GENETICS OF NEMATODE PHARYNGEAL MUSCLE EXCITABILITY

线虫咽肌兴奋性的遗传学

• 批准号: 2766767
• 负责人: Leon Avery
• 金额: $ 22.22万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-05 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2766767
• 关键词: Caenorhabditis elegans; Xenopus; action potentials; alleles; biological signal transduction; calcium ion; cholinergic receptors; electrophysiology; gene expression; gene interaction; gene mutation; lasers; membrane potentials; molecular cloning; muscle cells; muscle relaxation; nicotinic receptors; northern blottings; nucleic acid sequence; oral pharyngeal; phenotype; potassium channel; restriction fragment length polymorphism

The broad, long-term objective of this project is a detailed molecular understanding of the function of an electrically excitable cell, the pharyngeal muscle cell of Caenorhabditis elegans. Through our past research we have developed a model describing key ion channels active at each stage of the action potential, and identifying the genes controlling these channels. This model is incomplete: one channel (the negative spike channel) is not definitively identified, and we know that there must be other channels acting in the beginning and the middle of the action potential. The immediate object of this proposal is to complete the model. The hypotheses to be tested are: (1) exp-2 encodes a subunit of a negative spike potassium channel necessary for the fast repolarization of pharyngeal muscle. (2) Calcium-activated potassium channels contribute to the decay of membrane potential during the plateau phase of the pharyngeal muscle action potential. (3) One or more ion channels are specifically necessary for myogenic triggering of action potentials. (4) The activity of the myogenic system is regulated by a muscarinic acetylcholine receptor. The specific aims are: (1) Find genes necessary for function of the EXP-2 potassium channel. Loss-of-function mutations in other subunits of the channel will suppress an exp-2 gain-of-function phenotype. With the identification of these genes we can express the channel in Xenopus oocytes and determine if it is a negative spike channel (hypothesis 1). (2) Identify and disrupt calcium-activated potassium channel genes expressed in pharyngeal muscle. We will use reporter fusions to identify calcium-activated potassium channel genes found by analysis of genome sequence that are likely to be expressed in pharyngeal muscle. These genes will be knocked out and their effects on the action potential determined (hypothesis 2). (3) Test pharmacologically the contributions of acetylcholine receptors to controlling myogenic action potentials. We will measure the effect of acetylcholine agonists specific for nicotinic or muscarinic receptors on myogenic activity (hypothesis 4). (4) Identify and disrupt muscarinic receptor genes expressed in pharyngeal muscle. The methods of aim 2 will be used to measure the effects of pharyngeal muscarinic receptors on myogenic activity (hypothesis 4). (5) Screen for mutants with abnormal myogenic activity. We will screen for mutations that cause dauers, a diapause state with suppressed myogenic pumping, to pump, and then test whether they simultaneously relieve the normal dependence of myogenic activity on acetylcholine. These mutations may identify ion channels that trigger myogenic action potentials or proteins that interact with them (hypothesis 3). Diseases of the heart and skeletal muscle result from defects in the ion channels that control their excitability. This proposal identifies such genes and helps us understand how they contribute to cellular excitability.

这个项目的广泛的长期目标是详细的分子 了解电可兴奋细胞的功能， 秀丽隐杆线虫咽肌细胞 通过我们的过去 我们已经开发了一个模型，描述关键离子通道的活性 在动作电位的每个阶段， 控制这些渠道。这个模型是不完整的：一个通道（ 负尖峰通道）没有被明确识别，我们知道， 一定有其他渠道在开始和中间起作用， 动作电位 这项建议的直接目的是 完成模型。 待检验的假设是：（1）exp-2编码一个亚基， 快速复极化所必需的负峰电位钾通道 咽部肌肉 (2)钙激活钾通道 有助于在平台期膜电位的衰减 咽肌动作电位的变化 (3)一个或多个离子通道 是肌源性触发动作电位所必需的。 (4)生肌系统的活性由毒蕈碱调节， 乙酰胆碱受体 具体目的是：（1）寻找与该基因功能相关的基因， EXP-2钾通道。 其他亚基的功能丧失突变 将抑制exp-2功能获得性表型。 与 这些基因的鉴定我们可以表达的通道在非洲爪蟾 卵母细胞，并确定它是否是一个负尖峰通道（假设1）。 (2)识别和破坏钙激活钾通道基因 在咽肌中表达。我们将使用报告融合来识别 基因组分析发现钙激活钾通道基因 可能在咽肌中表达的序列。 这些 基因将被敲除，它们对动作电位的影响 假设（2）。 (3)测试贡献 乙酰胆碱受体来控制肌源性动作电位。 我们将测量乙酰胆碱激动剂的作用， 烟碱或毒蕈碱受体对生肌活性的影响（假说4）。 (4)鉴定和破坏在细胞中表达的毒蕈碱受体基因， 咽肌 目标2的方法将用于测量 咽部M受体对肌源性活动的影响 （假设4）。 (5)筛选具有异常生肌活性的突变体。 我们将筛选导致dauers的突变，dauers是一种滞育状态， 抑制肌源性泵送，泵，然后测试他们是否 同时缓解肌源性活动对 乙酰胆碱 这些突变可能会识别出触发 肌源性动作电位或与之相互作用的蛋白质 （假设3）。 心脏和骨骼肌的疾病是由于离子缺陷引起的。 控制其兴奋性的通道。 该提案确定了这样的 帮助我们了解它们是如何对细胞 兴奋性