STRUCTURE/FUNCTION STUDIES OF GAP JUNCTIONS

离子通道门控的潜在机制还不完全 明白了。分子遗传学和生物物理学的应用 技术应该导致对属性、组织 和蛋白质结构域的初级序列，这些结构域负责 间隙结的电压依赖关系。细胞间的调控 电压依赖的缝隙结的通信被认为是 在发育、神经信号和整合以及控制方面发挥作用 分泌物。在脊椎动物中，缝隙连接蛋白被认为是 由一个没有广泛序列同源性的小基因家族编码 和其他离子通道。几种缝隙连接蛋白(连接蛋白) 有哪些克隆的DNA已被证明与 不同的电压灵敏度和动力学，但它们的发散 初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 将把我们的初步研究重点放在两种脊椎动物连接蛋白Cx26和 Cx32。Cx26均聚物形成的缝隙结在以下方面有显著不同 电压依赖的形式和类型，当它们与 由紧密相关的蛋白质Cx32的均聚体形成的连接。 Cx26半通道与Cx26半通道结合产生的异型通道 Cx32半通道的独特之处在于它们显示的交叉点电流 当跨接电压VJ被 已申请。这种快速的主播依赖整改类似于所描述的 一些由神经系统中的缝隙连接形成的电子突触。 我们已经开发了一种形成基因嵌合体的新程序 并不依赖于两者之间是否存在序列同源性 域名。我们将使用这个程序来确定蛋白质序列 这是导致Cx26和Cx26电压依赖关系差异的原因 Cx32和异型通道的快速整改 由这些嵌合体成对的表达式形成的通道的属性 非洲爪哇卵母细胞。识别的蛋白质结构域在这一过程中的作用 电压依赖门控可以从生物物理分析和 测试使通道管腔排列成线，并形成调节离子流动的闸门。 如果这一假设得到证实，这个域与 在电压表达中起作用的分子的其他区域 我们将探讨依赖性。从长远来看，这些研究应该提供 关于电压过程的分子机制的说明 缝隙连接的浇口。对这种分子机制的描述可以 适用于其他电压相关离子通道的选通和 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.