Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor.
ABSTRACT Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.
Article: Efficacy of current drugs against soil-transmitted helminth infections: systematic review and meta-analysis.[show abstract] [hide abstract]
ABSTRACT: More than a quarter of the human population is likely infected with soil-transmitted helminths (Ascaris lumbricoides, hookworm, and Trichuris trichiura) in highly endemic areas. Preventive chemotherapy is the mainstay of control, but only 4 drugs are available: albendazole, mebendazole, levamisole, and pyrantel pamoate. To assess the efficacy of single-dose oral albendazole, mebendazole, levamisole, and pyrantel pamoate against A lumbricoides, hookworm, and T trichiura infections. A systematic search of PubMed, ISI Web of Science, ScienceDirect, the World Health Organization library database, and the Cochrane Central Register of Controlled Trials (1960 to August 2007). From 168 studies, 20 randomized controlled trials were included. Information on study year and country, sample size, age of study population, mean infection intensity before treatment, diagnostic method used, time between evaluations before and after treatment, cure rate (the percentage of individuals who became helminth egg negative following treatment with an anthelminthic drug), egg reduction rate, adverse events, and trial quality was extracted. Relative risk, including a 95% confidence interval (CI), was used to measure the effect of the drugs on the risk of infection prevalence with a random-effects model. Single-dose oral albendazole, mebendazole, and pyrantel pamoate for infection with A lumbricoides resulted in cure rates of 88% (95% CI, 79%-93%; 557 patients), 95% (95% CI, 91%-97%; 309 patients), and 88% (95% CI, 79%-93%; 131 patients), respectively. Cure rates for infection with T trichiura following treatment with single-dose oral albendazole and mebendazole were 28% (95% CI, 13%-39%; 735 patients) and 36% (95% CI, 16%-51%; 685 patients), respectively. The efficacy of single-dose oral albendazole, mebendazole, and pyrantel pamoate against hookworm infections was 72% (95% CI, 59%-81%; 742 patients), 15% (95% CI, 1%-27%; 853 patients), and 31% (95% CI, 19%-42%; 152 patients), respectively. No pooled relative risks could be calculated for pyrantel pamoate against T trichiura and levamisole for any of the parasites investigated. Single-dose oral albendazole, mebendazole, and pyrantel pamoate show high cure rates against A lumbricoides. For hookworm infection, albendazole was more efficacious than mebendazole and pyrantel pamoate. Treatment of T trichiura with single oral doses of current anthelminthics is unsatisfactory. New anthelminthics are urgently needed.JAMA The Journal of the American Medical Association 05/2008; 299(16):1937-48. · 30.03 Impact Factor
Article: Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole.[show abstract] [hide abstract]
ABSTRACT: 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 microm 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 alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-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 alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.Journal of Biological Chemistry 09/2004; 279(35):36372-81. · 4.77 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: We have characterized a small group of genes (13 loci) in the nematode Caenorhabditis elegans that, when mutated, confer resistance to the potent anthelmintic levamisole. Mutants at the 7 loci conferring the most extreme resistance generally possess almost identical visible and pharmacological phenotypes: uncoordinated motor behavior, most severe in early larval life, extreme resistance to cholinergic agonists and sensitivity to hypo-osmotic shock. Mutants with exceptional phenotypes suggest possible functions for several of the resistance loci. The most extreme mutants can readily be selected by their drug resistance (211 mutants, as many as 74 alleles of one gene). The more common resistance loci are likely to be unessential genes, while loci identified by only a few alleles may be essential genes or genes conferring resistance only when mutated in a special way. We propose that these mutants represent a favorable system for understanding how a small group of related genes function in a simple animal. The extreme drug resistance of these mutants makes them useful tools for the genetic manipulation of C. elegans. And, as the most resistant class of mutants might lack pharmacologically functional acetyl-choline receptors (LEWIS et al. 1980), these mutants may also be of some neurobiological significance.Genetics 09/1980; 95(4):905-28. · 4.01 Impact Factor
Eight genes are required for functional reconstitution
of the Caenorhabditis elegans levamisole-sensitive
Thomas Boulina,b,1, Marc Gielena,c,1, Janet E. Richmondd, Daniel C. Williamse, Pierre Paolettia,c,2, and Jean-Louis Bessereaua,b,3
aEcole Normale Supe ´rieure, Biology Department, 75005 Paris, France;bINSERM U789, Biologie Cellulaire de la Synapse, Paris 75005, France;cCNRS UMR 8544,
Laboratoire de Neurobiologie, Paris 75005, France;dDepartment of Biology, University of Illinois, Chicago, IL 60607; andeDepartment of Biology, University
of Utah, Salt Lake City, UT 84112
Edited by Cornelia I. Bargmann, The Rockefeller University, New York, NY, and approved October 8, 2008 (received for review July 30, 2008)
Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-
gated ion channels that mediate excitatory neurotransmission at the
neuromuscular junctions of nematodes. They constitute a major drug
nematode-specific cholinergic agonists such as levamisole. Genetic
sole toxicity identified genes that are indispensable for the biosyn-
thesis of L-AChRs. These include 5 genes encoding distinct AChR
subunits and 3 genes coding for ancillary proteins involved in assem-
bly and trafficking of the receptors. Despite extensive analysis of
L-AChRs in vivo, pharmacological and biophysical characterization of
these receptors has been greatly hampered by the absence of a
heterologous expression system. Using Xenopus laevis oocytes, we
were able to reconstitute functional L-AChRs by coexpressing the 5
distinct receptor subunits and the 3 ancillary proteins. Strikingly, this
system recapitulates the genetic requirements for receptor expres-
sion in vivo because omission of any of these 8 genes dramatically
impairs L-AChR expression. We demonstrate that 3 ?- and 2 non-?-
subunits assemble into the same receptor. Pharmacological analysis
reveals that the prototypical cholinergic agonist nicotine is unable to
activate L-AChRs but rather acts as a potent allosteric inhibitor. These
results emphasize the role of ancillary proteins for efficient expres-
in vitro screening of novel anthelminthic agents.
anthelminthic drug ? recombinant receptor expression
are infected by various intestinal nematode parasites (1). Among
the different anthelmintic drugs, cholinergic agonists such as le-
vamisole are widely used against intestinal nematodes (2). They
activate ligand-gated acetylcholine receptors present on muscle cell
membranes and cause spastic paralysis of the parasites. Levamisole
can be used in mammals because it does not activate the AChRs of
the infected host (3), yet the molecular basis of this specificity
Molecular identification of the levamisole target was achieved in
the nonparasitic nematode Caenorhabditis elegans (4). Acetylcho-
line is the main excitatory neurotransmitter in C. elegans, and ?29
genes encoding AChR subunits are predicted from its genome
sequence (5). Despite this complexity, genetic screens were able to
identify the genes coding for the subunits of levamisole-sensitive
AChRs (L-AChRs). Levamisole causes body-wall muscle hyper-
contraction, paralysis, and ultimately death of C. elegans at high
concentrations. By screening for mutant animals that survive
exposure to levamisole, mutations in 5 genes encoding AChR
subunits were found to confer partial or complete insensitivity to
levamisole (4). These include 2 non-?-subunits (LEV-1 and UNC-
29) and 3 ?-subunits (LEV-8, UNC-38, UNC-63) as defined by the
presence of a vicinal dicysteine in the primary sequence (6–8).
Consistently, electrophysiological analysis demonstrated a drastic
oil-transmitted helminth infections are a public health problem
of muscle AChR activated by nicotine (N-AChR) but insensitive to
levamisole (9). This receptor contains the subunit ACR-16, which
is closely related to the vertebrate ?7 gene. ACR-16 forms func-
L-AChRs and N-AChRs are partially redundant because disrup-
tion of either receptor causes no or weak locomotory defects,
whereas disruption of both receptors causes almost complete
paralysis of the animal (11, 12).
In addition to AChR subunits, genetic screens identified 3
ancillary proteins, RIC-3, UNC-50, and UNC-74, that are abso-
lutely required for the expression of L-AChRs in vivo. RIC-3 is an
endoplasmic reticulum transmembrane protein required for the
expression of at least 4 distinct AChRs in C. elegans, including
as a chaperone promoting AChR folding, assembly, or maturation
(reviewed in ref. 15). unc-74 was identified in early screens for
resistance to levamisole (4). It is predicted to encode a thioredoxin
closely related to the human TMX3 protein and seems to be solely
required for the expression of L-AChRs (D.C.W. and E. M.
Jorgensen, unpublished data; and ref. 16). unc-50 encodes a trans-
membrane protein that localizes mostly to the Golgi apparatus and
interacts with an ARF-GEF (guanine nucleotide exchange factor
for ADP-ribosylation factor GTPases) (17). In the absence of
UNC-50, L-AChRs but not N-AChRs are targeted to lysosomes
after they exit the endoplasmic reticulum and are degraded. unc-50
is evolutionarily conserved in most eukaryotes, including yeast,
plants, and mammals. However, its role for AChR expression has
not been tested so far in nonnematode species.
remains poorly characterized at the pharmacological level, and the
molecular basis for the action of levamisole is still unknown. Such
analysis was complicated by the inability to express recombinant
L-AChRs in a controlled heterologous system. Here, we demon-
strate that L-AChRs can be expressed in Xenopus oocytes by
providing not only the 5 receptor subunits but also the 3 ancillary
factors RIC-3, UNC-50, and UNC-74. This expression system was
Author contributions: T.B., M.G., P.P., and J.-L.B. designed research; T.B., M.G., J.E.R., and
P.P. performed research; T.B., M.G., and D.C.W. contributed new reagents/analytic tools;
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1T.B. and M.G. contributed equally to this work.
Neurobiologie, CNRS UMR 8544, 46, rue d’Ulm, 75005 Paris, France. E-mail: paoletti@
3To whom correspondence may be addressed at: Ecole Normale Supe ´rieure, De ´partement
de Biologie, INSERM U789, 46, rue d’Ulm, 75005 Paris, France. E-mail: jlbesse@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
November 25, 2008 ?
vol. 105 ?
no. 47 www.pnas.org?cgi?doi?10.1073?pnas.0806933105
used to characterize the biophysical and pharmacological proper-
ties of the L-AChR.
Eight Genes Are Required to Reconstitute L-AChRs in Xenopus Oocytes.
Because 8 genes are required in vivo for L-AChR expression, we
reasoned that the same set of genes may be necessary for
functional expression in a heterologous system. Xenopus oocytes
are particularly well suited to express multimeric receptors
oocyte cytoplasm. We injected in vitro-transcribed cRNAs cor-
responding to the 5 L-AChR subunit genes lev-1, lev-8, unc-29,
unc-38, and unc-63 and the 3 ancillary factors ric-3, unc-50, and
unc-74. Robust expression of L-AChR was obtained 1 or 2 days
after the injection. Perfusion of 100 ?M acetylcholine elicited
large inward currents in the 100 nA to several ?A range, with fast
activation and deactivation kinetics (limited by the speed of the
solution exchange), as expected for a receptor containing a
ligand-gated ion channel (Fig. 1A). The initial fast-desensitizing
component of the response was likely caused by the activation of
calcium-activated chloride channels endogenously expressed in
Xenopus oocytes (see below and Fig. 2A). Levamisole elicited
responses similar to ACh although of lower amplitude, whereas
nicotine was surprisingly ineffective, a feature that we later
subunits (black squares) and 3 ancillary factors (gray squares) is mandatory for robust expression of the L-AChR. Currents were measured at the peak. Average peak
current for coinjection of all 8 cRNAs was 4.1 ? 3.7 ?A (n ? 49). (C) A single oocyte coinjected with the acr-16 and ric-3 cRNAs displays large transient inward currents
elicited by ACh (500 ?M) or nicotine (500 ?M), but not by levamisole (500 ?M). (D) Functional expression of the homopentameric ACR-16 nicotine-sensitive AChR
6 ? 4.2 ?A (n ? 33). All recordings were made with 1 mM external CaCl2. Numbers above bars represent the number of oocytes recorded for each condition.
Expression of functional AChRs from C. elegans in X. laevis oocytes. (A) A single oocyte coinjected with the 8 cRNAs unc-29, unc-38, unc-63, lev-1, lev-8, ric-3,
amount of barium (0.3 mM) eliminates this peak and results in stable responses upon continuous application of ACh. (Inset) N-AChRs display profound macroscopic
desensitization upon continuous application of ACh (500 ?M) even after BAPTA injection (n ? 6). (B) Comparison of the I/V relationships of L-AChR responses elicited
by 100 ?M ACh in the presence of 1 mM or 10 mM extracellular CaCl2. Note that the L-AChR is potentiated by 10 mM extracellular calcium and that this potentiation
occurs over the whole voltage range (n ? 5, BAPTA-loaded oocytes). (C) Magnified view of the dash-boxed region in B showing the rightward shift of the reversal
potential induced by switching from 1 mM to 10 mM external Ca2?(1.6 mV to 3.4 mV for this cell).
The L-AChR is permeable to calcium and shows no macroscopic desensitization. (A) All traces are from a single oocyte expressing L-AChRs. In 1 mM external
Boulin et al.
November 25, 2008 ?
vol. 105 ?
no. 47 ?
characterized. Altogether, these data indicate that recombinant
L-AChRs can be efficiently expressed in Xenopus oocytes.
To test the relative contribution of each gene for the functional
expression of L-AChRs, we removed cRNAs from the injection
mixture, either singularly or in combination. Similar to the in vivo
situation, functional receptors were virtually eliminated when 1 or
more of the 5 receptor subunits were omitted, causing ?97%
of the 5 receptor subunits never yielded any measurable current
when the 3 ancillary proteins RIC-3, UNC-50, and UNC-74 were
absent. The requirement for individual ancillary proteins was then
tested by injecting the 5 L-AChR subunits and only 2 of the 3
amplitudes to ?10% of the response obtained with the full set of
cRNAs, whereas removal of RIC-3 gave slightly larger and more
variable responses (Fig. 1B). Hence, these data demonstrate the
functional L-AChRs in Xenopus oocytes.
Because the expression requirement of L-AChRs in Xenopus
oocytes resembles the in vivo situation, we tested whether this
would also be the case for the expression of N-AChRs. Injection of
ACR-16 cDNA alone was reported to produce functional AChRs
in Xenopus oocytes, yet yielding only small currents (usually ?200
nA at ?100 mV) (10, 18). Because ric-3 was demonstrated to be
essential in vivo for N-AChR expression (13), we coinjected acr-16
and ric-3 cRNAs. After only 1 day of expression, we recorded large
acetylcholine-induced currents (?1 ?A at ?60 mV) (Fig. 1C).
These recombinant receptors had the expected properties of N-
AChRs because they were activated by nicotine and insensitive to
levamisole. Strikingly, we could never measure any significant
current when the ric-3 cRNA was omitted (Fig. 1D). Because
UNC-50 and UNC-74 enhance the expression of L-AChRs in
Xenopus oocytes, they might also improve N-AChR expression.
However, coinjection of unc-50 and unc-74 cRNAs together with
ric-3 did not further enhance the N-AChR expression. This result
is consistent with the in vivo situation because mutations in unc-50
and unc-74 do not affect N-AChR expression in C. elegans (17)
(J.E.R. and E. M. Jorgensen, unpublished data). Altogether, these
be achieved in Xenopus oocytes by providing C. elegans ancillary
proteins in addition to the subunits of the AChR. Strikingly, the
expression requirement in the Xenopus oocyte recapitulates the
genetics of AChR expression in C. elegans.
L-AChRs Are Permeable to Calcium and Do Not Show Macroscopic
Desensitization. In vertebrates, AChRs form nonselective cation
channels with differing permeability to calcium ions. Whereas
muscle nAChRs are only weakly permeable to calcium, neuronal
AChRs display significantly higher calcium permeabilities that vary
depending on subunit composition (19–22). In C. elegans, calcium
permeability of L-AChRs remains unexplored.
To test whether calcium can permeate recombinant L-AChRs,
we replaced external Ca2?(1 mM) by Ba2?(0.3 mM) and observed
a strong reduction in the current amplitude together with a com-
Because Xenopus oocytes are known to express chloride channels
in 1 mM Ca2?a large fraction of the peak current evoked by ACh
is carried by endogenous chloride channels that are activated
secondary to the entry of Ca2?flowing through L-AChR channels.
This hypothesis was confirmed by demonstrating that intracellular
injection of the calcium chelator BAPTA totally eliminates the
peak response in 1 mM external Ca2?(Fig. 2A). Next, the relative
Ca2?permeability of L-AChRs was quantified by measuring
changes in the reversal potential of ACh-induced currents when
increasing the external Ca2?concentration from 1 mM to 10 mM.
In these conditions, reversal potentials shifted from 1.7 ? 0.3 mV
(n ? 4) to 3.7 ? 0.5 mV (n ? 4) (Fig. 2 B and C). According to the
a value intermediate between that of embryonic vertebrate muscle
receptors (0.1–0.3) and neuronal AChRs [?1–2 (22, 24)].
Inspection of the current traces also revealed that L-AChR
responses are enhanced by external calcium in a voltage-
independent manner (Fig. 2B). This modulatory effect is reminis-
neuronal AChRs (20, 21). An additional property of the L-AChR
is its absence of apparent macroscopic desensitization. Indeed,
steady responses are recorded upon ACh application in conditions
where Ca2?-activated chloride currents are silenced by BAPTA
injection (Fig. 2A). This is in striking contrast with the profound
desensitization observed with N-AChRs even after BAPTA injec-
tion (Fig. 2A Inset).
Pharmacology of the L-AChR. The pharmacological profile of re-
combinant L-AChRs was characterized by using cholinergic ago-
nists and antagonists. Levamisole (100 ?M) induced inward cur-
rents in all cells that were tested for their responsiveness to ACh
(Fig. 3A). However, responses to levamisole were consistently
smaller than the responses obtained in the same oocytes with 100
in activating L-AChRs. Pyrantel, another anthelminthic known to
act as a cholinergic agonist, was also able to activate L-AChRs.
However, the currents obtained with 100 ?M pyrantel were much
smaller than those induced by the same concentrations of levami-
sole or ACh (Fig. 3A). Surprisingly, nicotine, the prototypical
agonist of ionotropic AChRs, had almost no agonistic effect on the
L-AChR when applied at 100 or 500 ?M (Figs. 1A and 3A).
Nicotine-elicited currents were only detectable in oocytes express-
ing very high levels of L-AChRs. When ACh-evoked peak currents
reached 7–15 ?A, nicotine-induced responses were on average
?1% of the ACh-induced responses (0.55 ? 0.21%, n ? 4).
and levamisole, full dose-response experiments were performed
(Fig. 3B). Acetylcholine EC50was found to be 26.0 ? 3.2 ?M (n ?
13) and the Hill coefficient 1.05 ? 0.06. This EC50value was very
similar to that of N-AChRs [31 ? 0.5 ?M (n ? 6); supporting
information (SI) Fig. S1], yet the Hill coefficient was estimated to
be significantly higher for N-AChRs (2.4; Fig. S1). The levamisole
EC50was 10.1 ? 1.8 ?M (n ? 6), indicating that levamisole was
efficacy was markedly lower than that of ACh. At a saturating
levamisole concentration of 100 ?M, levamisole-induced current
(500 ?M), an additional antagonistic effect of levamisole could be
unmasked (Fig. 3B). This inhibition likely resulted from open-
26). To test this hypothesis, we performed voltage ramps and
demonstrated that 500 ?M levamisole produced a slight voltage-
dependent block of L-AChR responses (Fig. 3C). However, no
channel block was seen at 100 ?M levamisole, indicating that
channel block alone cannot account for the partial efficacy of
and levamisole responses was their washout kinetics (Fig. 3D).
independent washout kinetics, the washout time course of levami-
sole-evoked responses became increasingly slower with increasing
levamisole concentrations. Noticeably, at 500 ?M levamisole, cur-
rent washout was extremely slow, requiring minutes for complete
recovery. These long-lasting effects of levamisole are suggestive of
complex mode(s) of interaction of the levamisole molecule on
L-AChRs (see Discussion).
To determine the antagonist spectrum of L-AChR, we tested 5
compounds that inhibit ACh responses of different classes of
AChRs. D-Tubocurarine (dTC), methyllycaconitine (MLA), and
www.pnas.org?cgi?doi?10.1073?pnas.0806933105 Boulin et al.
hexamethonium (Hex) each inhibited ACh-triggered responses
(Fig. 4). In contrast, ?-bungarotoxin (?-BgTx) and dihydro-?-
erythroidine (DH?E) had very little antagonistic effects, similar to
(100 nM, 30-min incubation), which may be required to reach
binding equilibrium, were ineffective at inhibiting L-AChR re-
sponses [7.6 ? 1% (n ? 7)].
These results indicate that the L-AChR has a unique pharma-
cological profile and that levamisole has very distinct agonist
properties compared with the natural agonist acetylcholine.
Nicotine Is an Allosteric Inhibitor of L-AChRs. In the experiments
presented above, we observed that nicotine was unable to
activate recombinant L-AChRs (Figs. 1A and 3A). To test
whether this was an intrinsic characteristic of L-AChRs and not
a peculiar feature of our recombinant receptors, we analyzed the
nicotine response of L-AChRs on C. elegans muscle cells. We
used a double-mutant strain containing an acr-16-null mutation,
which removes nicotine-sensitive N-AChRs (12), and a mutation
in the unc-13 gene, which blocks presynaptic vesicle release (27).
We expect that in such a double-mutant strain, any current
recorded upon nicotine application can only be attributed to
postsynaptic L-AChR activation and not to activation of pre-
synaptic AChRs. When muscle cells of double-mutant animals
were recorded, no measurable response was detected after
nicotine application (Fig. 5A). In contrast, the response to
levamisole application was indistinguishable from the levamisole
response in control animals (Fig. 5B; see also refs. 11 and 26).
These results demonstrate that native and recombinant L-
AChRs share the inability to be activated by nicotine.
We next tested the possibility that nicotine might act as an
L-AChR antagonist. Strikingly, 500 ?M nicotine had a potent
inhibitory effect on currents evoked by ACh applications (Fig. 5C).
This inhibition appears to be almost entirely voltage-independent
(Fig. 5D), suggesting that it is not the result of direct channel block.
Alternatively, nicotine might act as a competitive antagonist of
ACh. However, performing acetylcholine dose–response experi-
little effect on the apparent affinity for ACh (?2-fold increase in
ACh EC50by 300 ?M nicotine; Fig. 5E). Taken together, these
results indicate that nicotine inhibits L-AChRs mainly through a
negative allosteric mechanism.
We demonstrate here that the C. elegans heteromeric levamisole-
sensitive AChR can be readily expressed in Xenopus oocytes by
providing 3 ancillary C. elegans proteins in addition to 5 distinct
AChR subunits. Our experiments led to the surprising conclusion
that 5 different subunits assemble into the same receptor complex
because removal of any of these 5 subunits reduces expression by
Acetylcholine, levamisole, and pyrantel, but not nico-
tine, activate the L-AChR. Concentrations of agonist are
indicated above each application. Traces are from a sin-
gle oocyte. (Right) Current relative to 100 ?M ACh (pla-
pyrantel 6.1 ? 0.7% (n ? 6), 500 ?M nicotine 0.55 ?
0.21% (n ? 4). (B) Dose–response curves for ACh and
levamisole. The value at 500 ?M levamisole has been
excluded from the fit because of voltage-dependent
block at this concentration. (C) High concentrations of
levamisole cause voltage-dependent channel block of
L-AChR. BAPTA-injected oocytes were subjected to volt-
age ramps in the presence of 100 ?M or 500 ?M levami-
sole (n ? 5). (Inset) Representative traces of L-AChR re-
sponses evoked by 100 ?M and 500 ?M levamisole. The
sole probably occurs because unbinding of levamisole
from its pore-blocking site is faster than unbinding of
levamisole from its agonist site. (D) Concentration-depen-
dent washout kinetics of levamisole-evoked responses.
ACh or levamisole traces obtained from a single oocyte at
different agonist concentrations were first normalized to
the current level measured just before agonist washout
and then superimposed. Note that although ACh-evoked
responses display classical concentration-independent
washout kinetics, the washout time course of levamisole-
dTC (96 ? 1%; n ? 7), 10 ?M MLA (39 ? 8%; n ? 5), and
1%, n ? 5; and 6 ? 1%, n ? 5; respectively).
Antagonist pharmacology of the L-AChR. L-
Boulin et al.
November 25, 2008 ?
vol. 105 ?
no. 47 ?
?97%. In contrast to earlier attempts that only used a subset of the
was efficient and robust in our system. ACh-elicited currents,
ranging from hundreds of nA to several ?A, could be measured in
almost all injected oocytes.
C. elegans RIC-3, UNC-50, and UNC-74 are critical for the
expression of L-AChRs in Xenopus oocytes. Although these 3
proteins are evolutionarily conserved, our results suggest they
may be absent or expressed at insufficient levels in oocytes.
Alternatively, species-specific determinants contained in L-
AChRs may require the presence of nematode ancillary proteins
for efficient expression. The orthologs of RIC-3 have been
characterized in insects and vertebrates. They are proposed to
act as chaperones that directly interact with various AChR or
5-HT3Asubunits and promote maturation and assembly of the
receptors (28). The human hRIC-3 is able to enhance the
expression of the C. elegans DES-2/DEG-3 AChR, demonstrat-
ing interspecies functionality (14). Hence, endogenous expres-
sion of RIC-3 in Xenopus oocytes might explain why low but
significant levels of L-AChRs could be expressed when the 2
other factors UNC-50 and UNC-74 were overexpressed.
UNC-50 was also conserved during evolution, and an UNC-50
ortholog can be readily identified in Xenopus laevis (17). Yeast,
nematode, and human UNC-50 proteins are all able to interact
is unable to rescue an unc-50 mutant in C. elegans (S. Eimer and
L-AChR expression in oocytes, it might be able to interact with
the vertebrate trafficking machinery but provide a nematode-
specific interface required for proper L-AChR trafficking to the
plasma membrane. The requirement of such ancillary factors
might be worth considering when recombinant receptor expres-
sion fails. For example, there is no system reported so far to
express native insect AChRs [except very inefficient expression
of the locust ?L1 (29)]. The fact that certain Drosophila AChR
?-subunits can only be expressed in Xenopus when they are
coinjected with vertebrate ?-subunits (30, 31) may indicate that
vertebrate ?-subunits are needed to recruit vertebrate ancillary
factors and promote receptor expression in the absence of
Drosophila-specific ancillary proteins.
In C. elegans, both levamisole- and nicotine-sensitive AChRs
are found at neuromuscular junctions [(11, 12, 32); M. Gendrel
and J.-L.B., unpublished data]. Based on the EC50 estimated
from our dose–response experiments, L-AChRs and N-AChRs
seem equally sensitive to their endogenous ligand ACh (26 ?M
and 31 ?M, respectively). However, their biophysical and phar-
macological differences are dramatically different. First, L-
(40 s), whereas ACR-16 homopentamers desensitize rapidly and
profoundly (?97% within 30 s). Second, even though L-AChRs
contain 3 ?-subunits that could contribute 3 ACh binding sites
per receptor molecule, there is no (or little) apparent ACh
binding cooperativity as suggested by the Hill coefficient close to
1. This contrasts with the Hill coefficient of 2.4 for ACR-16
homomers. Third, the calcium permeability of L-AChRs is
modest (PCa/PNa? 0.6), a value slightly higher than that found
for embryonic muscle AChRs at vertebrate neuromuscular
junctions. The calcium permeability of N-AChRs has not been
measured. However, ACR-16 resembles the neuronal ?7-like
homomer-forming subunits of vertebrates that are much more
permeable to calcium than heteromeric channels (19, 22, 24).
Fourth, L-AChRs are activated by levamisole, inhibited by
nicotine, and insensitive to DH?E, whereas N-AChRs are
activated by nicotine, partially inhibited by levamisole, and
blocked by DH?E. Finally, in vivo, both receptor subtypes
colocalize at neuromuscular junctions, yet distinct molecular
machineries are involved in the synaptic clustering of L- and
N-AChRs (11, 32). It is tempting to hypothesize that these
receptors might contribute different neuromuscular signaling
depending on locomotory regimes, but this still needs to be
Expression of L-AChRs provides a means to analyze the action
of levamisole in greater detail. This drug has been described as a
potent agonist of nematode AChRs. However, our dose–response
experiments demonstrate that levamisole is only a partial agonist,
?3-fold less efficient than ACh. At 500 ?M, levamisole behaves as
an open-channel blocker, as recently suggested by single-channel
recording of L-AChRs from C. elegans muscle membranes (ref. 26;
see Fig. 3C). However, the effectiveness of levamisole on parasitic
do not desensitize and should stay open as long as the drug is
the L-AChR. (A) Nicotine fails to activate L-AChR signifi-
cantly in vivo. In whole-cell recordings from C. elegans
body-wall muscles, a strain lacking both acr-16 and
tants [mean amplitudes of the response to 500 ?M nico-
tine were 21 ? 11 pA (n ? 4) and 420 ? 80 pA (n ? 4),
respectively]. (B) In contrast, responses to levamisole are
indistinguishable between these 2 strains [mean ampli-
tudes of the response to 500 ?M levamisole were 155 ?
37 pA (n ? 4) and 134 ? 22 pA (n ? 4), respectively]. (C)
Responses elicited by ACh are inhibited by nicotine in a
dose-dependent manner. Percentage inhibition of re-
ramps (?70 to ?50 mV) in 100 ?M ACh with or without
500 ?M nicotine. Note that nicotine inhibition occurs
over the whole voltage range (n ? 10, BAPTA-injected
oocytes). (E) Nicotine has a modest effect on ACh sensi-
respectively. In the presence of nicotine, EC50and nHfor
ACh are 58 ? 3 ?M and 1.23 ? 0.06 (n ? 5), respectively.
Nicotine acts mainly as an allosteric inhibitor of
www.pnas.org?cgi?doi?10.1073?pnas.0806933105 Boulin et al.
concentrations, washout kinetics are very slow, complete recovery
taking minutes compared with a few seconds for ACh-evoked
responses. Because levamisole is membrane-permeable, it is pos-
inherently tight ligand–receptor interaction but rather from slow
effects are reminiscent of the activation of GABAAreceptors by
neurosteroids. These compounds are thought to partition and
accumulate into the plasma membrane from where they access a
binding site on the receptor. The rate-limiting factor for deactiva-
tion may then be reservoir emptying rather than the intrinsic
kinetics of ligand dissociation (33). We propose that similar mech-
L-AChRs. Interestingly, the very slow-washout kinetics that we
observed are consistent with earlier reports that demonstrated that
levamisole caused a much more prolonged contraction of isolated
Ascaris muscle than ACh (34). This property likely contributes to
the anthelminthic efficacy of the drug.
Analysis of recombinant L-AChRs also revealed that nicotine,
the prototypical agonist of ionotropic acetylcholine receptors, is an
antagonist of L-AChRs. Only vertebrate ?9* AChR have been
reported to be nicotine-insensitive (35). On these receptors, nico-
tine actually behaves as a potent competitive antagonist. The
situation is different with L-AChRs because high concentrations of
nicotine do not dramatically affect ACh EC50. We also demon-
strated that this inhibition is not caused by open-channel block.
Hence, we concluded that nicotine is mainly a noncompetitive
L-AChR antagonist, possibly acting through allosteric inhibition.
Such a mechanism has been proposed for picrotoxin at GABAA
receptors (36), and for some AChR noncompetitive antagonists
such as the steroid promegestone that efficiently inhibits Torpedo
AChRs most probably by increasing the fraction of desensitized
In conclusion, the expression system that we have characterized
the unique L-AChR pharmacology. Understanding the molecular
basis for the agonist selectivity of levamisole on nematode but not
vertebrate AChRs might open the way for the rational design of
novel anthelminthic drugs.
Materials and Methods
For more detailed information, see SI Materials and Methods.
General Procedures and Strains. C. elegans strains were grown on nematode
growth medium by using standard conditions according to (38). The following
strains were used for electrophysiological recordings: BC168 unc-13(s69)I and
Electrophysiological Studies in X. laevis Oocytes. X. laevis oocytes were pre-
pared, injected, voltage-clamped, and superfused as described in ref. 39
except that gentamycin was omitted from the conservation medium because
prolonged treatments with this antibiotic can inhibit AChRs expressed in
Xenopus oocytes (40).
Electrophysiological Studies in C. elegans. Electrophysiological recordings on C.
elegans muscle cells were performed according to ref. 12.
ACKNOWLEDGMENTS. We thank Stuart Edelstein for critical reading of the
Utah, Salt Lake City). This work was supported by a European Molecular Biology
Organization Long-Term Fellowship and Institut National de la Sante ´ et de la
RechercheMe ´dicale(INSERM)JuniorContract(toT.B.),aMiniste `redelaRecher-
che fellowship (to M.G.), a genetics training grant (to D.C.W.), Association Fran-
c ¸aise Contre les Myopathies and ANR-07-NEURO-032-01 grants (to J.-L.B.), and
INSERM and Agence Nationale de la Recherche funding (for P.P.).
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