PERMSELECTIVITY PROPERTIES OF CONNEXINS

描述：这项赠款申请旨在建立 缝隙结沟道壁和沟道的精细结构 渗透性选择性。研究人员建议研究大鼠的特性 Cx26、Cx32、Cx37、Cx40、Cx43和Cx46在N2A中的表达 细胞。他们将通过分析阴离子荧光探针的扩散 三个基本参数的估计：细胞质扩散系数， 交界膜渗透性和渗漏渗透性。第二个具体问题 目的是用突变型连接蛋白转染鼠N2A细胞 在特定目标1下研究，并评估相同的渗透率 参数。通过这些研究，调查人员建议定位 网站(S)，构成了阴离子过滤器。诱变分析将会 重点关注四个跨膜结构域和两个细胞外环。 在具体目标三下，申请者将使用双重全细胞贴片 钳位技术，用于验证间隙结通道的存在，如果 找到通道，以确定么正电导和 正在研究的突变体的阳离子/阴离子渗透性。最后， 在具体目标四下，调查人员提议将一种 通道特性和染料转移之间的关系。后者可以允许 用于在通道水平上测定大阴离子的渗透性。

DESCRIPTION: This grant application is aimed at establishing a link between the fine structure of the gap junction channel wall and channel permselectivity. The investigators propose to study the properties of rat Cx26, Cx32, Cx37, Cx40, Cx43 and Cx46 when expressed in transfected N2A cells. They will analyze the spread of anionic fluorescent probes via estimations of three basic parameters: cytoplasmic diffusion coefficient, junctional membrane permeability and leak permeability. The second specific aim is to transfect mouse N2A cells with mutant versions of the connexins studied under Specific aim 1, and to evaluate the same permeability parameters. Through these studies, the investigators propose to locate the site(s) that constitute the anionic filter. The mutagenesis analysis will focus on the four transmembrane domains, and the two extracellular loops. Under specific aim three, the applicants will use the dual whole cell patch clamp technique to verify the existence of gap junction channels and, if channels are found, to determine the unitary conductance and the cation/anion permeability properties of the mutant under study. Finally, under specific aim four, the investigators propose to characterize a relation between channel properties and dye transfer. The latter may allow for determination of permeability of large anions at the channel level.