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PLoS Pathog
2014 Jan 30;101:e1003870. doi: 10.1371/journal.ppat.1003870.
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Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.
Buxton SK
,
Charvet CL
,
Neveu C
,
Cabaret J
,
Cortet J
,
Peineau N
,
Abongwa M
,
Courtot E
,
Robertson AP
,
Martin RJ
.
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Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.
Figure 2. Voltage-clamp of oocytes injected with O. dentatum Ode-unc-29 and Ode-unc-63 nAChR subunits.(A) Diagram of possible subunit arrangements of Ode-unc-29 and Ode-unc-63. X represents either UNC-63 or UNC-29 subunit. PyR, pyrantel; Tbd, tribendimidine, ACh, acetylcholine; Nic, nicotine; Bep, bephenium; The, thenium. (B) Representative traces showing the inward currents in oocytes injected with 1∶1 Ode-unc-29 and Ode-unc-63. (C) Bar chart (mean ± se) of agonists-elicited currents in the Ode-(29 - 63) Pyr-nAChR, (paired t-test, **p<0.01, ***p<0.001). All agonist responses have been normalized to the average 100 µM ACh currents. (D) Dose-response relationships for Pyr (inverted Δ, n = 6), Tbd (▴, n = 5) and ACh (•, n = 6) in the Ode-(29 - 63) Pyr-nAChR, (n = number of oocytes). (E) Bar chart (mean ± se) of normalized currents elicited by different agonists in 1∶5 Ode-unc-29∶Ode-unc-63 injected oocytes. Currents have been normalized to and compared with 100 µM ACh currents (paired t-test, **p<0.01, ***p<0.001). (F) Bar chart (mean ± se) of normalized currents elicited by different agonists in oocytes injected with 5∶1 Ode-unc-29∶Ode-unc-63. Currents normalized to and compared with 100 µM ACh currents (paired t-test, *p<0.05, **p<0.01).
Figure 4. Effects of derquantel on levamisole-activated and pyrantel-activated expressed Ode(29-63-8-38), the Lev-nAChR, and on pyrantel-activated and levamisole-activated Ode(29-63-38), the PyR/Trbd-nAChR subtypes.Der: derquantel; Lev: levamisole; Pyr: pyrantel. (A) Antagonistic effects of varying derquantel concentrations on levamisole currents of Ode(29-63-8-38). Levamisole evokes supramaximal normalized currents and derquantel competitively inhibited levamisole currents. (B) Derquantel antagonism of pyrantel currents of Ode(29-63-8-38). Here, derquantel produced mixed non-competitively competitive antagonism. Pyrantel did not activate supramaximal currents. (C) Antagonism of pyrantel by derquantel on the Ode(29-63-38), Pyr/Tbd-nAChR. Derquantel is a potent non-competitive antagonist. (D) Derquantel non-competitively antagonized levamisole responses on the Ode(29-63-38) receptor.
Figure 5. Ca2+ permeability of the O. dentatum receptor subtypes with large ACh currents.(A) Current-Voltage plot for oocytes injected with Ode(29–63–8), showing the change in current with voltage in 1 mM and 10 mM Ca2+ recording solutions. Insert: Magnified view of current-voltage plot from −20 mV to +40 mV showing the Erev in 1 mM and 10 mM extracellular Ca2+. (B) Current-Voltage plot for oocytes injected with Ode(29–63–38) showing the current changes in 1 mM and 10 mM Ca2+ recording solution under different voltages. Insert: Magnified view of current-voltage plot from −20 mV to +40 mV showing the Erev in 1 mM and 10 mM extracellular Ca2+. (C) Current-Voltage plot for oocytes injected with Ode(29–63–38–8) in 1 mM and 10 mM Ca2+ recording solutions. Insert: Magnified view of current-voltage plot from −20 mV to +40 mV showing the Erev in 1 mM and 10 mM extracellular Ca2+. The calcium permeability was calculated using the GHK equation (Text S1 Legend).
Figure 6. Single-channel properties of the Lev-nAChR subtype.(A) Representative current-voltage plot from oocyte-attached patch with 10 µM levamisole. Inserts are representative channel openings at +75 mV and −75 mV membrane potentials. Even with 10 µM levamisole, we sometimes observed ‘flickering’ channel block events; note the ‘flickering’ channel block shown by openings at −75 mV. (B) Bar chart (mean ± se) of the mean open times (τ) at the different patch potentials. (C) Bar chart (mean ± se) of the probability of channel opening, Po, at the different patch potentials.
Figure 1. Maximum likelihood tree showing relationships of acetylcholine receptor (nAChR) subunit cDNA sequences in Oesophagostomum dentatum (Ode, highlighted in red), Caenorhabditis elegans (Cel), Haemonchus contortus (Hco) and Teladorsagia circumcincta (Tci).The C. elegans acr-16 nAChR subunit sequence was used as an outgroup. Branch support was evaluated using the chi2 option of PhyML and values more than 0.9 were considered reliable.
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