CLONING AND EXPESSION OF A CARDIAC T TYPE CA CHANNEL

心脏 T 型 CA 通道的克隆和表达

• 批准号: 2735392
• 负责人: EDWARD PEREZ-REYES
• 金额: $ 27.07万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2735392
• 关键词: Xenopus; Xenopus oocyte; animal genetic material tag; calcium channel; complementary DNA; heart pharmacology; laboratory rat; molecular cloning; myocardium; oligonucleotides; protein structure function; recombinant proteins; tissue /cell culture; voltage gated channel

Intracellular calcium controls a variety of cellular functions such as contraction, secretion, proliferation, and gene expression. One of the major pathways of calcium influx into cardiac myocytes is through T- and L-type voltage-gated Ca2+ channels. T-type, or low voltage-activated (LVA) channels, open after small depolarizations of the membrane. T-type currents have been found in atrial myocytes and pacemaker cells, where they are involved in determining pacemaker activity. Numerous drugs block L-type channels, however, there are no selective blockers of T-type channels. One of the long-term objectives of this research is to provide an assay system that will allow the development of T-type channel blockers. Calcium channels are multisubunit complexes composed of a large, ion-conducting subunit, alpha1, and several accessory subunits(alpha 2delta, beta and gamma). Six genes encoding alpha1 subunits have been cloned. Cloning and expression of channels has allowed their electrophysiological and pharmacological classification. These studies established that all of these alpha1s are high voltage-activated channels. The aim of this project is to clone the cardiac low voltage-activated T- type Ca2+ channel. Exciting preliminary studies have identified a novel Ca2+ channel gene expressed in heart. The hypothesis is that this gene encodes a T-type channel. The specific aims of this project are to: 1) clone the full-length cDNA, 2) suppress the expression of native T-type channels in rat neonatal ventricular myocytes, 3) determine the tissue specific expression of this gene (Northerns and in situ hybridizations, 4) express the cDNA in heterologous expression systems and measure the biophysical properties of the channel (single channel conductance, voltage-dependence), and 5) determine the pharmacological properties of the expressed current. The research design uses recombinant DNA techniques to clone T-type channels from human heart tissue and express the cloned channels in both Xenopus laevis oocytes and HEK-293 cells. Electrophysiological methods are used to study the expressed channel at both the single channel and whole cell level. The hypothesis will then be tested by comparing the properties of the cloned channel to that observed from T-type channels in isolated cells.

细胞内钙控制多种细胞功能，例如 收缩、分泌、增殖和基因表达。 中的一个 钙流入心肌细胞的主要途径是通过T-和 L 型电压门控 Ca2+ 通道。 T型，或低电压激活 (LVA) 通道，膜小幅去极化后打开。 T型 在心房肌细胞和起搏细胞中发现了电流，其中 他们参与确定起搏器活动。 多种药物阻滞 L 型通道，但没有 T 型通道的选择性阻断剂 渠道。 这项研究的长期目标之一是提供 允许开发 T 型通道的检测系统 阻滞剂。 钙通道是由 大的离子传导亚基、α1 和几个附件 亚基（alpha 2delta、beta 和 gamma）。 编码 alpha1 的六个基因 亚基已被克隆。 通道的克隆和表达允许 它们的电生理学和药理学分类。 这些 研究证实所有这些 alpha1 都是高电压激活的 渠道。 该项目的目的是克隆心脏低压激活的 T- 型 Ca2+ 通道。 令人兴奋的初步研究发现了一种新颖的 Ca2+通道基因在心脏中表达。 假设该基因 编码T型通道。 该项目的具体目标是：1) 克隆全长cDNA，2）抑制天然T型表达 大鼠新生心室肌细胞中的通道，3) 确定组织 该基因的特异性表达（Northern 和原位杂交，4） 在异源表达系统中表达 cDNA 并测量 通道的生物物理特性（单通道电导， 电压依赖性），以及5）确定药理学特性 表示的电流。 研究设计使用重组DNA 从人类心脏组织中克隆 T 型通道并表达的技术 非洲爪蟾卵母细胞和 HEK-293 细胞中的克隆通道。 使用电生理学方法来研究表达通道 单通道和全细胞水平。 那么假设将是 通过将克隆通道的特性与观察到的特性进行比较来进行测试 来自分离细胞中的 T 型通道。