MOLECULAR ANALYSIS OF NEURONAL T TYPE CA++ CHANNELS

神经元 T 型 CA 通道的分子分析

• 批准号: 6540099
• 负责人: EDWARD PEREZ-REYES
• 金额: $ 33.55万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-05 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540099
• 关键词: Xenopus oocyte; anticonvulsants; antihypertensive agents; calcium channel; calcium channel blockers; chimeric proteins; divalent cations; electrophysiology; expression cloning; gene expression; molecular biology; molecular genetics; neurons; pharmacology; protein isoforms; protein structure function; tissue /cell culture; transfection; voltage gated channel

DESCRIPTION: (Applicant's Abstract) 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 cells is through voltage-activated Ca2+ channels. Low voltage-activated, T-type, Ca2+ channels can open after small depolarizations of the plasma membrane, leading to further depolarization of the membrane (pacemaker activity) and changes in intracellular Ca2+. T channels are also thought to play important roles in burst firing and in oscillatory behavior of neurons. Their ability to inactivate and recover quickly over a narrow voltage range are considered key properties of thalamic neurons, allowing them to show distinct firing patterns that correlate with sleep and wakefulness. Many antiepileptic drugs can block these channels in vitro, leading to the hypothesis that abnormal expression of T channels may be involved in epilepsy. Exciting preliminary studies demonstrate the cloning and expression of a new family of alpha1 subunits that encode T-type Ca2+ channels. Cloning of these channels has opened up new areas of research that should identify the physiology of this important class of ion channel. The specific aims of this project are to: 1) characterize the electrophysiological properties of these cloned T-type channels; 2) characterize their pharmacology, in particular their block by divalent cations, antihypertensives, and antiepileptics; 3) investigate structure-function relationships of T-type channels, focusing on inactivation properties; and 4) investigate the subunit structure of these channels, focusing on how Ca channels are regulated by their beta subunits. The research design uses recombinant DNA techniques to clone and modify T-type channels 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. These studies should provide significant insights into the functional diversity of voltage-activated Ca channels, their pharmacology, and how their structure determines their function in neuronal signaling.

描述：（申请人的摘要） 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。专业之一 钙流入细胞的途径是通过电压激活的Ca2+ 频道。低压激活，T型，Ca2+通道可以在小之后打开 质膜的去极化，导致进一步的去极化 膜（起搏器活性）和细胞内Ca2+的变化。 T通道 还被认为在爆炸和振荡中起着重要作用 神经元的行为。他们失活和迅速恢复的能力 狭窄的电压范围是丘脑神经元的关键特性， 允许他们显示出与睡眠和睡眠相关的独特触发方式 清醒。许多抗癫痫药可以在体外阻止这些通道， 导致假设T通道异常表达可能是 参与癫痫。 令人兴奋的初步研究证明了新的克隆和表达 编码T型Ca2+通道的alpha1亚基家族。这些克隆 渠道已经开放了新的研究领域，应确定 这一重要类离子通道的生理学。这个特定的目的 项目是：1）表征这些的电生理特性 克隆的T型通道； 2）特征其药理学，尤其是 由二价阳离子，抗高素质剂和抗癫痫药的阻塞； 3） 研究T型通道的结构功能关系，重点 灭活特性； 4）研究这些的亚基结构 渠道，重点介绍如何通过beta亚基调节CA渠道。 研究设计使用重组DNA技术来克隆和修改T型 通道并表达两个爪蟾卵母细胞和 HEK-293细胞。电生理方法用于研究表达的 在单个通道和整个单元格级别上的通道。这些研究应该 提供有关电压激活的功能多样性的重大见解 CA渠道，药理学以及它们的结构如何决定他们 神经元信号的功能。