VOLTAGE GATED ION CHANNELS--STRUCTURE & EXPRESSION

离子通道是神经和肌肉的基本成分 兴奋性，因此，是所有动物行为的基础。 对这些重要分子的全面了解 必须将分子结构和设计与生物物理和 这些蛋白质的生理作用。理想的分析系统 将允许克隆离子通道基因和 克隆基因在多种细胞类型中的重新导入 对合成和生物物理功能的各种研究。这位将军 这项建议的目的是开发这个全面的系统在 操纵和研究离子通道的果蝇。这个 这项建议的具体目标是： (1)DNA序列和基因组的阐明 可能的果蝇离子通道基因的组织。这个 第一个要研究的基因是用cdna探针分离出来的 脊椎动物的钠通道和部分测序 表明它与该通道有密切的同源性。这个 基因从细胞遗传学上映射到染色体区域 突变会导致行为和生理缺陷。 (2)A.将克隆的离子通道基因导入 P元件基因介导的果蝇生殖系 和B.离子通道的实验诱导。 通过构建一种杂交基因来表达基因 热休克调控下的离子通道结构基因 启动子，一种可以诱导基因表达的强大启动子 在大多数细胞类型中都有表达。动物被转化为 将对该基因进行以下检测：(A)mRNA的诱导性；(B) 蛋白质插入膜；(C)电压门控离子 电导。这些实验的一个最终目的是 是为了肯定地识别电压门控机制 这种分子；基因的一部分编码一串 正电氨基酸被认为是门控 电荷将通过定点突变而改变， 产生的生物物理表型由电压和 膜片钳技术。 (3)研究离子通道是成员的假设 一个扩大的基因家族，而且共享的同源性将 提供了一种在果蝇中克隆和分析品种的方法 离子通道基因。

Ion channels are the fundamental elements of nerve and muscle excitability and, as such, are fundamental to all animal behavior. A comprehensive understanding of these important molecules must relate molecular structure and design to the biophysical and physiological roles of these proteins. An ideal system of analysis would permit the cloning of ion channel genes and the reintroduction of the cloned genes in a variety of cell types for various studies of synthesis and biophyiscal function. The general aim of this proposal is to develop this comprehensive system in Drosophila for manipulating and studying ion channels. The specific aims of this proposal are: (1) The elucidation of the DNA sequence and genomic organization of a putative Drosophila ion channel gene. The first gene to be studied was isolated with a cDNA probe to the vertebrate sodium channel and partial sequencing has shown that it has close homology with this channel. The gene maps cytogenetically to a chromosomal region where mutations confer behavioral and physiological defects. (2) A. The introduction of a cloned ion channel gene into the germ line of Drosophila by P element-mediated gene transfer, and B. The experimental induction of ion channel gene expression by constructing a hybrid gene which puts an ion channel structural gene under control of the heat shock promoter, a strong promoter which can induce gene expression in most cell types. Animals transformed with this gene will be assayed for: (a) inducibility of mRNA; (b) insertion of protein into membranes; (c) voltage-gated ion conductance. One ultimate aim of these experiments will be to positively identify the voltage gating mechanism of this molecule; a portion of the gene coding for a string of positively charged amino acids hypothesized to be gating charges will be altered by site-directed mutagenesis and the resulting biophysical phenotypes analyzed by voltage and patch clamp techniques. (3) Investigating the hypothesis that ion channels are members of an extended gene family and that shared homology will provide a way in Drosophila to clone and analyze a variety of ion channel genes.