Pharmacological experiments on Ascaris suum have demonstrated the presence of three (N-, L-, and B-) subtypes of cholinergic receptor mediating contraction of body wall muscle in parasitic nematodes (Robertson et al., 2002). In the present study, these ionotropic acetylcholine (ACh) receptors (nAChRs) were activated by levamisole and bephenium under patch-clamp conditions and competitively antagonized by paraherquamide and 2-desoxoparaherquamide. A number of recordings exhibited three separate current amplitude levels, indicating the presence of small, intermediate, and large conductance subtypes of receptor. The mean conductance of the small conductance subtype, G25, was 22 ± 1 pS; the intermediate conductance channel, G35, was 33 ± 1 pS; and the large conductance channel, G45, was 45 ± 1 pS. The small channel was not antagonized significantly by paraherquamide and was identified as the N-subtype. The intermediate channel was preferentially activated by levamisole rather than bephenium and antagonized by paraherquamide: the intermediate channel was identified as the L-subtype. The large conductance channel was preferentially activated by bephenium, antagonized more by 2-desoxoparaherquamde than by paraherquamide and was identified as the B-subtype. These observations reveal that the three channel subtypes have different selectivity for cholinergic anthelmintics. The different selectivity of these compounds should be considered when dealing with drug resistant infections.

Nicotinic acetylcholine receptors (nAChRs) are important for synaptic neurotransmission. The roles of nAChR subtypes in the transmission are dependent on their channel properties and affinity to acetylcholine. In the nematode C. elegans the levamisole-sensitive nAChR is involved in many worm behaviors such as locomotion, egg laying and mating. Here we report the single-channel properties and kinetics of the levamisole receptor present at the neuromuscular junction (NMJ). In wild type animals nAChRs mostly aggregate at the NMJ. LEV-10 is a transmembrane protein that causes levamisole receptor clustering at the neuromuscular junction. In lev-10 knockouts, functional levamisole receptors are dispersed over the muscle cell body and are accessible for study using the patch-clamp technique. In this study, we present the first study of the single-channel properties of synaptic nAChRs in adult C. elegans . Acetylcholine and levamisole activated the receptor with a single-channel conductance of ∼30 pS and brief mean open-time. At high concentrations levamisole also behaved as an open channel blocker. We also tested for another neuromuscular nAChR subtype that is sensitive to nicotine. However, in lev-10 mutants no nicotine-sensitive receptor was detected from the extrasynaptic region. It demonstrates that the LEV-10 protein is only required for the clustering of levamisole-sensitive nAChRs but not nicotine-sensitive nAChRs. This provides an essential foundation for the understanding of synaptic transmission mediated by cholinergic receptors.

A large family of nAChR subunits has been discovered in C. elegans . The post-synaptic levamisole receptor, one nAChR present at the neuromuscular junction, is composed of essential subunits (UNC-63, UNC-38 and UNC-29) and non-essential subunits (LEV-1 and LEV-8). Behavioral studies indicated that knockout of any essential subunit leads to strong levamisole resistance. Knockout of either non-essential subunit leads to weak levamisole resistance. Previously we used lev-10 mutants to study the single-channel properties of muscle levamisole receptors. LEV-10 is a transmembrane protein that causes levamisole receptor clustering at the neuromuscular junction. In lev-10 knockouts, functional levamisole receptors are dispersed over the muscle cell body and are accessible for study using the patch-clamp technique. In this study, we used lev-1;lev-10 and lev-8;lev-10 double mutants to investigate the single-channel properties of receptors lacking these non-essential subunits. In both double mutants we detected levamisole activated single-channel currents. The single-channel conductance and mean open-time of the double mutants were similar to that of the lev-10 single mutant, but single-channel events occurred at a lower frequency. We found that in the lev-1;lev-10 mutant, less than 10% of the membrane patches were active and that channel opening was rare with NP_open = 0.0001. In the lev-8;lev-10 mutants, single-channel currents were present in ∼40% of the experiments and NP_open was significantly less than that observed from the lev-10 single mutant. These observations predict a reduction in current flow in response to levamisole and are in close agreement with previous studies using the whole-cell voltage-clamp technique.