Worm Breeder's Gazette 7(2): 57
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.
Putative acetylcholine receptor mutants of C. elegans can be selected as strains resistant to the cholinergic neurotoxic drug levamisole. At the last C. elegans meeting, one of us (J.A.L.) described a binding assay for detecting the wild type receptor activity and showed that same levamisole-resistant mutants were deficient in receptor binding activity. The assay was based on the retention of an insoluble, particulate [3H]-meta-aminolevamisole binding activity on a glass fiber filter after incubation of the radioactive ligand with total worm extract and filtration. Progress toward more detailed molecular studies of wild type and mutant receptors would require first that the presumably membrane bound receptor be solubilized in detergent solution without loss of binding activity and secondly that a suitable assay be found for detecting the solubilized activity. We have now shown that the levamisole receptor is soluble and active in 1% solutions of the detergents Triton X-100, Lubrol PX, or sodium cholate. In Triton X-100, activity can be detected, in order of increasing efficiency, by the bovine Y-globulin- polyethylene glycol precipitation method of Cuatrecasas, by retention on DEAE filter paper, or by centrifugation through a mini-column (1ml) of G-25 Sephadex. A serendipitous result of detergent solubilization in 1% Triton X-100 is that the affinity of the solubilized receptor for [3H] MAL is increased roughly 100 fold due to a corresponding increase in the rate of association between ligand and receptor. The equilibrium dissociation constant for solubilized receptor is thus ~10+E-10 M as compared to ~10+E-8 M for the 'native' receptor found in untreated extracts. A much lower amount of radioactive ligand is thus required to assay solubilized receptor, resulting in more than a four fold improvement in the signal-to-noise ratio of the assay. Whereas the filter assay of insoluble receptor requires assays in quadruplicate for accuracy, single determinations of solubilized receptor activity by the Sephadex assay will suffice for most purposes. These vastly improved assay capabilities should allow fine distinctions to be made between wild type and mutant receptor properties and make purifying the receptor much easier as well. Previously, we had shown by assay of total, insoluble activity that mutants at 5 of the 7 genetic loci associated with extreme resistance to levamisole had some deficiency in receptor binding activity, ranging from about a 50% decrease (2 loci) to around a 90% decrease (3 loci). To confirm the authenticity of the solubilized binding activity, we showed that the unc-29(x29) levamisole resistant mutant, one of those ~90% deficient in insoluble activity, was also similarly deficient in solubilizable activity as detected by all three assay methods. Mutants at the two resistance loci showing apparently normal levels of insoluble activity were tested for detergent-extractable activity. These mutants, unc-63(x37) and lev-1(x21), had about a tenth and a third of the expected wild type detergent-soluble activity. In the wild type, about 20% of the total activity is not detergent- extractable and remains in the pellet. The unc-63 and lev-1 mutants had wild type amounts of nonextractable activity as did the unc-29 mutant 90% deficient in total and detergent-extractable receptor activities. The nonextractable activity is thus likely to be a secondary (artifactual?) activity unrelated to the detergent-soluble receptor. The levamisole receptors made by the unc-63 and lev-1 mutants are probably more unstable or much less active in detergent than the wild type receptor. With these results, same degree of receptor abnormality is now known for mutants at all 7 of the loci producing extreme levamisole resistance.