We have cloned several structural subunits of the levamisole- acetylcholine receptor and an additional as yet uncharacterized nicotinic acetylcholine receptor. A lambda genomic library was screened under low stringency conditions using a drosophila nonalpha acetylcholine receptor subunit (ARD). Hybridizing clones were localized by fingerprinting to eight distinct locations on the physical map. Three of these have been shown to encode the genes for
unc-29,
unc-38, and lev-l . The complete cDNA and genomic sequences have been determined for these genes. An unexpected result has been the finding that
unc-38 is the alpha (ligand-binding) subunit rather than
unc-29 as had been previously predicted by us. This could have striking implications for studies of the structuraVfunctional relationships of the acetylcholine receptor subunits as both
unc-29 and
unc-38 gene products are required to form the ligand binding site. The
unc-29 transcript contains the C.elegans spliced leader sequence while the
unc-38 and lev-l transcripts appear to lack it. Unc-29 has been rescued with a single lambda clone. Comparitive quantitation of
unc-29 and
unc-38 transcript levels has been determined for N2 and levamisole resistant mutants (
unc-29, unc- 38,
unc-50 and
unc-74) by competitive PCR. Several of the other clones which fingerprinted to mapped contigs encode acetylcholine receptor subunit homologs that do not appear to be part of the levamisole-type receptor. They may represent the receptor responsible for the nicotinic sensitivity in the pharynx that remains in extremely levamisole resistant mutants. This work represents the only known, viable, ligand-gated ion channel mutants characterized to date in any species. We have been limited by an inability to carry out electrophysiological experiments in situ, due to the small size of the organism. In an attempt to circumvent the problem, we have investigated C. elegans acetylcholine and GABA receptors, initially from normal (N2) worms, by injecting mixed stage total RNA into oocytes of Xenopus laevis. At concentrations above a threshold of around 1 .OX 106M, levamisole acts on an expressed C. elegans acetylcholine receptor that gates a cation channel. Levamisole is the most potent agonist tested to date and is much more effective than nicotine. Levamisole-induced depolarizations are reversibly reduced in amplitude by 1.0X104M mecamylamine. At concentrations above a threshold of approximately 1.0X10-6M., GABA- induced responses are also detected in oocytes injected with C.elegans RNA. [See Figure]