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、长期的目标是一个详细的分子 了解电可兴奋细胞的功能， 秀丽隐杆线虫咽肌细胞。穿越我们的过去 研究我们开发了一个描述关键离子通道活动的模型 在动作电位的每个阶段，并识别基因 控制着这些频道。这种模式是不完整的：一个渠道( 负峰电流通道)未被明确识别，我们知道 在开始和中期一定有其他渠道在起作用 动作电势。这项建议的直接目的是 完成模型。 要检验的假设是：(1)EXP-2编码一个亚单位 负峰电流钾通道是快速复极所必需的 咽部肌肉。(2)钙激活钾通道 在平台期有助于膜电位的衰减 咽部肌肉动作电位。(3)一个或多个离子通道 是肌源性动作电位触发所必需的。 (4)生肌系统的活动受毒扁豆碱调节。 乙酰胆碱受体。 具体的目标是：(1)寻找细胞功能所必需的基因 EXP-2钾通道。其他亚基的功能缺失突变 该通道的激活将抑制EXP-2的功能增益表型。使用 这些基因的鉴定可以在非洲爪哇中表达该通道 并确定它是否是负峰电流通道(假设1)。 (2)鉴定和阻断钙激活钾通道基因 在咽部肌肉中表达。我们将使用记者融合来识别 基因组分析发现的钙激活钾通道基因 可能在咽部肌肉中表达的序列。这些 基因将被敲除，它们对动作电位的影响 已确定(假设2)。(3)药理试验 控制肌源性动作电位的乙酰胆碱受体。 我们将测量乙酰胆碱激动剂对 烟碱或毒扁豆碱受体对生肌活动的影响(假设4)。 (4)鉴定和干扰M受体基因在 咽部肌肉。目标2的方法将被用来测量 咽部M受体对肌源性活动的影响 (假设4)。(5)筛选生肌活动异常的突变体。 我们将筛选导致Dauers的突变，一种滞育状态 抑制肌源性泵浦，进行泵浦，然后测试它们是否 同时减轻对肌源性活动的正常依赖 乙酰胆碱。这些突变可能识别触发的离子通道 肌源性动作电位或与其相互作用的蛋白质 (假设3)。 心脏和骨骼肌疾病是由离子缺陷引起的 控制其兴奋性的通道。这项提案确定了这样的 基因，并帮助我们了解它们如何对细胞 兴奋性。