Molecular Basis of the Differential Sensitivity of Nematode and Mammalian Muscle to the Anthelmintic Agent Levamisole
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m403096200
ISSN1083-351X
AutoresDiego Rayes, Marı́a José De Rosa, Mariana Bartos, Cecilia Bouzat,
Tópico(s)Insect and Pesticide Research
ResumoLevamisole 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 were transfected with mouse α (wild-type or mutant), β, δ, and ϵ cDNAs using calcium phosphate precipitation at a subunit ratio of 2:1:1:1 for α:β:δ:ϵ, respectively, mainly as described previously (13Bouzat C. Bren N. Sine S. Neuron. 1994; 13: 1395-1402Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 14Bouzat C. Roccamo A.M. Garbus I. Barrantes F.J. Mol. Pharmacol. 1998; 54: 146-153Crossref PubMed Scopus (76) Google Scholar). A plasmid encoding green fluorescent protein (pGreen lantern) was also included for recordings to allow identification of transfected cells under fluorescence optics. Mutant subunits were constructed using the QuikChange™ site-directed mutagenesis kit (Stratagene). Restriction mapping and DNA sequencing confirmed all constructs. Cells were used for patch clamp recordings 48 h after transfection. Patch Clamp Recordings and Kinetic Analysis—Recordings were obtained in the cell-attached configuration (15Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15149) Google Scholar) at 20 °C (13Bouzat C. Bren N. Sine S. Neuron. 1994; 13: 1395-1402Abstract Full Text PDF PubMed Scopus (134) Google Scholar). The bath and pipette solutions contained 142 mm KCl, 5.4 mm NaCl, 1.8 mm CaCl2, 1.7 mm MgCl2, and 10 mm HEPES (pH 7.4). Acetylcholine (ACh), levamisole (Sigma), or both drugs were added to the pipette solution. Single channel currents were recorded using an Axopatch 200 B patch clamp amplifier (Axon Instruments, Inc., CA), digitized at 5-μs intervals with the PCI-611E interface (National Instruments, Austin, TX), recorded to the hard disk of a computer using the program Acquire (Bruxton Corporation, Seattle, WA), and detected by the half-amplitude threshold criterion using the program TAC 4.0.10 (Bruxton Corporation, Seattle, WA) at a final bandwidth of 10 kHz. Open and closed time histograms were plotted using a logarithmic abscissa and a square root ordinate and fitted to the sum of exponentials by maximum likelihood using the program TACFit (Bruxton Corp., Seattle, WA). Clusters were identified as a series of closely spaced events preceded and followed by closed intervals longer than a specified duration (tcrit); this duration was taken as the point of intersection of the predominant closed time component and the succeeding one in the closed time histogram. Clusters showing double openings were discarded. For each recording, clusters were selected on the basis of their distribution of open probability (Popen), mean open duration, and mean closed duration (16Wang H.L. Auerbach A. Bren N. Ohno K. Engel A.G. Sine S.M. J. Gen. Physiol. 1997; 109: 757-766Crossref PubMed Scopus (117) Google Scholar, 17Bouzat C. Barrantes F.J. Sine S.M. J. Gen. Physiol. 2000; 115: 663-672Crossref PubMed Scopus (84) Google Scholar, 18Bouzat C. Gumilar F. del Carmen Esandi M. Sine S.M. Biophys. J. 2002; 82: 1920-1929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Popen distributions of the αG153E mutant AChR show two clear components, which were defined as high Popen (HPopen) and low Popen (LPopen) mode. Clusters of each mode were first selected to allow a separate kinetic analysis for each mode. For the analysis, we only used clusters that showed only one gating mode. 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 (16Wang H.L. Auerbach A. Bren N. Ohno K. Engel A.G. Sine S.M. J. Gen. Physiol. 1997; 109: 757-766Crossref PubMed Scopus (117) Google Scholar, 17Bouzat C. Barrantes F.J. Sine S.M. J. Gen. Physiol. 2000; 115: 663-672Crossref PubMed Scopus (84) Google Scholar, 18Bouzat C. Gumilar F. del Carmen Esandi M. Sine S.M. Biophys. J. 2002; 82: 1920-1929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The resulting open and closed intervals from the selected clusters were analyzed according to kinetic schemes using an interval-based full-likelihood algorithm (www.qub.buffalo.edu) (QuB suite, State University of New York, Buffalo). The dead time was typically 30 μs. 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. (19Qin F. Auerbach A. Sachs F. Biophys. J. 1996; 70: 264-280Abstract Full Text PDF PubMed Scopus (372) Google Scholar). Calculated rates were accepted only if the resulting probability density functions correctly fitted the experimental open and closed duration histograms. The other approach was applied to the low Popen gating mode of the αG153E AChR and to wild-type AChRs activated by levamisole, in which less clear or no clusters were observed. We analyzed the behavior of bursts of single channel activity elicited by low agonist concentrations, where no clusters can be seen and block is insignificant, on the basis of the classical activation Scheme 1, where two agonists (A) bind to the receptor (R) in the resting state with association rates k+1 and k+2 and dissociate with rates k–1 and k–2. AChRs occupied by two agonist molecules open with rate β and close with rate α. Bursts at low agonist concentrations contain information about the open state and the immediately adjacent closed state (16Wang H.L. Auerbach A. Bren N. Ohno K. Engel A.G. Sine S.M. J. Gen. Physiol. 1997; 109: 757-766Crossref PubMed Scopus (117) Google Scholar). Therefore, estimates of β, α, and k–2 can be obtained from the mean duration of the briefer component of the closed time histogram (τc), its relative area (Aτc), and the mean burst duration (τb) as follows: τc = 1/(β + k–2); Aτc = β/(β + k–2); τb = (1 + β/k–2) (1/α). Macroscopic Current Recordings—For outside-out patch recordings, the pipette solution contained 140 mm KCl, 5 mm EGTA, 5 mm MgCl2, and 10 mm HEPES (pH 7.3). Extracellular solution contained 150 mm NaCl, 5.6 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH 7.3). The patch was excised in this configuration and moved into position at the outflow of a perfusion system as described before (20Spitzmaul G. Dilger J.P. Bouzat C. Mol. Pharmacol. 2001; 60: 235-243Crossref PubMed Scopus (26) Google Scholar, 21Gumilar F. Arias H.R. Spitzmaul G. Bouzat C. Neuropharmacol. 2003; 45: 964-976Crossref PubMed Scopus (59) Google Scholar). The perfusion system allows for a rapid (0.1–1 ms) exchange of the solution bathing the patch. A series of applications of extracellular solution containing ACh, levamisole, or both drugs were applied to the patch during 150 ms. Macroscopic currents were filtered at 5 kHz, digitized at 20 kHz, and stored on the hard disk. Data analysis was performed using the IgorPro software (WaveMetrics Inc., Lake Oswego, OR). The ensemble mean current was calculated for 5–10 individual current traces. Mean currents were usually fitted by a single exponential function: I(t) = I0 exp(–t/τd) + I∞ where I0 and I∞ are the peak and the steady state current values, respectively, and τd is the decay time constant that measures the current decay due to desensitization. Current records were aligned with each other at the point where the current reached 50% of its maximum level. Single Channel Currents Activated by Levamisole—Levamisole 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 present study, we evaluated if this anthelmintic drug also acts on mammalian muscle AChRs. To this end, we first recorded single channels from cells expressing adult muscle AChRs (Fig. 1). As shown in this figure, levamisole is capable of activating mammalian AChRs. However, channel openings are significantly briefer than those activated by the endogenous neurotransmitter ACh. Open time distributions of 1 μm levamisole-activated AChRs can be well fitted by a main component of 220 ± 20 μs (relative area >0.7) (Fig. 1). The duration of the main open component is 4-fold briefer than that observed at 1 μm ACh (860 ± 80 μs, Fig. 1) (13Bouzat C. Bren N. Sine S. Neuron. 1994; 13: 1395-1402Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 14Bouzat C. Roccamo A.M. Garbus I. Barrantes F.J. Mol. Pharmacol. 1998; 54: 146-153Crossref PubMed Scopus (76) Google Scholar). Increasing levamisole concentration from 1 to 300 μm does not produce the typical clustering observed with full agonists, such as ACh. At ACh concentrations higher than 10 μm, wild-type AChRs open in clusters of well defined activation episodes (17Bouzat C. Barrantes F.J. Sine S.M. J. Gen. Physiol. 2000; 115: 663-672Crossref PubMed Scopus (84) Google Scholar) (Fig. 1). Each activation episode begins with the transition of a single receptor from the desensitized to the activable state and terminates by returning to the desensitized state. At 300 μm ACh, the probability of channel opening within a cluster is ∼1 (17Bouzat C. Barrantes F.J. Sine S.M. J. Gen. Physiol. 2000; 115: 663-672Crossref PubMed Scopus (84) Google Scholar; Fig. 1). In contrast, when AChRs are activated by levamisole, even at concentrations as high as 300 μm, clusters are not observed (Fig. 1). These results suggest that levamisole opens mammalian AChRs with greater latency and closes them faster than ACh. Increasing levamisole concentration from 1 to 300 μm leads to a significant reduction of open durations. Open time histograms of AChRs activated by 300 μm levamisole can be fitted by a single exponential with a mean open time of 80 ± 9 μs (Fig. 1). Such a concentration-dependent decrease in the mean open time indicates that in addition to its capability of activating mammalian AChRs, levamisole may act as an open channel blocker (see below). Macroscopic Currents Activated by Levamisole—To evaluate the efficacy of levamisole in activating mammalian AChRs, we recorded macroscopic currents from outside-out patches rapidly perfused with levamisole. Fig. 2 shows ensemble currents obtained from a single outside-out patch exposed to brief applications of 100 μm ACh (control) and 100 μm levamisole. In control data, the current reaches the peak after 0.1–1 ms and then decays with a time constant (τd) of about 20–30 ms due to desensitization (Fig. 2) (20Spitzmaul G. Dilger J.P. Bouzat C. Mol. Pharmacol. 2001; 60: 235-243Crossref PubMed Scopus (26) Google Scholar). The peak current is only about 3% when the same patch is exposed to 100 μm levamisole. Moreover, in many patches only single channels were observed after perfusion with levamisole. These results confirm the low efficacy of the drug to activate mammalian AChRs. Given that the single channel recording experiments suggest that levamisole may also block AChRs (Fig. 1), we studied its action as a channel blocker in the absence and presence of ACh. Increasing levamisole concentration systematically displaces to briefer durations the open time distributions of AChR channels activated either by 1 μm ACh (Fig. 3) or by levamisole (Fig. 1). We used the classical linear blocking model to describe the action of levamisole as an open channel blocker as shown in Scheme 2, where C indicates closed, O indicates open, and OB indicates blocked states. In agreement with Scheme 2, a linear relationship between the reciprocal of the mean open time and levamisole concentration ([B]) is observed (Fig. 4a). The calculated value for the forward rate constant of the blocking reaction (k+b in Scheme 2), given by the slope of the curve, is 30 × 106 and 25 × 106m–1 s–1 for ACh- or levamisole-activated channels. Thus, AChRs activated by either ACh or levamisole are blocked by levamisole at a similar rate. The apparent closing rate, calculated from the intercept with the y axis, is 5600 s–1 for levamisole-activated AChRs and 1600 s–1 for ACh-activated channels. This value agrees with the closing rate of ACh-activated channels calculated by kinetic analysis (17Bouzat C. Barrantes F.J. Sine S.M. J. Gen. Physiol. 2000; 115: 663-672Crossref PubMed Scopus (84) Google Scholar, 18Bouzat C. Gumilar F. del Carmen Esandi M. Sine S.M. Biophys. J. 2002; 82: 1920-1929Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).Fig. 4Characterization of levamisole blockade. a, AChR channels were recorded in the absence (•) and presence (○) of 1 μm ACh plus levamisole at different final concentrations. The mean open times were obtained from the corresponding open time histograms. The data are fitted by the equation 1/mean open time = α + k+b [levamisole], where k+b is the association rate of levamisole for channel block, and α is the apparent channel closing rate. Data are shown as mean ± S.D. of 3–5 patches. b, AChR currents were recorded in the presence of 1 μm ACh plus levamisole. The mean blocked time (•) and its relative area (○) were obtained from the corresponding closed time histograms. Data are shown as mean ± S.D. of 3–5 different recordings for each condition. Membrane potential, –70 mV. c, relationship between the mean blocked time and membrane potential. Levamisole concentration, 50 μm. Data are shown as mean ± S.D. of 3–5 patches. d, rapid perfusion currents activated by 1 mm ACh (control) alone and together with 100 μm levamisole (+ levamisole). Currents were recorded at –70 mV (downward currents) and +70 mV (upward currents). Each current represents the average of 5–10 records.View Large Image Figure ViewerDownload (PPT) To analyze the blockade by levamisole, we studied the closed time distributions of AChRs activated by 1 μm ACh in the presence of levamisole. In its absence, closed time histograms corresponding to 1 μm ACh-activated channels show a main component whose duration is dependent on the number of channels in the patch (17Bouzat C. Barrantes F.J. Sine S.M. J. Gen. Physiol. 2000; 115: 663-672Crossref PubMed Scopus (84) Google Scholar) (Fig. 3). The presence of levamisole significantly changes the closed time distributions of ACh-activated channels, and a new closed component of about 175 μs is systematically observed (Fig. 3). The duration of this component does not change with levamisole concentration, but its area increases as a function of its concentration (Fig. 4b). It is therefore possible to assume that this closed component corresponds to the blockade by levamisole of the ACh-activated channels (1/k–b in Scheme 2). From the duration of this closed component (1/k–b) a value of 5700 s–1 is obtained for k–b. Thus, the apparent dissociation constant for the blocking process, KB = k–b/k+b, is 190 μm at a membrane potential of –70 mV. At levamisole concentrations higher than 100 μm, the blocked area does not increase as a function of concentration. The values for the closed components and relative areas are 175 ± 12 μs and 0.32 ± 0.03, and 220 ± 15, and 0.33 ± 0.08 μs for 100 and 300 μm levamisole, respectively. Therefore, at higher concentrations the channel block mechanism deviates from Scheme 2. The duration of the blocked periods increases with higher negative membrane potentials, indicating that the unblocking process is voltage-dependent (Fig. 4c). The voltage dependence of the effect is confirmed by outside-out patch recordings (Fig. 4d). At positive membrane potentials, 100 μm levamisole does not affect the decay constant of currents elicited by 1 mm ACh, and the data can be fitted by a single exponential decay similar to the control (22.5 ms). In contrast, at –70 mV, an initial fast decay precedes desensitization. This fast component (about 0.5 ms) is due to open channel blockade. The slow decay time constant, which corresponds to desensitization, is 19.4 ± 1.8 ms. The peak current is not affected, suggesting that at the ratio of concentrations that are used, levamisole cannot compete with ACh for channel activation. In short, the characterization of the blockade indicates that levamisole acts as a typical open channel blocker at concentrations below 100 μm. Sequence comparison reveals key residues that are differentially conserved between mammalian and nematode muscle α subunits (Fig. 5). To identify if these residues are involved in the differential behavior of anthelmintic drugs at parasite and mammalian muscle, we replaced them by the equivalent residues in nematodes, cotransfected cells with the mutant α and wild-type non-α subunits, and we evaluated channel activity elicited by levamisole. The efficacy of levamisole to activate mammalian AChRs is greatly increased when the residue αGly-153 is replaced by a glutamic acid. As shown in Fig. 6, channel activity appears now in easily recognizable clusters at 50 μm levamisole. In contrast, no changes are observed in the activation by levamisole (1–100 μm) of AChRs carrying either the mutations αD152S, αS154N, or the insertion of a proline after αTyr-190 (α191insP).Fig. 6Activation of mutant AChRs by levamisole. Channels activated by 50 μm levamisole were recorded from HEK cells expressing AChRs containing wild-type and the mutant αD152S, αG153E, αS154N, and α191insP subunits. Left, currents are displayed at a bandwidth of 9 kHz with channel openings as upward deflections. Membrane potential, –70 mV. Right, closed time histograms of AChRs carrying the specified mutant subunit. The data are representative of 4–6 recordings for each condition.View Large Image Figure ViewerDownload (PPT) Clusters of αG153E AChR can be identified at concentrations higher than 10 μm. The clustering of opening events is accompanied by important changes in the closed time histograms. The main component of the closed time distributions, which corresponds to closings within clusters, is displaced to briefer durations as a function of levamisole concentration (Fig. 7). To uncover the mechanistic consequences of the presence of a glutamic acid at α153, we recorded channels activated by a range of levamisole concentrations (0.1 nm to 300 μm) and analyzed the activity of single channel openings in clusters. In parallel, we compared the kinetics of activation by the full agonist ACh. When examined in detail, it can be observed that clusters of αG153E activated by either ACh or levamisole are not homogeneous, indicating that this mutant receptor activates in distinct kinetic modes (Fig. 8). The distribution of Popen values showed two different components (see "Experimental Procedures"). We therefore classified the clusters as belonging to two main gating modes: an HPopen and an LPopen mode. At all agonist concentrations, the HPopen mode consisted of openings that were significantly longer and closings within clusters that were briefer than those of the LPopen mode (Table I). Based on the Popen distributions, we selected the clusters corresponding to each mode and analyzed them as a function of agonist concentration (Table I and Fig. 8).Table IChannel properties of αG153E mutant AChRs activated by ACh or levamisoleKinetic modeAChLevamisoleτoτcPopenμmmsHPopen0.00011.560.10.930.011.71 ± 0.240.08 ± 0.0070.98 ± 0.0090.11.78 ± 0.050.07 ± 0.0050.96 ± 0.002100.380.320.52500.260.320.451000.210.260.460.012.40 ± 0.200.05 ± 0.0020.98 ± 0.00113.20 ± 0.760.05 ± 0.0080.98 ± 0.002104.30 ± 0.670.05 ± 0.0030.98 ± 0.002502.70 ± 0.660.05 ± 0.0140.97 ± 0.0091002.80 ± 0.570.04 ± 0.0080.97 ± 0.005LPopen0.010.43 ± 0.07NDND10.31 ± 0.10NDND100.23 ± 0.0717.00 ± 6.500.019 ± 0.008500.19 ± 0.0210.60 ± 1.900.027 ± 0.0021000.16 ± 0.025.08 ± 0.800.037 ± 0.0043000.08 ± 0.0053.50 ± 1.000.027 ± 0.00510.9 ± 0.1NDND101.1 ± 0.31.51 ± 0.150.42 ± 0.13501.1 ± 0.20.32 ± 0.050.83 ± 0.051001.2 ± 0.10.25 ± 0.020.88 ± 0.02 Open table in a new tab High Popen Clusters—At very low concentrations of ACh or levamisole (0.1 nm to1 μm), only clusters corresponding to the high Popen gating mode can be observed (Fig. 8). The rest of the openings appear as isolated events. The Popen calculated for these clusters is about 1 at 1 nm of either agonist. Most interestingly, there are no significant differences in the properties between ACh- or levamisole-activated clusters (Fig. 8 and Table I). Therefore, at low agonist concentrations, levamisole activation of the αG153E AChR seems to be kinetically indistinguishable from ACh activation. Clusters of the HPopen mode are also observed at higher concentrations of both agonists. However, the mean channel duration as well as the Popen decrease at higher concentrations of levamisole due to channel blockade (Table I). Low Popen Clusters—As the agonist concentra
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