MOLECULAR ANALYSIS OF NEURONAL T TYPE CA++ CHANNELS

描述：（申请人摘要） 细胞内钙控制着多种细胞功能，例如 收缩、分泌、增殖和基因表达。的一个主要 钙离子流入细胞的途径是通过电压激活的Ca 2 + 渠道低电压激活，T型，Ca 2+通道可以在小 细胞膜的去极化，导致细胞膜的进一步去极化。 膜（起搏器活性）和细胞内Ca 2+的变化。T通道 也被认为在突发放电和振荡放电中起重要作用。 神经元的行为他们的能力，恢复和迅速超过一个 窄电压范围被认为是丘脑神经元的关键特性， 使它们能够显示出与睡眠相关的独特的放电模式， 清醒许多抗癫痫药物可以在体外阻断这些通道， 这导致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，克隆和表达一种新的 编码T型Ca 2+通道的α 1亚基家族。克隆这些 渠道开辟了新的研究领域，应该确定 这类重要的离子通道的生理学。具体目标是 项目是：1）表征这些电生理特性 克隆的T型通道; 2）表征其药理学，特别是其 被二价阳离子、抗高血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;和4）研究这些亚基结构 通道，重点是如何钙通道的β亚基调节。 本研究设计利用重组DNA技术克隆和修饰T型 通道，并在非洲爪蟾卵母细胞中表达克隆通道， HEK-293细胞。采用电生理学方法研究表达的 在单个通道和整个细胞水平上的通道。这些研究应 提供了重要的见解的功能多样性的电压激活的 钙通道，它们的药理学，以及它们的结构如何决定它们的 在神经元信号传导中起作用。

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.