Abstract

We describe a glass fiber filter binding assay for the levamisole receptor, a putative acetylcholine receptor of the nematode Caenorhabditis elegans, and we show that receptor detected in vitro binds both levamisole derivatives and cholinergic agonists with the pharmacological specificity expected of the physiologically functional nematode receptor. The receptor is detected by the binding of tritiated meta-aminolevamisole ([3H]MAL, 27 Ci/mmol). In extracts of the wild-type nematode, there is a saturable, high affinity binding activity for [3H]MAL (Kd approximately 5-10 nM). Well fed wild-type worms contain as much as 3 fmol of high affinity binding activity per mg of extract protein (0.14 pmol/g of wet weight of worms) and dauer larvae, a special juvenile stage, contain as much as 15 fmol of activity per mg of protein. Specific binding activity per mg of protein is highest in larval stages and decreases severalfold in the adult worm. The rates of formation and dissociation of the [3H]MAL-receptor complex are relatively slow (dissociation half-life, 17 min), in agreement with physiological studies of levamisole on Ascaris muscle strips. Levamisole derivatives and cholinergic agonists have the same relative potencies in inhibiting [3H]MAL binding as they do in causing nematode muscle contraction. Vertebrate cholinergic antagonists do not inhibit [3H]MAL binding, but several antagonists (mecamylamine, alpha-bungarotoxin, and cobra venom) potentiate the binding of [3H]MAL and can be used to demonstrate more clearly the presence of a second, lower affinity binding activity whose ligand-binding affinity is also potentiated by these agents. Both high and low affinity wild-type binding components are missing in the extremely levamisole-resistant mutant unc-74(x19).