Abstract
Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.
Highlights
At the neuromuscular junction, acetylcholine (ACh)1 mediates fast neurotransmission by activating nicotinic receptors (AChRs)
To identify residues involved in this different selectivity, we combined site-directed mutagenesis at residues differentially conserved between muscle ␣ subunits from nematodes and vertebrates, and we evaluated the changes in levamisole activation
Single Channel Currents Activated by Levamisole—Levamisole is a full agonist of the nematode muscle AChR [1]
Summary
Site-directed Mutagenesis and Expression of AChR—HEK293 cells were transfected with mouse ␣ (wild-type or mutant), , ␦, and ⑀ cDNAs using calcium phosphate precipitation at a subunit ratio of 2:1:1:1 for ␣::␦:⑀, respectively, mainly as described previously [13, 14]. Clusters were selected on the basis of their distribution of open probability (Popen), mean open duration, and mean closed duration (16 –18). For the high Popen mode activated by ACh and levamisole, the kinetic analysis was restricted to clusters, each reflecting the activity of a single AChR (16 –18). The resulting open and closed intervals from the selected clusters were analyzed according to kinetic schemes using an interval-based full-likelihood algorithm Probability density functions of open and closed durations were calculated from the fitted rate constants and instrumentation dead time and superimposed on the experimental dwell time histogram as described by Qin et al [19]. The other approach was applied to the low Popen gating mode of the ␣G153E AChR and to wild-type AChRs activated by levamisole, in which less clear or no clusters were observed.
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