Gap Junction Channel Permeability: A Mutagenic Approach

描述(由申请人提供)：我们假设有具体的 优先影响分子渗透的电荷基团 第二信使的大小和收费范围，但对 一价阳离子选择性。我们假设有多个站点 能够影响间隙结通道的渗透选择性。具体目标 包括： 目的：将突变型基因导入小鼠N2A细胞或大鼠胰岛素瘤细胞(RIN) Cx43、Cx40和Cx37的不同版本，并通过Western和 Northern印迹分析。我们还将使用GFP标签策略来确定 蜂窝分布。我们选择了Cx43和Cx40，因为它们无处不在 在房室结的心脏，因此它们的潜在影响心脏 心律不齐。Cx37是血管内皮细胞的主要连接蛋白。 目的1)检测Kc1，Csc1， NaCI和TEACI使用对偶整体评估阳离子/阴离子选择性的变化 细胞膜片钳。 目的2)监测组成一个半通道的单通道电导 一个是突变体连接蛋白(muCx43或muCx4O)，另一个是由野生型 在KCL、CSCI、氯化钠和TEAC1中键入Cx43或Cx40的连接蛋白以评估 阳离子/阴离子选择性。 我们将使用半胱氨酸扫描突变来确定突变的位点是否连接到 可怜的墙。目的3)同时测定结电导和 野生型和突变型带电荧光探针的连接通透性 连接蛋白(同型和异型)。这将允许下定决心 带电探针在0.8-1.2 nm尺寸范围内的相对渗透率 致K/Cs。惰性探针(荧光黄、DAPI、羧基荧光素)是探针 选择是因为它们不会像这种情况那样迅速从细胞质中去除 对于lP3(Tau小于60s)。

DESCRIPTION (provided by applicant): We hypothesize that there are specific charge groups that preferentially influence the permeation of molecules in the size and charge range of second messengers while having little influence on monovalent cation selectivity. We hypothesize that there are multiple sites able to affect the permselectivity of gap junction channels. The specific aims are: Aim la) Transfect mouse N2A cells or rat insulinoma cells (RIN) with mutant versions of Cx43, Cx40, and Cx37 cDNA and determine expression via Western and Northern blot analysis. We will also use the GFP tag strategy to determine cellular distribution. We have chosen Cx43 and Cx40 because of their ubiquity in the SA an AV nodes of the heart and hence their potential to affect cardiac arrhythmias. Cx37 is the major connexin of endothelium. Aim ib) Monitor single channel conductance in homotypic mutants in KC 1, CsCI, NaCI and TEACI to assess changes in cation/anion selectivity using dual whole cell patch clamp. Aim 2) Monitor single channel conductance in which one hemichannel is composed of a mutant connexin (muCx43 or muCx4O) and the other is composed of a wild type connexin of Cx43 or Cx40 in Kcl, CsCI, NaCl and TEAC1 to assess changes in cation/anion selectivity. We will use cysteine scanning mutation to determine if a mutated site lines the poor wall. Aim 3) Simultaneously determine junctional conductance and junctional permeability to charged fluorescent probes for wild type and mutant connexins (homotypic and heterotypic forms). This will allow the determination of the permeation rates of charged probes in the 0.8-1.2 nm size range relative to K+/Cs+. Inert probes (Lucifer Yellow, DAPI, carboxyfluorescein) are probes of choice because they are not rapidly removed from the cytosol, as is the case for lP3 (Tau less than 60s).