Rapid pharyngeal pumping is one of the behavioral responses of a wild type worm to bacteria. Laser ablation experiments have shown that the pharyngeal motor neuron MC is responsible for rapid pumping (Neuron 3: 473). Genetic and pharmacological evidence suggest that the MC neurotransmitter is acetylcholine (ACh). Mutants with reduced acetylcholine neurotransmission or synthesis, such as
snt-1 and
unc-17, have reduced pumping rates. Antagonists of nicotinic acetylcholine receptors reversibly reduce the pumping rate of a dissected pharynx whereas nicotine stimulates pharyngeal contraction (Genetics 141: 1365). We identified
eat-2 in a screen for mutations that eliminate neurotransmission from MC.
eat-2 worms are incapable of rapid pharyngeal pumping. Two results implicate
eat-2 in MC neurotransmission. First, electrical recordings from the pharynxes of
eat-2 mutants were similar to recordings from worms in which MC had been ablated. Also, when MC was ablated in two
eat-2 loss of function alleles, pharyngeal pumping rate did not decrease when compared to the same mutants in which MC was intact (Genetics 141: 1365). Fourteen independent recessive mutations were identified in
eat-2 that all caused a reduced pumping rate. There is complex intragenic complementation among the 14
eat-2 alleles as well as allele specific interactions between
eat-2 and a semidominant allele of another slow pumping mutant,
eat-18 (Genetics 141: 1365).
eat-18 is MC- by the same criteria as
eat-2. A model consistent with these data is that
eat-2 and
eat-18 gene products interact in a protein complex that is involved in ACh neurotransmission from MC. We mapped
eat-2 to the end of the right arm of LGII near
unc-52, a region of the genome that is currently being sequenced. A recent sequence update contained a nicotinic ACh receptor subunit in an area consistent with the map position of
eat-2 (Eric Jorgenson, personal communication). Transformation of
eat-2 mutants with a YAC containing the nicotinic receptor subunit rescued the slow pumping phenotype. To determine if the nicotinic receptor was responsible for rescuing
eat-2 we used PCR to amplify a 10kb genomic region that contained the coding region of the receptor and 4kb of upstream sequence. This 10kb PCR product was sufficient to rescue
eat-2, showing that the nicotinic receptor is encoded by
eat-2. We also found that the
eat-2 allele
ad451 contains a missense mutation that changes a highly conserved glutamate to a lysine in the amino-terminal extracellular region of the receptor. Furthermore, we were able to rescue MC neurotransmission in an
eat-2 mutant by expressing an
eat-2 genomic clone in pharyngeal muscle using the
myo-2 promoter. Rescue by this construct shows activity of
eat-2 in pharyngeal muscle is sufficient to restore MC neurotransmission. Comparison of
eat-2 with other nicotinic receptor subunits shows it is most similar to vertebrate alpha7 subunits of neuronal nicotinic ACh receptors. However it does not have the vicinal cysteines present near the ACh binding site characteristic of all alpha subunits and is therefore a non-alpha subunit.