CLONING AND EXPESSION OF A CARDIAC T TYPE CA CHANNEL

细胞内钙离子控制多种细胞功能，如 收缩、分泌、增殖和基因表达。其中一个 钙内流到心肌细胞的主要途径是通过T-和 L型电压门控性钙通道。T型，或低电压激活 (LVA)通道，在膜的小去极化后开放。T型 在心房肌细胞和起搏器细胞中发现了电流，其中 它们参与确定起搏器的活动。众多药物封杀 L型通道，但没有T型选择性阻滞剂 频道。这项研究的长期目标之一是提供 一种允许T-型通道发展的检测系统 拦截者。钙通道是由多个亚单位组成的复合体 大的离子传导亚基，α1和几个附件 亚基(α2β、β和伽马)。编码α1的六个基因 亚基已经被克隆。克隆和表达通道已经允许 他们的电生理和药理学分类。这些 研究证实，所有这些字母1都是高压激活的 频道。 本项目的目的是克隆心脏低电压激活的T细胞。 键入钙离子通道。令人兴奋的初步研究确定了一部小说 钙通道基因在心脏中表达。假设是这个基因 对T型通道进行编码。本项目的具体目标是：1) 克隆全长cDNA2)抑制天然T型基因的表达 新生大鼠心室肌细胞的通道，3)确定组织 该基因的特异性表达(Northerns和原位杂交，4) 在异源表达系统中表达该基因并检测其活性 通道的生物物理属性(单通道电导， 电压依赖性)，以及5)确定其药理特性 所表达的电流。研究设计采用重组DNA 人心脏组织中T型通道的克隆及表达技术 非洲爪哇卵母细胞和HEK-293细胞中的克隆通道。 使用电生理学方法研究表达的通道在 无论是单通道级别还是整个细胞级别。那么这个假设就是 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 来自隔离细胞中的T-型通道。

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.