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.
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