Occurrence of muscarinic acetylcholine receptors in wild types and cholinergic mutants of Caenorhabditis elegans

Crude homogenates of the nematode worm Caenorhabditis elegans were shown to bind the cholinergic antagonists [³H]N-methylscopolamine and quinuclidinyl benzilate ([H]QNB) with high affinity. The dissociation constant for [³H]N-methylscopolamine binding determined from equilibrium saturation experiments (K(D)=3.7 x 10^-10 M) was in good agreement with that determined from forward and reverse rate constants (k(D) = k(off)/k(on) = 5 x 10^-10M. These binding sites were blocked stereospecifically by the (+) enantiomer of the muscarinic antagonist benzetimide, as would be expected of true muscarinic receptors. Furthermore, competition experiments with unlabeled cholinergic agonists and antagonists indicate that [³H]QNB and [³H]N-methylscopolamine bind to the same sites in nematode homogenates and that these sites are similar but not identical to muscarinic receptors in vertebrates. The concentration of [³H]N-methylscopolamine and [³H]QNB binding sites in adult populations of wild-type nematodes was approximately 10 fmol/mg of protein. This was approximately 4-fold lower than the concentration of binding sites in young (L1 and L2 stage) juveniles. These stage-specific differences in binding site concentrations parallel differences in acetylcholinesterase activity in larval and adult nematodes. Three methods of elevating cholinergic agonist levels in vivo were attempted in order to determine whether regulation of muscarinic receptors occurs in nematodes as it does in vertebrates. Two methods involved prolonged in vivo inhibition of acetylcholinesterase activity, while the third method involved in vivo treatment with the potent agonist levamisole. All three methods failed to reveal down regulation of muscarinic receptors, suggesting that this regulatory mechanism may not exist in nematodes. Finally, phenotypic revertants of acetylcholinesterase-deficient double mutants as well as a class of agonist-resistant mutants were screened for possible alterations in [³H]QNB or [³H]N-methylscopolamine binding levels. None of these mutants exhibited gross deficiencies in [³H]QNB or [³H]N-methylscopolamine binding, although partial deficiencies might have gone undetected by the methods used here. Our binding studies show that muscarinic receptors, which mediate a large proportion of cholinergic signaling in advanced vertebrates, are also present in the very simple nematode system.