Gap Junction Channel Permeability: A Mutagenic Approach

描述（由申请人提供）：我们假设有特定的 优先影响分子在膜中渗透的电荷基团 第二信使的大小和电荷范围，而对 单价阳离子选择性。我们假设有多个地点 能够影响间隙连接通道的选择透过性。具体目标 为： 目的la）用突变体转染小鼠N2 A细胞或大鼠胰岛素瘤细胞（RIN）， Cx43、Cx40和Cx 37 cDNA的不同版本，并通过Western和 北方印迹分析。我们还将使用GFP标签策略来确定 细胞分布我们选择Cx43和Cx40是因为它们的普遍性 在心脏的SA和AV结中，因此它们可能影响心脏 心律不齐Cx 37是内皮细胞的主要连接蛋白。 目的ib）监测KC 1，CsCl， NaCl和TEACI，以评估使用双全氟氯化钠的阳离子/阴离子选择性的变化 细胞膜片钳 目的2）监测由一个半通道组成的单通道电导 一个是突变型连接蛋白（muCx 43或muCx 4 O），另一个是野生型连接蛋白。 型连接蛋白Cx43或Cx40在KCl，CsCl，NaCl和TEAC 1中的变化，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位点是否符合 可怜的墙。目的3）同时测定结电导和 野生型和突变体对带电荧光探针的连接通透性 连接蛋白（同型和异型形式）。这将有助于确定 在0.8-1.2 nm尺寸范围内的带电探针的渗透速率相对 K+/Cs+。惰性探针（荧光黄，DAPI，羧基荧光素）是探针 因为它们不会像在这种情况下那样迅速地从胞质中被清除 对于IP 3（τ小于60 s）。

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).