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Gap Junction Channel Permeability: A Mutagenic Approach

间隙连接通道渗透性：诱变方法

基本信息

批准号：
6936576
负责人：
PETER R BRINK
金额：
$ 25.02万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2007-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6936576
关键词：
biological signal transduction cell cell interaction cell line electrical conductance gap junctions gene expression gene mutation laboratory mouse laboratory rat membrane channels membrane permeability membrane structure northern blottings protein structure function site directed mutagenesis transfection voltage /patch clamp western blottings

项目摘要

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).

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电荷范围，而影响不大单价阳离子选择性。我们假设有多个站点能够影响间隙连接通道的通透选择性。具体目标是：目标la)用突变体转染小鼠N2A细胞或大鼠胰岛素瘤细胞(RIN) Cx43、Cx40 和 Cx37 cDNA 版本，并通过 Western 和 Northern印迹分析。我们还将使用 GFP 标签策略来确定细胞分布。我们选择 Cx43 和 Cx40 因为它们无处不在位于心脏的 SA 和 AV 结中，因此它们有可能影响心脏心律失常。 Cx37 是内皮细胞的主要连接蛋白。目标 ib) 监测 KC 1、CsCI、同型突变体的单通道电导 NaCl 和 TEACI 使用双整体评估阳离子/阴离子选择性的变化细胞膜片钳。目标 2) 监测由一个半通道组成的单通道电导一个突变连接蛋白（muCx43 或 muCx4O），另一个由野生连接蛋白组成在 Kcl、CsCI、NaCl 和 TEAC1 中键入 Cx43 或 Cx40 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位点是否符合可怜的墙。目标 3) 同时确定结电导和野生型和突变体对带电荧光探针的连接渗透性连接蛋白（同型和异型形式）。这将允许确定带电探针在 0.8-1.2 nm 尺寸范围内的相对渗透率至 K+/Cs+。惰性探针（荧光黄、DAPI、羧基荧光素）是探针选择，因为它们不会像实际情况那样快速从细胞质中去除对于 lP3（Tau 小于 60 秒）。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Recreating the biological pacemaker.

重建生物起搏器。

DOI：
10.1002/ar.a.20073
发表时间：
2004
期刊：
The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology.
影响因子：
0
作者：
Rosen,MichaelR;Robinson,RichardB;Brink,Peter;Cohen,IraS
通讯作者：
Cohen,IraS

Non-stationary fluctuation analysis of macroscopic gap junction channel records.

宏观间隙连接通道记录的非平稳波动分析。

DOI：
10.1007/s00232-005-0765-4
发表时间：
2005
期刊：
The Journal of membrane biology
影响因子：
0
作者：
Ramanan,SV;Valiunas,V;Brink,PR
通讯作者：
Brink,PR

Cloning and functional expression of invertebrate connexins from Halocynthia pyriformis.

梨状盐藻无脊椎动物连接蛋白的克隆和功能表达。

DOI：
10.1016/j.febslet.2004.09.071
发表时间：
2004
期刊：
FEBS letters.
影响因子：
0
作者：
White,ThomasW;Wang,Huan;Mui,Rickie;Litteral,Jennifer;Brink,PeterR
通讯作者：
Brink,PeterR

The effect of low-frequency electromagnetic field on human bone marrow stem/progenitor cell differentiation.

DOI：
10.1016/j.scr.2015.04.009
发表时间：
2015-07
期刊：
Stem cell research
影响因子：
1.2
作者：
Ross CL;Siriwardane M;Almeida-Porada G;Porada CD;Brink P;Christ GJ;Harrison BS
通讯作者：
Harrison BS

Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.