PERMSELECTIVITY PROPERTIES OF CONNEXINS

连接蛋白的选择性渗透特性

• 批准号: 2701798
• 负责人: PETER R BRINK
• 金额: $ 18.74万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701798
• 关键词: cell line; gap junctions; gene expression; laboratory mouse; laboratory rat; membrane channels; membrane permeability; membrane structure; protein structure function; site directed mutagenesis; transfection

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.

描述：本拨款申请旨在建立两者之间的联系 间隙连接通道壁与通道的精细结构 渗透选择性。 研究人员提议研究大鼠的特性 在转染的 N2A 中表达时的 Cx26、Cx32、Cx37、Cx40、Cx43 和 Cx46 细胞。 他们将通过以下方式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， 交界膜通透性和渗漏通透性。 第二具体 目的是用连接蛋白的突变版本转染小鼠 N2A 细胞 根据具体目标 1 研究，并评估相同的渗透率 参数。 通过这些研究，研究人员建议找出 构成阴离子过滤器的位点。 诱变分析将 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，申请人将使用双全细胞贴片 钳位技术来验证间隙连接通道的存在，并且，如果 找到通道，以确定单一电导和 所研究的突变体的阳离子/阴离子渗透特性。 最后， 在具体目标四下，研究人员提议描述 通道特性和染料转移之间的关系。 后者可能允许 用于测定通道水平大阴离子的渗透性。