Molecular Basis of the Differential Sensitivity of Nematode and Mammalian Muscle to the Anthelmintic Agent Levamisole

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 μm 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 α subunit that differ between vertebrates and nematodes. The replacement of the conserved αGly-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 αGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs. 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 μm 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 α subunit that differ between vertebrates and nematodes. The replacement of the conserved αGly-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 αGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs. At the neuromuscular junction, acetylcholine (ACh) 1The abbreviations used are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; Popen, channel open probability; HEK cells, human embryonic kidney cells. mediates fast neurotransmission by activating nicotinic receptors (AChRs). AChRs in nematode muscle are targets for anthelmintic chemotherapy. Levamisole and pyrantel are two widely used anthelmintic drugs. By binding to the AChR they lead to a depolarization of the somatic muscle of nematodes. The efficacy of these drugs is based on their ability to act as full agonists of AChRs in nematodes (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). Contractility and membrane potential measurements have shown that the nematode axial muscle is 10–100 times more sensitive to the acute action of pyrantel and levamisole than the rat muscle (2Atchison W.D. Geary T.G. Manning B. VandeWaa E.A. Thompson D.P. Toxicol. Appl. Pharmacol. 1992; 112: 133-143Crossref PubMed Scopus (22) Google Scholar). The molecular bases of this selectivity have not been yet elucidated. The kinetics of activation of nematode AChRs by levamisole has been studied in several preparations from parasite muscle (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar, 3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar), but its action on mammalian muscle AChRs has not been described to date. The effects of levamisole on human neuronal α3β2 and α3β4 AChRs have been studied recently (4Levandoski M.M. Piket B. Chang J. Eur. J. Pharmacol. 2003; 471: 9-20Crossref PubMed Scopus (35) Google Scholar) with the voltage clamp method. It was shown that levamisole behaves as a weak partial agonist, an allosteric modulator, and an open channel blocker of neuronal AChRs (4Levandoski M.M. Piket B. Chang J. Eur. J. Pharmacol. 2003; 471: 9-20Crossref PubMed Scopus (35) Google Scholar). ACh is responsible for neuromuscular transmission in nematodes (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). In Caenorhabditis elegans muscle, levamisole-activated AChRs are composed of the unc-38 subunit, which encodes an α subunit, and lev-1 and unc-29, which encode non-α subunits (5Fleming J.T. Squire M.D. Barnes T.M. Tornoe C. Matsuda K. Ahnn J. Fire A. Sulston J.E. Barnard E.A. Sattelle D.B. Lewis J.A. J. Neurosci. 1997; 17: 5843-5857Crossref PubMed Google Scholar). Expression studies in Xenopus oocytes have shown that both unc-38 and unc-29 subunits are necessary for AChR function (5Fleming J.T. Squire M.D. Barnes T.M. Tornoe C. Matsuda K. Ahnn J. Fire A. Sulston J.E. Barnard E.A. Sattelle D.B. Lewis J.A. J. Neurosci. 1997; 17: 5843-5857Crossref PubMed Google Scholar). Both subunits are required for the expression of levamisole-sensitive receptors in body wall muscles of these nematodes (6Richmond J.E. Jorgensen E.M. Nat. Neurosci. 1999; 2: 791-797Crossref PubMed Scopus (356) Google Scholar). Other nematode α subunits have been cloned from the parasitic nematodes Trichostrongylus colubriformis (7Wiley L.J. Weiss A.S. Sangster N.C. Li Q. Gene (Amst.). 1996; 182: 97-100Crossref PubMed Scopus (22) Google Scholar), Haemonchus conturtus (8Hoekstra R. Visser A. Wiley L.J. Weiss A.S. Sangster N.C. Roos M.H. Mol. Biochem. Parasitol. 1997; 84: 79-187Crossref Scopus (46) Google Scholar), and Ascaris suum (9Le Novère N. Changeux J.P. Nucleic Acid Res. 1999; 27: 340-342Crossref PubMed Scopus (83) Google Scholar), showing a 91.6, 91, and 76% similarity with unc-38, respectively. The main structural features of the AChR subunits are strikingly conserved in phylogeny from higher organisms to the nematode. However, residues differentially conserved between mammalian and nematode AChRs may lead to a differential pharmacological action of anthelmintics at AChRs. In this study, we explore for the first time the interaction of levamisole with mammalian muscle AChRs at the single channel and macroscopic current levels. Our results reveal that levamisole shows an extremely low efficacy for channel activation. At high levamisole concentrations, channel blockade also contributes to maintain a low probability of channel opening. In contrast, levamisole has been shown to act as a potent agonist of different nematode muscle AChRs (3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar, 10Lewis J.A. Wu C.H. Levine J.H. Berg H. Neuroscience. 1980; 5: 967-989Crossref PubMed Scopus (191) Google Scholar, 11Harrow I.D. Gration K.F. Pestic. Sci. 1985; 16: 662-672Crossref Scopus (99) Google Scholar, 12Martin R.J. Pennington A.J. Duittoz A.H. Robertson S. Kusel J.R. Parasitology. 1991; 102: 41-58Crossref PubMed Scopus (37) Google Scholar). Thus, this anthelmintic compound therapeutically exploits differences by selectively activating the AChR of the parasite and not that of the host. To identify residues involved in this different selectivity, we combined site-directed mutagenesis at residues differentially conserved between muscle α subunits from nematodes and vertebrates, and we evaluated the changes in levamisole activation. Our results reveal that the glutamic acid at position 153, which is highly conserved in all nematode α subunits cloned to date, may be involved in the potent activation of nematode AChRs by levamisole. The elucidation of the molecular basis of anthelmintic activation of AChRs will greatly contribute to the development of more selective therapies against parasites and to the understanding of how parasites develop resistance to the anthelmintics. In addition, it pinpoints determinants of function. Site-directed Mutagenesis and Expression of AChR—HEK293 cells with α or and at a subunit of for as described C. N. S. PubMed Scopus Google Scholar, C. I. Mol. Pharmacol. PubMed Scopus Google Scholar). was also for to of cells subunits the site-directed mutagenesis and all used for clamp and in the A. B. PubMed Scopus Google Scholar) at C. N. S. PubMed Scopus Google Scholar). The and and levamisole or both drugs to the channel an clamp at with the to the of a the and by the the at a of and time a and a and to the of by the as a of and by than a this was as the of of the time and the in the time showing openings clusters on the basis of their of open probability open and A. N. K. J. 1997; PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, C. J. PubMed Scopus (46) Google Scholar). of the AChR two which as high and low of mode first to a kinetic for the we used clusters that the high mode activated by ACh and the kinetic was to the activity of a single AChR A. N. K. J. 1997; PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, C. J. PubMed Scopus (46) Google Scholar). The open and from the clusters to kinetic an of The time was of open and from the and time and on the time as described by A. J. 1996; PubMed Scopus Google Scholar). the probability the open and The was to the low mode of the AChR and to AChRs activated by in which or clusters the of of single channel activity by low agonist concentrations, clusters be and block is on the basis of the activation two agonists to the receptor in the with and and with and AChRs by two agonist open with and with at low agonist about the open and the A. N. K. J. 1997; PubMed Scopus Google Scholar). of and be from the of the briefer of the time its and the as recordings, the and and The was in this and position at the of a as described J.P. C. Mol. Pharmacol. PubMed Scopus Google Scholar, C. 2003; PubMed Scopus Google Scholar). The for a of the the of of ACh, or both drugs to the at at and on the was performed the The current was for current by a single and are the peak and the current and is the time that the current to with at the the current of its Channel by is a full agonist of the nematode muscle AChR (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). In the study, we evaluated this anthelmintic also acts on mammalian muscle AChRs. To this we first single from cells muscle AChRs shown in this levamisole is of activating mammalian AChRs. However, channel openings are significantly briefer than those activated by the ACh. time of μm levamisole-activated AChRs be by a main of The of the main open is briefer than that at μm ACh C. N. S. PubMed Scopus Google Scholar, C. I. Mol. Pharmacol. PubMed Scopus Google Scholar). levamisole from to μm not the with full as ACh. At ACh higher than AChRs open in clusters of activation C. J. PubMed Scopus Google Scholar) activation with the of a single receptor from the to the and by to the At μm ACh, the probability of channel a is C. J. PubMed Scopus Google In contrast, when AChRs are activated by at as high as clusters are not results that levamisole mammalian AChRs with and than ACh. levamisole from to μm to a of open time of AChRs activated by μm levamisole be by a single with a open time of a in the open time that in to its of activating mammalian levamisole may act as an open channel blocker by the efficacy of levamisole in activating mammalian we macroscopic from with levamisole. shows from a single to of μm ACh and μm levamisole. In the current the peak and with a time of about to J.P. C. Mol. Pharmacol. PubMed Scopus Google Scholar). The peak current is about 3% when the is to μm levamisole. in single with levamisole. results the low efficacy of the to activate mammalian AChRs. that the single channel that levamisole may also block AChRs we studied its action as a channel blocker in the and of ACh. levamisole to briefer the open time of AChR activated by μm ACh or by levamisole used the to the action of levamisole as an open channel blocker as shown in and In with a between the of the open time and levamisole is The for the of the in by the of the is and for or levamisole-activated channels. Thus, AChRs activated by ACh or levamisole are by levamisole at a similar The apparent from the with the is for levamisole-activated AChRs and for channels. with the of by kinetic C. J. PubMed Scopus Google Scholar, C. J. PubMed Scopus (46) Google of levamisole AChR in the and of μm ACh levamisole at different concentrations. The open times from the open time The are by the open time α is the of levamisole for channel and α is the apparent channel are shown as of AChR in the of μm ACh levamisole. The time and its from the time are shown as of different for –70 between the time and membrane Levamisole are shown as of activated by ACh and with μm levamisole at –70 and current the of To the blockade by we studied the time of AChRs activated by μm ACh in the of levamisole. In its time to μm a main is on the of in the C. J. PubMed Scopus Google Scholar) The of levamisole significantly changes the time of and a of about is The of this not with levamisole but its increases as a function of its It is to that this to the blockade by levamisole of the in the of this a of is for Thus, the apparent for the is μm at a membrane potential of –70 At levamisole higher than the not as a function of The for the and are and and and for and μm respectively. at higher the channel block from The of the increases with higher membrane that the is The voltage of the effect is by At membrane μm levamisole not the of by ACh, and the be by a single similar to the In contrast, at –70 an fast fast is to open channel The time which to is The peak current is not suggesting that at the of that are levamisole with ACh for channel activation. In the of the blockade that levamisole acts as a open channel blocker at residues that are differentially conserved between mammalian and nematode muscle α subunits To identify these residues are involved in the differential of anthelmintic drugs at parasite and mammalian muscle, we by the residues in cells with the α and non-α and we evaluated channel activity by levamisole. The efficacy of levamisole to activate mammalian AChRs is greatly when the αGly-153 is by a glutamic shown in channel activity in clusters at μm levamisole. In contrast, changes are in the activation by levamisole of AChRs the or the of a of AChRs by levamisole. activated by μm levamisole from HEK cells AChRs and the and are at a of with channel openings as –70 time of AChRs the The are of for of AChR be at higher than The of is by changes in the time The main of the time which to is to briefer as a function of levamisole To the of the of a glutamic acid at we activated by a of levamisole to and the activity of single channel openings in In we the kinetics of activation by the full agonist ACh. in it be that clusters of activated by ACh or levamisole are not that this receptor activates in kinetic The of two different the clusters as to two main an and an At all agonist concentrations, the mode of openings that significantly and clusters that briefer than those of the mode on the we the clusters to mode and as a function of agonist and of AChRs activated by ACh or in a low of ACh or levamisole clusters to the high mode be The of the openings appear as The for these clusters is about at of are differences in the between or levamisole-activated clusters and at low agonist concentrations, levamisole activation of the AChR to be from ACh activation. of the mode are also at higher of both However, the channel as as the at higher of levamisole to channel blockade the agonist is clusters of the mode be In to we for the high the low differences between ACh and levamisole and In the of ACh, low clusters are at higher than The of these clusters increases as a function of ACh and a of about at μm ACh In addition, the open channel and the clusters as ACh is that they to activation levamisole is used as an agonist, low clusters be also at higher than The openings these clusters as a function of as for ACh However, to the open channel the open and the as the is The of the low clusters are significantly for levamisole than for AChRs To the of the mode to levamisole we clusters and the of activation to this between clusters or the of a single at μm of the clusters to the and of all clusters at activation activation of AChRs in the mode may significantly contribute to the selective action of anthelmintics on nematode AChRs. channel activity from cells was in the of agonist in the that mode results from AChR activation. from the of AChRs activated by levamisole a in the in and a in the as with AChRs activated by ACh Thus, the that levamisole is a weak agonist of the AChR to a low efficacy for activation. The also that the for the low mode of activated by levamisole is higher than that of AChRs for and AChRs activated by ACh and in a the we clusters for both and levamisole-activated and we kinetic to the open and time of these In this it is that an from the activation of a single that open and time of the of both and levamisole-activated AChRs we the to the activation and open of the AChR. The is for all ACh as as for levamisole than open blockade is not used the at of ACh and levamisole than μm and respectively. on this the kinetic for the the activation for both ACh and levamisole. from the open and time showing that activation is described by the The are similar for and levamisole-activated AChRs It be concluded that shows a mode which is highly sensitive to the agonist and is activated by levamisole and ACh. has been in a channel K. C. A. PubMed Scopus Google Scholar). studied the activation of this AChR by μm levamisole and it with that of At μm single channel clusters of that levamisole is more to activate than AChRs. However, are differences with to levamisole activation of high clusters are when is activated by μm levamisole. clusters with a open channel of a time of and a of are similar to those of clusters of the AChR at the agonist clusters of the at levamisole higher than but the for clusters is μm for AChRs. it that both the position and the of acid are in AChRs highly sensitive to anthelmintics. Levamisole the in C. and produces of A. suum muscle cells, and activates all nematode muscle AChRs (3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar, 10Lewis J.A. Wu C.H. Levine J.H. Berg H. Neuroscience. 1980; 5: 967-989Crossref PubMed Scopus (191) Google Scholar, J. R. Br. J. Pharmacol. PubMed Scopus Google Scholar). of muscle and membrane potential have shown that the by levamisole is similar to that by ACh in the muscle of H. which is 10–100 times more sensitive to the acute action of levamisole than the rat muscle (2Atchison W.D. Geary T.G. Manning B. VandeWaa E.A. Thompson D.P. Toxicol. Appl. Pharmacol. 1992; 112: 133-143Crossref PubMed Scopus (22) Google Scholar). elucidated mammalian muscles sensitivity to the widely used anthelmintic agent levamisole than parasitic muscle, and we residues involved in differential Our studies at the molecular for the first time that levamisole is a weak agonist of mammalian muscle AChRs and reveal the basis of the low probability of mammalian AChR by an extremely low efficacy for channel activation. In addition, the low efficacy of levamisole is as a function of its to channel By site-directed mutagenesis we that which is conserved in all α subunits of muscle be involved in the high efficacy of levamisole at nematode AChRs. The weak activation of mammalian AChRs by levamisole is as the of a of levamisole concentrations, AChR activated by do not at clusters of activation at ACh higher than In contrast, it has been shown that both ACh and levamisole activate in the in nematode muscle (3Robertson S.J. Martin R.J. Br. J. Pharmacol. 1993; 108: 170-178Crossref PubMed Scopus (80) Google Scholar). The current by with levamisole with that activated by ACh. similar has been described for the partial agonist which produces a peak current of about that activated by the of ACh J.P. 1993; PubMed Scopus Google Scholar). Thus, in to is in the kinetics of levamisole-activated mammalian AChRs greatly differ from those of AChRs activated by the ACh. The increases the efficacy of levamisole as an agonist of mammalian AChRs. different be in this AChR on the basis of their probability of channel opening. kinetics have been in AChRs 1993; PubMed Scopus Google Scholar), and the and of kinetic have been shown to in AChRs K. R. PubMed Scopus Google Scholar, K. J. A. A.P. K. J. PubMed Scopus Google Scholar). The that at low to channel the kinetics of activation of the high mode is almost for ACh or the of levamisole in against The has been in by a K. C. A. PubMed Scopus Google Scholar). channel kinetic of AChRs has a of ACh which the AChR to open ACh K. C. A. PubMed Scopus Google Scholar). it has been that the to in the homologous position of the subunit high affinity binding for agonists with an in N. S. Changeux J.P. J. 2003; PubMed Scopus Google Scholar). of this receptor that the the of activation between the and open channel of the AChR N. S. Changeux J.P. J. 2003; PubMed Scopus Google Scholar). 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Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). is similar to the for mammalian AChRs in the activation by levamisole is strikingly different between mammalian and nematode muscle the open channel blockade to be that blockade is not involved in the differential selectivity of anthelmintics on both of Levamisole and pyrantel are to the of action they have different is a levamisole is an By single channel recordings, we have shown C. PubMed Scopus Google Scholar) that pyrantel acts as a low agonist and a high affinity open channel blocker of the mammalian muscle AChRs. Our results that levamisole is potent than pyrantel to activate as as to block the mammalian AChR C. PubMed Scopus Google Scholar). The affinity for levamisole is higher than that of pyrantel μm at –70 to the weak agonist activity of pyrantel at mammalian it is that its effects are by its Our which that the ability of pyrantel is higher than are in with the that pyrantel has more effects than levamisole (1Martin R.J. Valkanov M.A. Dale V.M. Robertson A.P. Murray I. Parasitology. 1996; 113: S137-S156Crossref PubMed Google Scholar). resistance to the anthelmintics pyrantel and levamisole is an in nematode that in the mammalian AChR be to the nematode AChR, and this in the nematode AChR, it be to at are involved in the development of resistance of the parasite against anthelmintics. for and