MOLECULAR PHYSIOLOGY OF CLONED ION CHANNELS

克隆离子通道的分子生理学

基本信息

批准号：
2210031
负责人：
Gordon Frank Tomaselli
金额：
$ 7.98万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-04-01 至 1995-03-31
项目状态：
已结题

项目摘要

The nicotinic acetylcholine receptor channel (nAChRC) is the best understood ion channel, it transduces the chemical signal at the neuromuscular junction and is the target of pathogenic antibodies in myasthenia gravis. The goal of this proposal is to better understand how the channel protein structure produces its functional properties, specifically, how electrical charge on the surface of the protein influences ion transport. A series of receptor channels with single charged amino acid mutations will be created by site-directed mutagenesis using a uracil-enriched template cDNA. Channel protein expression in Xenopus oocytes and single channel recording under conditions of varied ionic strength and permeant ion concentration will be used to determine the conductance of the channel. The conductance and the shape of the current- voltage relations of the mutant receptor channels will be compared to the wild type receptor. These data will be interpreted in terms of a computational model of the electric potential profile of the nAChRC. From this analysis the relative electrical distances of the point charges to the pore mouth will be calculated. Examination of the sidedness of the effect of changes to the pore mouth will be calculated. Examination of the sidedness of the effect of changes in ionic strength and the rectification of the current-voltage relation will allow the localization of the charge change to the cytoplasmic or extracellular side of the pore complementary to what is currently known about the transmembrane topology from protein chemistry and antibody analysis. The candidate, through work on this project, hopes to refine his skills in ion channel expression and single channel recording, and acquire new skills in DNA cloning and site-directed mutagenesis. He will ultimately apply these techniques to the study of voltage-dependent ion channels from cardiac tissue, specifically the sodium channel from ventricular muscle. He hopes to study questions related to the molecular nature of the local anesthetic antiarrhythmic drug binding site in this channel.

烟碱乙酰胆碱受体通道（NACHRC）是最好的理解的离子通道，它在神经肌肉连接，是致病性抗体的靶标肌无力重症。该提议的目的是更好地了解通道蛋白结构产生其功能特性，具体而言，如何在蛋白质表面上电荷影响离子运输。一系列具有单一的受体通道带电的氨基酸突变将由位置定向诱变创建使用富含尿嘧啶的模板cDNA。通道蛋白表达在各种条件下离子强度和渗透离子浓度将用于确定通道的电导。电流的电导和形状将突变受体通道的电压关系与野生型受体。这些数据将用 NACHRC电势轮廓的计算模型。从该分析点电荷的相对电距离将计算毛孔口。检查效果的侧面将计算毛孔口的变化。检查离子强度变化效应的侧面性和矫正的效果电流 - 电压关系将允许电荷定位改变孔补充的细胞质或细胞外侧目前对蛋白质跨膜拓扑的了解化学和抗体分析。候选人通过该项目的工作希望完善他的技能离子频道的表达和单频道记录，并获得新技能在DNA克隆和定点诱变中。他最终将申请这些技术用于研究电压依赖性离子通道的心脏组织，特别是心室肌肉的钠通道。他希望研究与局部分子性质有关的问题该通道中的麻醉性抗心律失常药物结合位点。