The levamisole activated acetylcholine receptor in C.elegans is now known to constitute four major subunits- LEV-1, UNC-29, UNC-38 and UNC-63. Mutants for these genes are strongly resistant to the anthelminthic, levamisole. However, regulators of receptor subunit function have not all been fully characterised. Mutations in lev-9 have been shown to confer partial resistance to both levamisole and nicotine, but do not affect levamisole binding in vitro (Lewis et al ., 1987). Thus, it has been suggested that lev-9 is a possible modulator of levamisole receptor function. We have cloned lev-9 and shown that it encodes a molecule homologous to the transmembrane glycoprotein, alpha sarcoglycan. In humans, the sarcoglycans are a major component of the dystrophin glycoprotein complex (DGC) and are associated with autosomal recessive forms of limb girdle muscular dystrophy. LEV-9 is mainly expressed in the muscles of the head, pharynx, body and vulva. Using fluorescently labeled a -bungarotoxin, we have shown that lev-9 exhibits decreased surface receptor expression compared to wild type. Preliminary results using in vivo calcium imaging from vulval muscles in dissected worm preparations also suggest a difference between lev-9 and wild type in the rate of calcium influx, in response to nicotine. Further experiments using fluorescent labeling to determine how LEV-9 affects the levamisole receptor surface expression and electron microscopic comparison of lev-9 and wild types are in progress. P> P> Lewis JA, et al . (1987) J. Neurosci . 7(10), 3059-71

Barnes TM et al. (1991) International C. elegans Meeting "THE MOLECULAR GENETICS OF THE LEVAMISOLE ACETYLCHOLINE RECEPTOR"

Barnes TM, Fleming JT, Squire MD, Lewis JA

[

International C. elegans Meeting,

1991]

The drug levamisole is a potent anthelminthic causing nematode muscles to hypercontract, which eventually leads to death. In C. elegans, mutants resistant to the cholinergic agonist levamisole define 9 genes postulated to encode both musde acetylcholine receptor ( AChR) subunits and, perhaps more interestingly, products required for the regulation and assembly of this receptor. Three of these genes ( lev-8, lev-9 and lev-10) may affect receptor function only indirectly, while the other six (lev-l, unc-29, unc-38, unc-50, unc-63 and unc-74) have more direct effects. Two types of resistant mutants are seen: strong (receptor nulls) and weak (partial receptor function). One of these genes, lev-l, is distinct in two respects: the null phenotype is weak resistance, with strongly resistant mutants being much rarer; and these strongly resistant mutants are semidominant. Previous genetic analysis and binding studies using extracts from both classes of lev-l mutants have suggested that the lev-l product is not essential for the function of the receptor, but rather has a peripheral modifying role. Similar types of evidence has led to the notion that unc-29 and unc-38 may encode structural subunits, while unc-50, unc-63 and unc-74 may be involved in receptor biogenesis. Using a combination of RFLP mapping and transposon tagging the genomic location of the lev-l gene has been established. The gene sequence has been determined from both genomic and cDNA clones, and surprisingly its putative product is a homologue of the non-ligand-binding (non-a) subunits of vertebrate and invertebrate AChRs. The gene has fewer introns than its vertebrate counterparts, but the positions of two introns have been conserved in nematode, insect and vertebrate genes. The unc-29 and unc-38 genes have also recently been cloned by transposon tagging (see Fleming et al. abstract) and both appear to encode structural subunits, in accord with the earlier predictions. A comparison of the three subunits with other known insect and vertebrate subunits shows that the unc-29 and lev-l products are very similar to each other but not to anything else. The unc-38 product is similarly very different from other a subunits. This is not consistent with subunit specialization predating phylogenetic divergence. That lev-l encodes a subunit leaves three questions: l) Why is weak resistance the null phenotype? 2) Why does only lev-l mutate to semidominance? 3) What is the basis of the semidominance? Reinterpretation of previous data concerning lev-l mutants leads to a model where the position in the receptor normally occupied by the lev-l product uniquely has fewer structural requirements for receptor assembly than do other subunits, which property may be intimately tied to the normal function of this subunit.

Fleming JT et al. (1991) International C. elegans Meeting "The cloning and expression of C.elegans acetylcholine receptors."

Fleming JT, Sattelle DB, Lewis JA, Riina HA

[

International C. elegans Meeting,

1991]

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]

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