MOLECULAR ANALYSIS OF NEURONAL T TYPE CA++ CHANNELS

描述：(申请人摘要) 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。其中一个主要的 钙离子内流途径是电压激活的钙离子通道 频道。低电压激活的T型钙通道可以在较小的时间内开放 质膜的去极化，导致进一步的去极化 膜(起搏器活性)和细胞内钙的变化。T形通道 也被认为在爆发式发射和振荡中起着重要作用 神经元的行为。它们能够在一年内灭活并快速恢复 窄电压范围被认为是丘脑神经元的关键特性， 使他们能够表现出与睡眠和睡眠相关的不同的放电模式 保持清醒。许多抗癫痫药物在体外可以阻断这些通道， 导致假设T通道的异常表达可能是 与癫痫有关。 令人兴奋的初步研究表明，一种新的基因克隆和表达 编码T型钙通道的α1亚基家族。克隆这些基因 频道开辟了新的研究领域，应该能够识别 这类重要的离子通道的生理学。这样做的具体目的是 项目是：1)表征这些物质的电生理特性 克隆的T型通道；2)鉴定它们的药理作用，特别是它们的 二价阳离子、降压药和抗癫痫药阻断；3) 研究T型通道的结构-功能关系，重点是 失活特性；以及4)研究这些分子的亚基结构 通道，重点是钙通道是如何被其β亚基调节的。 研究设计使用重组DNA技术来克隆和修饰T型 通道在非洲爪哇卵母细胞和非洲爪哇卵母细胞中表达 HEK-293细胞。用电生理学方法研究表达的 单通道和整个小区级别的通道。这些研究应该 提供对电压激活的功能多样性的重要见解 钙通道，它们的药理作用，以及它们的结构如何决定它们的 在神经元信号传递中的作用。

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.