STRUCTURE AND REGULATION OF CHLORIDE ION CHANNELS

氯离子通道的结构和调节

• 批准号: 2143463
• 负责人: ERNEST G PERALTA
• 金额: $ 23.8万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143463
• 关键词: Xenopus oocyte; antibody; beta adrenergic receptor; calcium; calcium flux; chimeric proteins; chloride channels; cyclic AMP; cystic fibrosis; gene expression; genetic library; genetic manipulation; human genetic material tag; human tissue; laboratory rabbit; membrane proteins; messenger RNA; molecular cloning; molecular pathology; muscarinic receptor; nucleic acid sequence; protein sequence; protein structure; second messengers; site directed mutagenesis; transfection; voltage /patch clamp

At the cellular level, the principal defect in cystic fibrosis (CF) appears to be the abnormal regulation of Cl- transport across the apical membrane of airway epithelia. Apical CI- channels on the epithelia of CF patients are insensitive to stimulation by the Ca 2, or cAMP- dependent pathways which activate Cl- conductances in normal cells. Although the most common genetic defect in CF occurs within the cystic fibrosis transmembrane regulator (CFTR), the relationship between this protein and the regulation of apical Cl- conductances is unknown. The proposed research will employ techniques of molecular genetics and electrophysiology to isolate molecular clones encoding Cl- channels and study the regulatory interactions between Cl- channels and the cAMP and Ca2+ second messenger pathways. These studies will provide important information regarding the structure of Cl- channels and the mechanisms by which these channels are regulated in normal and CF afflicted cells. The primary structures of CI- channels remain unknown. To obtain molecular clones encoding CI- channels, size-fractionated mRNA, isolated from a cell line expressing these Cl- channels, has been identified which directs the expression of Cl- conductances following injection and two electrode voltage clamp of Xenopus oocytes. A cDNA expression library derived from this mRNA will be transcribed in vitro and similarly analyzed in oocytes: this will allow the identification of clones encoding Cl- channels.These studies will provide the first information regarding the primary structure and potential regulatory domains of Cl- channels. The molecular mechanisms by which Cl- channels are regulated are poorly understood. To determine the mechanisms of channel regulation, molecular clones encoding Cl- channels will be expressed alone or in the presence of cloned beta-adrenergic or muscarinic acetylcholine receptors The effect of beta-adrenergic stimulation (coupled to cAMP increase), muscarinic stimulation (coupled to Ca2+ increase) and membrane potential on Cl- channels will be analyzed by patch clamp of transfected mammalian cells or voltage clamp of Xenopus oocytes. In tandem, CF-affected skin fibroblast cells will be studied under patch clamp to assess the physiologic relevance of expression studies. Site-directed mutagenesis of the cloned Cl- channel will ultimately allow identification of the critical structures involved in channel regulation.

在细胞水平上，囊性纤维化（CF）的主要缺陷 似乎是对顶端cl传输的异常调节 气道上皮的膜。 CF上皮上的顶部CI-通道 患者对CA 2或cAMP依赖的刺激不敏感 激活正常细胞中CL-电导的途径。 虽然 CF中最常见的遗传缺陷发生在囊性纤维化中 跨膜调节剂（CFTR），该蛋白与 顶端电导率的调节尚不清楚。 提议 研究将采用分子遗传学技术和 电生理学分离分子克隆，编码CL-通道和 研究Cl渠道与营地之间的监管相互作用以及 CA2+第二通路。 这些研究将提供重要的 有关Cl通道结构和机制的信息 这些通道在正常和CF折磨细胞中受到调节。 CI-通道的主要结构尚不清楚。 获得 编码CI-通道的分子克隆，大小分级mRNA，分离 从表达这些Cl通道的细胞系中已经确定了 指导注射后Cl-电导的表达和两个 异武卵母细胞的电极电压夹。 cDNA表达式库 源自此mRNA的体外和类似地转录 在卵母细胞中分析：这将允许识别克隆 这些研究将提供第一个信息 关于Cl-的主要结构和潜在调节域 频道。 调节Cl-通道的分子机制是 理解不佳。 为了确定通道调控的机制， 编码Cl-通道的分子克隆将单独或在 克隆的β-肾上腺素能或毒蕈碱乙酰胆碱受体的存在 β-肾上腺素能刺激的影响（耦合到营地增加）， 毒蕈碱刺激（耦合到Ca2+增加）和膜电位 在Cl-通道上将通过转染的哺乳动物的斑块夹进行分析 爪蟾卵母细胞的细胞或电压夹。 在串联，受CF影响的皮肤 成纤维细胞将在斑块夹下进行研究，以评估 表达研究的生理相关性。 定向诱变 克隆的cl通道最终将允许识别 涉及通道调控的关键结构。