PERMSELECTIVITY PROPERTIES OF CONNEXINS

描述：本补助金申请旨在建立联系， 差距连接通道壁和通道的精细结构 选择渗透性 研究人员建议研究老鼠的特性 Cx 26、Cx 32、Cx 37、Cx40、Cx43和Cx46在转染的N2 A中表达时 细胞 他们将分析阴离子荧光探针的传播， 三个基本参数的估计：细胞质扩散系数， 连接膜通透性和渗漏通透性。 第二特定 目的是用连接蛋白的突变体转染小鼠N2 A细胞， 根据具体目标1进行研究，并评估相同的渗透性 参数 通过这些研究，研究人员提出， 构成阴离子过滤器的位点。 诱变分析将 集中在四个跨膜结构域和两个细胞外环上。 根据具体目标三，申请人将使用双全细胞贴片 钳位技术，以验证间隙连接通道的存在，如果 通道被发现，以确定单位电导和 研究中的突变体的阳离子/阴离子渗透特性。 最后， 在具体目标四下，调查人员建议描述一种 通道特性与染料转移的关系。 后者可能允许 用于测定大阴离子在通道水平的渗透性。

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.