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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.
Williamson SM
,
Robertson AP
,
Brown L
,
Williams T
,
Woods DJ
,
Martin RJ
,
Sattelle DB
,
Wolstenholme AJ
.
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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 approximately 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-38ratioAsu-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-38ratioAsu-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.
Figure 1. The translated sequence of Asu-UNC-29 and Asu-UNC-38 aligned with the C. elegans and B. malayi equivalents (UNC-29 only).In panel A), the conserved regions used in the design of the degenerate oligonucleotides are highlighted in blue. For both panels, the predicted signal peptide is in lower case, the transmembrane regions are highlighted in yellow, the ligand binding residues [39] in red, the glutamic acid and aspartic acid residues conferring cation specificity to the ion channel [40] are in highlighted in green. The N-terminal subunit-specific peptides used to raise antisera are highlighted in black. In panel B), the amino-acid residues identified by Rayes et al [24] as essential for levamisole activation are highlighted in purple.
Figure 2. Immunolocalisation of nAChR subunits in isolated A. suum muscle cells.A) Confocal microscope images showing indirect immunofluorescent labelling of Ascaris suum muscle with primary antibody against Asu-UNC-38 and FITC conjugated secondary antibody. A whole muscle cell is shown, with Asu-UNC-38 localised to the cell membrane over the entire cell surface. Arrow A indicates the muscle cell arm, B the bag region of the cell, and C the contractile region. B) Asu-UNC-38 localised to the muscle arms where contact with the nerve cord is made to form the neuromuscular junction (indicated by arrows). No labelling of the nerve cord itself was observed. C) Co-localisation of Asu-UNC-29 and Asu-UNC-38 on the muscle cell arm (region A in panel A).
Figure 3. Two-electrode voltage clamp electrophysiological experiments on Xenopus oocytes.A) Oocytes injected with Asu- unc-38 or Asu-unc-29 cRNAs alone produced no functional receptors, whereas oocytes injected with both subunit cRNAs in an equimolar ratio gave robust responses to application of 100 µM acetylcholine (ACh). B) Oocytes injected with an equimolar ratio of Asu-unc-38 and Asu-unc-29 cRNA responded to 100 µM acetylcholine (ACh), 100 µM levamisole (lev) and 100 µM nicotine (nic). These responses could all be reversibly blocked by application of 10 µM mecamylamine. C) Dose-response relationships for the agonists levamisole (lev, grey circles), acetylcholine (ACh, black diamonds) and nicotine (nic, black triangles) from oocytes injected with an equimolar ratio of Asu-unc-38 and Asu-unc-29. Responses are all normalized to the response to 100 µM acetylcholine. N = 3 (where N is batches of oocytes) and n = 6 (where n is the number of individual oocytes) minimum for each data point.
Figure 4. Levamisole and nicotine responses from oocytes injected with cRNAs at 5∶1 and 1∶5 ratios.A) Levamisole and nicotine responses from oocytes injected with a 1∶5 ratio of Asu-unc-38 and Asu-unc-29. The currents evoked by levamisole application are larger than those produced by nicotine application. Agonist application is indicated by a bar above the trace, and the concentrations are indicated in µM. The dose-response relationships for levamisole and nicotine are derived from N = 3 n = 6 minimum for each data point, where n = number of oocytes tested, N = number of frogs from which the oocytes were obtained. Levamisole acts as a full agonist on this receptor subtype, whereas nicotine acts as a partial agonist. All responses were normalized to the response to 100 µM acetylcholine: an example of such a response is shown as the inset. B) Nicotine and levamisole responses from oocytes injected with a 5∶1 Asu-unc-38∶Asu- unc-29 ratio. The currents evoked by nicotine application are larger than those produced by levamisole application. Agonist application is indicated by a bar above the trace, concentrations are given in µM. The dose-response relationships for levamisole and nicotine, where N = 3 n = 6 minimum for each data point. Nicotine acts as a full agonist on this receptor subtype, whereas levamisole acts as a partial agonist. All responses were normalized to the response to 100 µM acetylcholine, an example of which is shown.
Figure 5. Responses to the anthelmintics pyrantel and oxantel.A) Electrophysiological recording from an oocyte injected with a 1∶5 Asu-unc-38∶Asu-unc-29 ratio in response to the application of pyrantel (upper trace) and oxantel (lower trace). Pyrantel clearly acts as an agonist at lower concentrations (<1 µM) and a channel block effect reduces the response seen at higher concentrations; in comparison to this oxantel only produced small currents at any concentration. Agonist application is indicated by a bar above the trace and the concentrations are given in µM. The dose-response relationships for pyrantel and oxantel from oocytes injected with a 1∶5 ratio of Asu-unc-38 and Asu-unc-29 are shown: N = 3 n = 6 minimum for each data point. All responses were normalized to the maximal response (i.e. to the response to 1 µM pyrantel), which was comparable to the response to 100 µM acetylcholine (not shown). B) Electrophysiological recording from oocytes injected with a 5∶1 Asu-unc-38∶Asu-_unc-29 ratio in response to the application of oxantel (upper trace) and pyrantel (lower trace). Oxantel clearly acts as an agonist at lower concentrations (<10 µM) and a channel block effect reduces the response seen at higher concentrations; in comparison to this pyrantel only produced small currents at any concentration. Agonist application is indicated by a bar above the trace. Agonist concentrations and the concentrations are given in µM. The Dose-response relationships for pyrantel and oxantel from oocytes injected with a 5∶1 ratio of Asu-unc-38 and Asu-unc-29 are based on N = 3 n = 6 minimum for each data point. All responses were normalized to the maximal response (i.e. to the response to 10 µM oxantel), which was comparable to the response to 100 µM acetylcholine (not shown).
Figure 6. Possible combinations of the two subunits in the oocyte membrane.The possible combination of Asu-UNC-38 and Asu-UNC-29 that might be present in the injected oocytes are shown. Agonist binding sites, which, by analogy with mammalian receptors should be at an interface between the two subunits, [38] are indicated by black circles. The Figure is based on a similar discussion for mammalian receptors [32].
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