PHYSIOLOGICAL ROLE OF MULTIPLE K+ CHANNELS

描述(改编自申请人的摘要)： 这四个基因编码了大量的电压敏感K通道 在Shaker家族中。在果蝇中，这些基因中的三个经历了 对它们的mRNA进行选择性剪接以产生多种蛋白质 不同的生物物理特性。此外，单个基因的剪接变体 基因可以相互作用形成异多聚体通道。生理学 这种钾通道多样性的作用还不是很清楚。它是 很难将特定的基因与细胞中的离子电流联系起来， 并展示该基因产物如何帮助形成电生理 细胞的属性和细胞所在的神经网络 功能。这项提议的目的是用小的， 甲壳类口胃神经节内有清晰的幽门网络。 这个网络的14个神经元很容易识别，它们独特的 生理特性是众所周知的。我们正在克隆人的cDNA Shaker家族中的四个基因(shaker、shab、shaw和shal)，以及 确定它们在这14个确定的细胞中的每一个中的功能 幽门网络。为了理解振荡器家族电流在 幽门网络运动模式的产生，我们提出如下建议 目标： 1)克隆Shaker、Shab、Shaw和Shal形式的选择性剪接变体 龙虾。2)在非洲爪哇卵母细胞和龙虾神经元中表达这些变体， 并将它们的生物物理特性与体内的内生电流进行比较 口胃神经元。3)将这些变体的表达映射到 使用我们已有的单细胞聚合酶链式反应方法 最近开发出来的。4)人为改变Shaker家族的表达 通过将正义或反义RNA注射到已识别的STG神经元中，以及 确定或增加或降低特定电流对 细胞和网络特性以及细胞对单胺的反应 调制器。 这项工作的最终目标是回答为什么有这么多基因和 电压依赖K通道的剪接变体。我们的研究将允许 美国将为特定的K通道基因分配生理角色 已识别神经元和运动的固有放电特性 幽门网络产生的模式。

DESCRIPTION (Adapted from applicant's abstract) : A large number of voltage-sensitive K+ channels are coded by the four genes in the Shaker family. In Drosophila, three of these genes undergo alternative splicing of their mRNA to generate multiple proteins with different biophysical properties. In addition, splice variants of a single gene can interact to form heteromultimeric channels. The physiological roles of this potassium channel diversity are not well understood. It is difficult to associate a particular gene with an ionic current in a cell, and to show how that gene product helps to shape the electrophysiological properties of the cell and the neural network within which the cell functions. This proposal is aimed at filling this gap using the small, well-defined pyloric network in the crustacean stomatogastric ganglion. This network's 14 neurons are easily identified, and their unique physiological properties are well known. We are cloning the cDNAs of the four genes in the Shaker family (shaker, shab, shaw and shal), and determining their functions in each of the 14 identified cells in this pyloric network. To understand the roles of Shaker family currents in the generation of the pyloric network's motor pattern, we propose the following goals: 1) Clone the alternative splice variants of shaker, shab, shaw and shal form lobster. 2) Express these variants in Xenopus oocytes and lobster neurons, and compare their biophysical properties to endogenous currents in the stomatogastric neurons. 3) Map the expression of these variants in the individual identified neurons using a single cell PCR method that we have recently developed. 4) Artificially alter the expression of Shaker family genes by injecting sense or antisense RNA into identified STG neurons, and determine the effect or raising or lowering specific currents on the cellular and network properties and cellular responses to monamine modulators. The ultimate goal of this work is to answer why there are so many genes and splice variants for voltage-dependent K+ channels. Our studies will allow us to assign physiological roles to specific K+ channel genes in determining both the intrinsic firing properties of identified neurons and the motor pattern generated by the pyloric network.