Occurrence of muscarinic acetylcholine receptors in wild type 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 ([3H]QNB) with high affinity. The dissociation constant for [3H]N-methylscopolamine binding determined from equilibrium saturation experiments (KD = 3.7 x 10-l’ M) was in good agreement with that determined from forward and reverse rate constants (KD = k&k,, = 5 x 10-l’ M). 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 C3H]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]Z’V-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 (Ll and L2 stage) juveniles. These stage-specific differences in binding site concentrations parallel differences in acetylcholinesterase activity in larval and adult nematodes (Culotti, J., G. von Ehrenstein, M. R. Culotti, and R. L. Russell (1981) Genetics 97: 281-305). Three methods of elevating cholinergic agonist levels in viva were attempted in order to determine whether regulation of muscarinic receptors occurs in nematodes as it does in vertebrates (Klein, W. L., N. Nathanson, and M. Nirenberg (1979) Biochem. Biophys. Res. Commun. 90: 506-512). Two methods involved prolonged in viva inhibition of acetylcholinesterase activity, while the third method involved in viva 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 (Lewis, J. A., C. H. Wu, H. Berg, and J. H. Levine (1980a) Genetics 95: 905-928) were screened for possible alterations in [3H]QNB or [“H]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. A promising organism for the study of neurotransmitter-related behavioral and developmental phenomena is I This research was supported by National Institutes of Health Grant NS16510 to J. C. , a Muscular Dystrophy Association grant to J. C. , and National Institutes of Health Grant NS15299 to W. K. Special thanks to Mary Carton and Teresa Perney for their excellent technical assistance and to William James and Chris Decrease for their preliminary experimental results on nematode receptors. We are grateful to Drs. Ariens and Van Bever of Janssen Pharmaceutics for their generous gift of the stereoisomers of benzetimide. ‘To whom correspondence should be addressed at Department of the free-living nematode worm Caenorhabditis elegans. This organism is genetically and biochemically tractable and exhibits an anatomical simplicity that has allowed an extensive characterization of its embryonic and postembryonic development (Deppe et al., 1978; von Ehrenstein and Schierenberg, 1980; Sulston and Horvitz, 1977; E. Schierenberg and J. Sulston, personal communication). These studies have provided a nearly complete picture of the cell lineages that give rise to the relatively Neurobiology and Physiology, 4-140 Hogan Hall, Northwestern University, Evanston, IL 60201.