STRUCTURE/FUNCTION STUDIES OF GAP JUNCTIONS

Cx26和Cx32形成的缝隙结在分子上不同 渗透性和对跨结电压的敏感度。这些差异 将被用来定义电压背后的分子机制 细胞间通道和膜通道的依赖性和离子通量 连接蛋白基因家族的蛋白质。从长远来看，数据的整合 来自生物物理学、分子遗传学、核磁共振和计算机模拟的研究将 有助于了解这种膜蛋白的构象变化 还有一些动态地与它们的功能以及固定电荷如何调节有关 离子通量。在拟议的研究中，Cx32航道的衬孔序列 将被进一步定义，野生菌氨基末端的原子结构 类型和突变的Cx32将通过高分辨率核磁共振来解决。的地位 电压传感器及其与交叉电场的关系将 被探索。有人建议进行研究，以检验这一假设 脯氨酸扭结基序的构象柔性是构象的基础 电压依赖门控所需的更改。还建议进行研究，以 区分协同和单个亚基门控模式。数据来自 离子通量的分子和电生理学研究将使用 用Chen和Eisenberg的电扩散模型进一步确定 带电氨基酸在决定缝隙结渗透性和选择性中的作用 频道。X连锁Charcot-Marie-Tooth病(CMTX)是由 人类Cx32和Cx26突变所致的非综合征性耳聋。生物物理学和 分子研究表明，Cx32通透性的变化可能会 CMTX的病因学基础。拟议的研究将推进基本的 定义这个和其他分子基础所需的科学知识 连接蛋白相关疾病，以及缝隙连接的生物学作用。

Gap junctions formed by Cx26 and Cx32 differ in molecular permeability and in sensitivity to transjunctional voltage. These differences will be exploited to define the molecular mechanisms that underlie voltage dependence and ionic fluxes of intercellular and membrane channels formed by proteins of the connexin gene family. In the long term, integration of data from biophysical, molecular genetic, NMR and computer modeling studies will lead to understanding of how conformational changes in this membrane protein and others dynamically relate to their function and how fixed charges modulate ion flux. In the proposed studies the pore-lining sequence of the Cx32 channel will be further defined and the atomic structure of the amino terminus of wild type and mutated Cx32 will be solved by high resolution NMR. The position of the voltage sensor and its relation to the transjunctional electric field will be explored. Studies are proposed to test the hypothesis that the conformational flexibility of a proline kink motif underlies the conformational changes required for voltage dependent gating. Studies are also proposed to distinguish between concerted and individual subunit gating models. Data from molecular and electrophysiological studies of ionic flux will be examined using the electrodiffusive model of Chen and Eisenberg to further define the roles of charged amino acids in determining permeation and selectivity of gap junction channels. X-linked Charcot-Marie-Tooth disease (CMTX) is caused by mutations of human Cx32, and nonsyndromic deafness by mutations of Cx26. Biophysical and molecular studies have shown that changes in permeability of Cx32 are likely to underlie the etiology of CMTX. The proposed studies will advance the basic scientific knowledge required to define the molecular basis of this and other connexin-related diseases, and the biological role of gap junctions.