Cholinergic receptor mutants of the nematode Caenorhabditis elegans

Abstract

Potential acetylcholine receptor (AChR) mutants of the nematode are selectable by resistance to the neurotoxic drug levamisole, a probable cholinergic agonist. To determine which mutants may have achieved resistance through loss of levamisole receptor function, we have assayed mutant extracts for specific 3H-meta-aminolevamisole binding activity in the presence and absence of mecamylamine. We find that mutants in 3 of the 7 genes associated with extreme levamisole resistance are obviously deficient in saturable specific 3H-meta-aminolevamisole binding activity. Mutants of the 4 other genes have abnormal binding activities that fail to undergo the apparent allosteric activation of saturable specific 3H-meta-aminolevamisole binding activity caused by mecamylamine. Thus, all 7 genes appear to be required to produce a fully functional levamisole receptor. Mutants of several other genes associated only with partial resistance to levamisole have at least grossly normal receptor binding activities.

Caenorhabditis elegansLevamisole Resistance Geneslev-1,unc-29, andunc-38Encode Functional Nicotinic Acetylcholine Receptor Subunits

“…Both UNC-29 and LEV-1 are about equally similar to neuronal ␣ and non-␣ of the rat and chick (rat ␣2-␣4 and ␤2-␤4, chick ␣2 and ␣4 and ␤2 and ␤4) with UNC-29 showing slightly greater over all sequence similarity, ϳ55 versus ϳ50% for LEV-1. Consistent with these results, many aspects of the in situ pharmacology of muscle nAChRs of C. elegans and Ascaris resemble those of vertebrate neuronal nAChRs, including insensitivity to ␣-bungarotoxin, block by mecamylamine (Lewis et al, 1987a(Lewis et al, , 1980bFleming et al, 1993), and, in the case of Ascaris suum muscle nAChRs, block by -bungarotoxin (Colquhoun et al, 1993).…”

“…The number of weak alleles isolated thus mainly reflects their relative ease of isolation. This table summarizes discussion of previously reported work described in the text (Lewis et al, 1980a(Lewis et al, , 1987b; work cited in other footnotes). Moerman et al (1988).…”

“…unc-29 mutants had little specific [ 3 H]MAL binding, whereas unc-38 mutants retained detectable binding (Lewis et al, 1987b). Levamisole seems to act like nicotine, competing with acetylcholine for binding to the receptor (Lewis et al, 1980b;Harrow and Gration, 1985;Lewis et al, 1987a).…”

“…3 H]meta-aminolevamisole-binding studies using membrane fragments (Lewis et al, 1987a) and cut worm muscle contraction assays (Lewis et al, 1980b). Cytoplasmic co-injection of cRNAs encoding UNC-29, LEV-1, and UNC-38 resulted in inwardly directed currents (holding potential, Ϫ60 mV) in response to levamisole (1 ϫ 10 Ϫ4 M), whereas oocytes injected separately with message encoding a single subunit or the equivalent volume of distilled water showed no such responses (Fig.…”

“…The null phenotype of unc-29 has yet to be defined, but both homozygotes of the unc-29(x29) and unc-29(e1072) mutations have been shown previously to contain little or no detectable specific high-affinity [ 3 H]M AL binding (Lewis et al, 1987b). Although the focus of unc-38 and lev-1 expression has not been defined in this study, the great similarity in mutant phenotypes of unc-38 mutants and lev-1 semidominants to unc-29 mutants makes it likely that muscle is also the major focus for the expression of these genes as well.…”