Worm Breeder's Gazette 8(2): 41
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have previously hypothesized and offered preliminary evidence that some levamisole-resistant mutants, particularly the most resistant kind, might be receptor-deficient mutants that survive the neurotoxic effects of levamisole because they no longer have a sufficiently normal receptor to be fatally stimulated by the drug. Here we outline our results showing that indeed all seven genes giving rise to extreme levamisole resistance when mutated produce an abnormal levamisole receptor as detected by the in vitro binding of [3H] meta- aminolevamisole ([3H] MAL). Several other genes which only seem to be mutable to partial levamisole resistance and mutants of the two twitcher genes unc-22 and lev-11 appear to produce a normal receptor as detected by [3H] MAL binding. A key element in determining whether receptor activity in these mutants is wild-type or mutant has been the effect of mecamylamine on levamisole receptor binding activity. Mecamylamine is a vertebrate cholinergic blocking agent and seems to also act as a cholinergic blocking agent on living worms to stop the muscle contracting effects of levamisole and cholinergic agonists in a noncompetitive fashion, (i. e., the blocking effect is not easily competed away by jacking up the concentration of levamisole). Curiously enough, in [3H] MAL binding assays, mecamylamine acts as an apparent allosteric activator of [3H] MAL receptor binding activity. The observation of potentiation instead of inhibition is still consistent with a noncompetitive physiological blocking effect if the several fold higher affinity [3H] MAL binding state observed in the presence of mecamylamine is an inactive state of the receptor. Upon assaying the wild-type and levamisole-resistant mutants for specific [3H] MAL binding in the presence and absence of 1 mM mecamylamine, our results are as follows: The binding activity of the wild-type appears to be composed of a true, saturable high affinity receptor binding activity and another unrelated, linearly increasing nonsaturable binding activity. Mutants of three resistance loci, unc- 29, y the nonsaturable component and lack the saturable high affinity activity. The residual nonsaturable component shows no mecamylamine effect. Mutants of the other four levamisole-resistant unc loci, unc-38, lev-1 and lev-7, all show substantial amounts of saturable high affinity receptor binding activity. The most important distinguishing feature of the receptor activities in these mutants is that alleles of all four loci show no mecamylamine effect. The affinity of the receptor found in these mutants also seems to be consistently much higher than the affinity of the wild-type receptor for [3H] MAL. Perhaps the nascent state of the [3H] MAL binding subunit is one of high affinity and formation of a complete, intact receptor converts the [3H] MAL binding subunit to the lower affinity state seen in the wild-type. Except for the lev-7 locus, we've assayed two extremely levamisole-resistant mutants for each locus and obtained consistent results between alleles, suggesting we've identified the usual receptor phenotype of extreme mutants at each locus. The lev-1 locus is more complicated than this simple picture and that's described below. Thus, we have two tests: One test, [3H] MAL binding activity, is pretty one-dimensional and probably reveals mostly whether the [3H] MAL binding peptide(s) is present in any receptor that survives in a mutant. The second test, the mecamylamine effect, is a more sophisticated multi-dimensional test of receptor activity, demanding that other wheels and pulleys be functional beyond the [3H] MAL binding peptide(s). Lack of a levamisole response can be attributed to receptor deficiency in mutants of all seven extreme resistance loci as evidenced by lack of a mecamylamine effect even though mutants at four of the seven loci still possess substantial amounts of [3H] MAL binding activity. The results have been summarized as generally as possible because the lev-1 locus represents an important qualification. Striking differences in receptor binding activity exist between extremely and partially resistant lev-1 mutants. Extremely resistant mutants of lev- 1 are difficult to isolate. Only two such mutants have been found. The binding activity in these mutants is pretty wild-type despite their extreme levamisole resistance. Unlike the other extremely resistant, uncoordinated mutants tested, the binding activity in these mutants is activated by mecamylamine. The mutants are abnormal in that both contain about twice as much receptor activity as found in the wild-type and one of the mutants seems to have a higher affinity for [3H] MAL in the absence of mecamylamine. Partially resistant lev- 1 mutants, which by contrast are easy to isolate, contain a much different receptor activity. The receptor present is not activated by mecamylamine and is not elevated above the wild-type amount as found for the rare, extremely resistant lev-1 isolates. Both extremely resistant lev-1 isolates can be 'reverted' to partial resistance and the receptor activity in both mutants 'reverts' to the same type of activity found in partially resistant isolates of lev-1, (i.e., the receptor activity exhibits no mecamylamine effect and is present in just wild-type amounts). The results are consistant with our interpretation that the rare and semi-dominant extremely resistant isolates result from an altered lev-1 gene product and the partially resistant lev-1 mutants lacking mecamylamine effect probably represent the null phenotype of the lev-1 locus. The analysis of the lev-1 locus basically shows that there are additional dimensions to levamisole receptor function than are revealed by whether a mecamylamine effect exists. The mecamylamine effect, for instance, may be a good probe of molecular function but it certainly does not tell you where the receptor is found in the living worm. We find upon testing one mutant for each of the following loci that partially resistant mutants of lev-8, cher mutants of unc-22 and lev-11 produce a pretty normal levamisole receptor as judged by [3H] MAL binding and the mecamylamine effect. The receptor in both twitcher mutants seems to occur in about twice the wild-type amount. The increase in receptor content might be a trivial nonspecific consequence of the twitcher phenotype (i.e., scrawny worms or whatnot) or perhaps represent a 'derepression' of receptor in response to deficiencies of the muscle contraction process in twitchers.