The Nicotinic Acetylcholine Receptors of the Parasitic Nematode Ascaris suum: Formation of Two Distinct Drug Targets by Varying the Relative Expression Levels of Two Subunits

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Abstract
Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics. Ascarid nematodes are major pathogens of humans and livestock. The major method of control is by the use of anthelmintic drugs, many of which target the nervous system. Drugs such as levamisole, pyrantel and oxantel target the nicotinic acetylcholine receptors present on muscle. Nematodes have several such receptors, and until now these have been best understood in the model species Caenorhabditis elegans. We have started to characterise the nicotinic receptors of Ascaris suum, and find that the genetics and pharmacology of the A. suum receptors differ from C. elegans. In both species, nicotine and levamisole preferentially activate different forms of the nicotinic receptor, the N- and L-type, respectively. In C. elegans, the L-type receptor is made up of five subunits, whereas the N-type is a homomer of a sixth subunit. We can recapitulate many of the properties of the A. suum N- and L-type receptors, including their sensitivity to two other important anthelmintics, pyrantel and oxantel, by expressing just two subunits at varying ratios. This has implications for the use of drug combinations and for cross-resistance between nicotinic anthelmintics. It may start to give an explanation for the varying effectiveness of nicotinic drugs against different parasites.

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