The long-term objective is to understand the function of an excitable cell, the pharyngeal muscle cell. The pharynx is a tubular pump responsible for sucking bacteria into the worm, concentrating them, and grinding them up. It consists of epithelial cells, marginal cells, and muscle cells. A basal lamina surrounds the entire pharynx and isolates it from the rest of the worm. Pharyngeal neurons lie under the basal lamina in indentations of the muscle cells. Aside from the connections to the mouth and the intestine, there are only two holes in the basal lamina, through which the processes of a pair of neurons pass to connect to the pharyngeal nervous system. The pharynx pumps even when the entire pharyngeal nervous system has been killed by laser microsurgery, and pharyngeal muscle motions remain synchronized.As in the vertebrate heart, the nervous system regulates the timing and rate of pumping. Acetylcholine is probably one of the neurotransmitters used, since acetylcholine agonists promote contraction and inhibit relaxation, and a mutant unable to synthesize acetylcholine will only pump in the presence of acetylcholine agonists. Dr. Avery's previous work has shown that genes with abnormal pharyngeal muscle excitability can be identified, and their electrophysiological effects studied. Pharyngeal motions are fast and the effects of some mutations are subtle, so he has developed the means for millisecond analysis of muscle motions. There are three hypotheses to be tested. First, genes that affect pharyngeal muscle relaxation act within the muscle cell to control membrane currents. The proposed experiments will test whether the eat-6, eat-11, eat-12, egl-30, or exp-2 genes (at least two of which are known to affect pharyngeal muscle excitability) act on pharyngeal muscle, and what their electrophysiological effects are. The second hypothesis is that some or all of these genes encode components of a signal transduction pathway within the muscle cell that controls the timing of relaxation. This will be tested by determining whether certain genes encode known signal transduction proteins. The third hypothesis is that the timing of relaxation is modulated by controlling the outward current that returns the membrane potential to resting levels. This will be tested by measuring the current in the presence of drugs and mutations that influence relaxation timing. Relaxation current will be measured by extracellular or intracellular recording. There are five specific aims. First, laser microsurgery experiments will determine whether genes that affect pharyngeal muscle excitability act in the muscle or in the nervous system. Second, two genes, eat-6 and eat-11, are to be cloned, their expression patterns are to be determined, and mutant alleles are to be sequenced. Third, the null phenotypes of these two genes will be determined. Fourth, new mutations that enhance or suppress existing pharyngeal excitability mutant phenotypes will be isolated. Fifth, pharyngeal muscle membrane potential will be recorded from wild type and mutant pharynxes using intracellular electrodes or patch-clamp techniques. The genes to be studied affect the electrical properties of pharyngeal muscle in various ways. For example, the eat-5 mutation uncouples corpus and terminal bulb motions. eat-6 mutations inhibit pharyngeal relaxation, and eat-4 affects the timing of relaxation. The eat-5 gene has been located within a 3.8 kb genomic fragment that rescues the eat-5 mutant phenotype. Sequencing is in progress. The eat-4 gene all falls within a region that has already been sequenced by the C. elegans genome project. It has been specifically localized by means of an RFLP associated with an eat-4 mutation, and lies within a cosmid that rescues the eat-4 mutant phenotype. The GENEFINDER program has identified seven possible genes within this cosmid, two of which overlap the restriction fragment mutated in eat-4. Neither gene encodes a protein with significant similarity to known proteins, and further work is aimed at pinning down which gene is eat-4.
Showing the most recent 10 out of 35 publications
