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.

该项目的广泛长期目标是详细的分子 理解电气激发电池的功能， 秀丽隐杆线虫的咽肌细胞。 通过我们的过去 研究我们已经开发了一个描述关键离子渠道活动的模型 在动作电位的每个阶段，并识别基因 控制这些渠道。该模型不完整：一个通道（ 负面的尖峰频道）并未明确确定，我们知道 一开始和中间必须有其他渠道 动作潜力。 该提议的直接目的是 完成模型。 要测试的假设是：（1）EXP-2编码A的亚基 快速复制所需的负峰值钾通道 咽肌。 （2）钙激活的钾通道 在高原阶段有助于膜电位的衰减 咽部肌肉动作潜力。 （3）一个或多个离子通道 对于动作电位的肌源性触发是特别需要的。 （4）肌源系统的活性由毒蕈碱调节 乙酰胆碱受体。 具体目的是：（1）找到功能所需的基因 EXP-2钾通道。 其他亚基的功能丧失突变 该通道将抑制EXP-2功能获得的表型。 和 这些基因的识别我们可以在xenopus中表达通道 卵母细胞并确定它是否是负尖峰通道（假设1）。 （2）识别和破坏钙激活的钾通道基因 在咽肌肉中表达。我们将使用记者融合来识别 通过基因组分析发现钙激活的钾通道基因 可能在咽肌中表达的序列。 这些 基因将被淘汰，它们对动作电位的影响 确定（假设2）。 （3）在药理上测试贡献 乙酰胆碱受体的控制肌源作用电位。 我们将衡量乙酰胆碱激动剂特有的影响 在肌源性活性上的烟碱或毒蕈碱受体（假设4）。 （4）识别和破坏在 咽肌。 AIM 2的方法将用于测量 咽毒蕈碱受体对肌原性活性的影响 （假设4）。 （5）筛查具有异常肌源活性的突变体。 我们将筛选导致Dauers的突变，这是一个滞育状态 抑制肌源性抽水，泵送，然后测试它们是否 同时缓解肌原活性的正常依赖性 乙酰胆碱。 这些突变可能识别触发的离子通道 与之相互作用的肌源作用电位或蛋白质 （假设3）。 因离子缺陷而导致心脏和骨骼肌疾病 控制其兴奋性的渠道。 该提案确定了这样的 基因并帮助我们了解它们如何对细胞有助于 兴奋性。