ION CHANNELS

快速交流，通常是在遥远的神经区域之间 系统，是由编码和传输的可兴奋细胞完成的 通过电信号提供信息。电压门控钠通道在 脊椎动物细胞电兴奋性的关键作用 无脊椎动物的神经系统。带着理解的长期目标 电兴奋性在正常发育和发育中的调节 病理改变的情况下，目前的提议利用 果蝇，一种生物体，其中生理和 可以使用分子技术来检查功能和 电压门控钠通道的调节，由parA基因编码，在 初级神经元。我们的初步研究表明，替代方案 对钠通道前体信使核糖核酸的剪接 第一个细胞质环，导致产生四个不同于 它们被蛋白激酶A(PKA)磷酸化的可能性。相关性 用全细胞电生理记录的parm RNA图谱，在 单个胚胎神经元，表明其中一个亚型包含一个 PKA位点(由外显子a编码)是钠电流表达所必需的。 这项提案中的第一项研究将确定钠通道的活性 在果蝇中，神经元可以通过磷酸化来调节，如果这是 与编码特异性PKA的parmRNA异构体表达的相关性 网站。在第二个系列的实验中，我们将检查 选择性地减少或消除所涉及的特定剪接变体 在调节通道功能时，对钠电流有表达作用。这将是 通过产生表达热休克的转基因果蝇完成的 靶向特定剪接变异体或暴露于 培养神经元以拼接变异型特异性反义寡核苷酸。 已知可改变cAMP依赖的突变体的钠通道功能 磷酸化，将被检测以确定内源性的影响 改变了通道功能上的磷酸化水平。在第三节 部分，相关研究，以确定其他替代领域 参与功能的副基因的剪接将被启动。自.以来 我们的长期利益是对电刺激性的监管，最后一个 一系列研究的目的是识别编码神经元的基因 钾和钙通道。表情分析技术将会 被用来筛选候选离子通道基因的表达 胚胎神经元。一旦我们确定了编码钙的基因 和钾通道，前几节中开发的技术将 应用于研究这些电流的调节。要研究如何 电压门控离子通道基因的表达受协同调控 导致细胞特异性激发特性，mRNA表达谱 具有独特激发特性的细胞将被比较。这些研究的结果 研究将增加我们对替代的作用的理解 剪接和磷酸化在介导离子通道功能中的作用 离子通道基因表达的协调调节如何影响 发育中的神经系统中的细胞通过 电脉冲。

Rapid communication, often between widely separated regions of the nervous system, is accomplished by excitable cells that encode and transmit information via electrical signals. Voltage-gated sodium channels play a key role in electrical excitability of cells in vertebrate and invertebrate nervous systems. With the long term goal of understanding the regulation of electrical excitability during normal development and in pathologically altered conditions, the current proposal utilizes Drosophila, an organism in which a combination of physiological and molecular techniques can be employed, to examine the function and regulation of voltage-gated sodium channels, encoded by the para gene, in primary neurons. Our preliminary studies have shown that alternative splicing of the para sodium channel pre-mRNA, in the region encoding the first cytoplasmic loop, results in generation of four mRNAs that differ in their potential for phosphorylation by protein kinase A (PKA). Correlation of para mRNA profiles with whole cell electrophysiological recordings, in single embryonic neurons, suggests that one of the isoforms containing a PKA site (encoded by exon a) is necessary for sodium current expression. The first study in this proposal will determine if sodium channel activity in Drosophila neurons can be modulated by phosphorylation and if this is correlated with expression of para mRNA isoforms encoding specific PKA sites. In a second series of experiments we will examine the effects, of selectively decreasing or eliminating specific splice variants implicated in regulating channel function, on sodium current expression. This will be accomplished by generating transgenic flies expressing heat-shock inducible ribozymes targeted to specific splice variants or exposure of cultured neurons to splice variant-specific antisense oligonucleotides. Sodium channel function, in mutants known to alter cAMP-dependent phosphorylation, will be examined to determine the effects of endogenously altered levels of phosphorylation on channel function. In the third section, correlative studies to identify additional regions of alternative splicing in the para gene involved in function, will be initiated. Since our long term interest is regulation of electrical excitability, the last series of studies is aimed at identification of genes encoding neuronal potassium and calcium channels. The technique of expression profiling will be used to screen for expression of candidate ion channel genes in embryonic neurons. Once we have identified the genes encoding the calcium and potassium channels, techniques developed in the previous sections will be applied to the study of regulation of these currents. To examine how expression of voltage-gated ion channel genes is coordinately regulated to result in cell-specific firing properties, mRNA expression profiles of cells with unique firing properties will be compared. The results of these studies will increase our understanding of the roles of alternative splicing and phosphorylation in mediating ion channel function, as well as how coordinate regulation of ion channel gene expression influences the ability of cells in the developing nervous system to communicate through electrical impulses.