一级蛋白质序列足以防止识别 表示电压依赖性所需的区域。 我们 将集中我们的初步调查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以下方面显著不同： 电压依赖性的形式和类型， 由密切相关的蛋白质Cx32的均聚物形成的连接。 由Cx26半通道与Cx26半通道结合产生的异型通道 Cx32半通道是独特的，因为它们显示连接电流， 当跨接电压Vj 应用。 这种快速Vj相关整流类似于所描述的整流。 神经系统中缝隙连接形成的电子突触。 我们开发了一种新的基因嵌合体形成方法， 不依赖于两者之间存在序列同源性 域. 我们将使用这个程序来确定蛋白质序列 这是造成Cx26的电压依赖性差异的原因， Cx32和异型通道的快速整流 通过成对表达这些嵌合体形成的通道的性质， 非洲爪蟾卵母细胞。 已鉴定的蛋白质结构域在此过程中的作用 电压依赖性门控可以从生物物理分析中推断出 测试线的通道内腔，并形成一个门，调节离子流。 如果这一假设得到验证，那么这个域与 分子的其他区域在电压表达中起作用 将探索依赖性。 从长远来看，这些研究应该提供 电压过程的分子机制 缝隙连接的门控。 这些分子机制的描述可能 适用于依赖于离子通道的其它电压的门控， 应提供有关蛋白质之间关系的信息 结构及其功能。

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.