Worm Breeder's Gazette 13(4): 76 (October 1, 1994)
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.
|1||MGH Cancer Center, Dept. of Pediatrics, Harvard University, Charlestown, MA|
|2||The Babraham Institute Laboratory of Molecular Signalling, Dept. of Zoology, Cambridge, UK|
|3||Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX|
Lev-1 is a non-alpha subunit of the nicotinic acetylcholine receptor. Of the several hundred levamisole resistant alleles that were isolated in several screens only lev-1 ( x21 and x61 )were dominant when crossed into wild-type animals. In order to determine the mutations that result in the 2 lev-1 ( x21 , x61 )alleles with the unusual (for a lev-1 mutant) unc phenotype and dominance over wild-type, the 1.4 kb cDNAs were amplified by PCR and directly sequenced. It was found that the genes were both mutated within MII. (see figure) The mutant subunit is able to negate the effect of the cationic ion-flow that should be induced by a receptor containing the wild-type copy of the gene. Two possible explanations for this effect are: (1) if the mutant subunit is preferred over the wild-type in the final receptor assembly (in effect diluting out the wild-type receptor), or (2) if the receptor containing the mutant subunit is able to neutralize the effect of the cationic ion flow of the normal levamisole receptor. The two lev-1 dominant mutations found are located at M2 equivalent positions that have already been studied in the alpha-7 neuronal nAChR by in vitro mutagenesis followed by expression in Xenopus oocytes (Galzi, et al., 1992; Revah, et al., 1991; Bertrand, et al., 1992). Substitutions and/or additions of amino acids at the glutamic acid found mutated in the lev-1 ( x21 )dominant allele were shown to be sufficient to convert the alpha-7 ion-channel selectivity from cationic to anionic. The fewest changes in the alpha-7 nAChR that was able to reverse the ion flow of the channel involved (1) substituting an alanine for the glutamic acid (alpa-7 position 237), (2) inserting an additional proline or adenine in front of the glutamine and (3) replacing the valine with a threonine at alpha-7 position 251. While the substitution found in lev-1 ( x21 )only involves a single amino acid, it replaces the negatively charged glutamic acid (corresponding to alpha-7 glu-237 )with a positively charged lysine rather than a neutral alanine as is the case with alpha-7. This reversal of polarity may well be sufficient to switch the channel flow and not require additional substitutions as is the case for alpha-7 where this amino acid has been more conservatively substituted. The anionic alpha-7 nAChR mutants also displayed high affinity for Ach which is consistent with our finding that the lev-1 dominants had twice the high affinity levamisole binding activity of wild-type (Lewis, et. al, 1987b). The lev-1 ( x61 )dominant contains an insertion of a leucine in M2 that is at the equivalent position of leucine-247 in alpha-7. Replacement of alpha-7 Leu-247 with either a threonine or a serine results in a channel with abolished current rectification, reduced desensitization and increased affinity for ACh (Revah, et al., 1991). The alpha-7 nAChR is unique among vertebrate nAChR subunits in being able to form a functional homo-oligomeric channel when expressed in Xenopus oocytes. The effects of the mutations reported for alpha-7 are thus the result of the same mutation in all 5 subunits of the receptor. It is striking that the phenotype of the lev-1 dominants in C. elegans is caused by a single non-a subunit while the remaining subunits lining the channel are all wild-type. The ability of lev-1 , unc-29 and unc-38 to express functional channels when coinjected into Xenopus oocytes will allow the characterization of the channel properties of these dominant mutations. In addition to in vitro studies on expressed vertebrate alpha-7 receptors, a field-isolated population of Drosophila resistant to the insecticide dieldrin was found to have a single conserved (alanine-302 to serine) amino acid substitution in the M2 of a GABA receptor subunit in between the locations of the 2 lev-1 dominants (ffrench-Constant, et aL, 1993). Expression of this subunit in Xenopus oocytes gave normal channel properties in response to GABA but was insensitive to the channel block induced by picrotoxin or dieldrin (ffrench-Constant, et al., 1993). Our results and the findings in Drosophila demonstrate the importance of a single amino acid substitution in one gene in the development of drug resistance and also point to M2 in ligand-gated ion channels as a key site for resistance to anthelmintic drugs and insecticides. We are also hoping to use the lev-1 dominant cDNA as a positive selectable marker in transgenic experiments.