Levamisole was identified in 1966 by Paul Janssen and collaborators as a potent broad spectrum anthelminthic. It activates acetylcholine receptors (AChR) at the neuromuscular junction, which causes worm hypercontraction and irreversible paralysis at high concentrations. Genetic screens conducted over 4 decades for mutants either insensitive to the drug (resistance) or able to recover movement after initial paralysis (adaptative-resistance) have been instrumental to identify subunits and regulators of the levamisole-sensitive AChR (L-AChR). Yet, the precise physiological mechanisms of the levamisole response in the wild type (WT) and in adaptative-resistant mutants remain elusive. Using a combination of electrophysiological, Ca2+ imaging and genetic tools we have identified a complex sequence of events that underlie the behavioral features observed at the whole-organism level. First, when worms are dropped onto plates containing 1 mM levamisole, their muscles hypercontract within a few minutes. This initial response is similar in WT and adaptative-resistant mutants, and abolished in mutants lacking L-AChRs. We show that this early contraction is not due to the entry of Ca2+ only through L-AChR, but requires the activation of the voltage-gated Ca2+ channels (VGCC) EGL-19. Second, after 10 minutes on levamisole, WT start to relax, whereas adaptative-resistant mutants such as lev-10 remain hypercontracted. This long-lasting contraction is correlated with the maintenance of a high intracellular Ca2+ concentration in muscle cells. The difference of behavior between WT and lev-10 worms is puzzling: in lev-10 mutants L-AChRs are spread over the muscle surface instead of being clustered at post-synaptic sites, but the total number of L-AChRs is unchanged (Gally 2004). How can levamisole have an effect dependent on L-AChR subcellular localization? We show that the difference between WT and mutants is explained by VGCC properties. In WT, L-AChRs remain opened on levamisole and the muscle depolarization causes a complete inactivation of VGCC. In lev-10 worms, muscle cells are less depolarized on levamisole and a fraction of VGCC is still open, explaining why lev-10 mutants remain hypercontracted when WT worms relax over time. Third, after 8 hours of levamisole exposure, WT worms are still paralyzed whereas lev-10 mutants partially recover locomotion. In these mutants, we show that the membrane resting potential is back to normal, suggesting that L-AChRs are not functional anymore. Our results demonstrate that slightly reducing the magnitude of muscle depolarization during the acute phase of levamisole exposure is the key to explain adaptative-resistance. A new large-scale screen conducted in our laboratory has demonstrated that levamisole-resistance screens are not saturated and could still reveal new molecular players involved in the implementation of L-AChR activation.

Affiliation:
- INMG, Universite Lyon 1, CNRS 5310 INSERM U1217, France