STRUCTURE AND FUNCTION OF A NEURONAL CALCIUM CHANNEL

电压门控性钙通道调节钙离子进入神经元 从而控制各种Ca2+依赖性过程， 神经递质释放、轴突生长、兴奋性和基因 表情 电生理实验表明，多种类型的 Ca2+通道（分为T，N，L和P）已经进化到执行 在细胞的不同部位有专门的功能。 实验 本建议中概述的内容旨在了解 这种功能多样性的结构基础。 我们已经分离出 重叠的cDNA编码一本小说的整个开放阅读框架 Ca2+通道（doe-4），来自从电叶制备的文库， 海洋射线Discopyge ommata。 基于氨基酸序列分析， DOE-4与骨骼肌和心肌大约40%同源 L-型钙离子通道和大约75%同源的神经元 P型通道。 我们还克隆了另外两个Ca2+通道， 电子波瓣，doe-2和doe-3，几乎与 哺乳动物L型和P型通道。 因此我们怀疑 该制剂中最丰富克隆Doe-4是N型Ca2 + 频道 这一点得到了我们先前研究的支持， Discopyge ommata电器官是一个丰富的结合位点的来源， N-型Ca2+通道拮抗剂，（omegaCgTx. 这个项目的总体目标是 项目是将分子生物学和生物化学中的联合收割机技术结合起来， 表征这一重要的结构和功能特性 频道 我们将使用doe-4 cDNA作为起始材料，在多种 解决结构-功能问题的实验。 表征 的一些结构特性的渠道，我们建议开发 一系列针对氨基酸的抗融合蛋白抗体 特异于DOE-4的序列。 初步实验将针对 确定组织分布、亚细胞定位，以及 通道复合物的亚基组成。 我们亦会研究 的两种交替剪接形式的分布特性， 频道 这些实验的目的是为了更好地理解 Ca2+通道在决定突触中的作用 专业化 此外，我们还将研究 在哺乳动物细胞系中表达的DOE-4。 我们将使用抗体作为 探针，以确定不同结构域的功能重要性， 蛋白质，并研究与其他亚基和 调节蛋白 我们还将开发一种大规模的检测方法， 药理学化合物的筛选。 的理解 神经元钙通道功能的分子细节将提供新的 深入了解神经元兴奋性的复杂性， 神经递质释放，并可能导致发现更有效的 用于治疗多种神经递质相关病症的疗法。

Voltage-gated Ca2+ channels regulate the entry of Ca2+ ions into neurons and thereby control a variety of Ca2+-dependent processes such as neurotransmitter release, neurite outgrowth, excitability and gene expression. Electrophysiological experiments suggest that multiple types of Ca2+ channels (classified as T, N, L, and P) have evolved to perform specialized functions in different parts of the cell. The experiments outlined in this proposal are directed toward understanding the structural basis for this functional diversity. We have isolated overlapping cDNAs that code for the entire open reading frame of a novel Ca2+ channel (doe-4) from a library prepared from the electric lobe of the marine ray Discopyge ommata. Based on amino acid sequence analysis, doe-4 is approximately 40% homologous to skeletal and cardiac muscle L-type Ca2+ channels and approximately 75% homologous to a neuronal P-type channel. We have also cloned two other Ca2+ channels from the electric lobe, doe-2 and doe-3, that are nearly identical to the mammalian L-type and P-type channels, respectively. We suspect therefore that doe-4, the most abundant clone in the preparation, is an N-type Ca2+ channel. This is supported by our previous studies showing that Discopyge ommata electric organ is a rich source of binding sites for the N-type Ca2+ channel antagonist, (omegaCgTx. The overall goal of this project is to combine techniques in molecular biology and biochemistry to characterize the structural and functional properties of this important channel. We will use doe-4 cDNA as the starting material in a variety of experiments addressing structure-function questions. To characterize some of the structural properties of the channel, we propose to develop an array of anti-fusion protein antibodies directed against amino acid sequences specific to doe-4. Initial experiments will be aimed at determining the tissue distribution, subcellular localization, and subunit composition of the channel complex. We will also study the distribution properties of two alternatively spliced forms of the channel. The goal of these experiments is to gain a better understanding of the role that Ca2+ channels play in determining synaptic specialization. Moreover, we will study the functional properties of doe-4 expressed in a mammalian cell line. We will use antibodies as probes to determine the functional importance of various domains of the protein, and to study direct interactions with other subunits and regulatory proteins. We will also develop an assay for large scale screening of pharmacological compounds. An understanding of the molecular details of neuronal Ca2+ channel function will provide new insights into the complexities of neuronal excitability and neurotransmitter release, and may lead to the discovery of more effective therapy for treatment of a variety of neurotransmitter related disorders.