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Originally published In Press as doi:10.1074/jbc.M404370200 on August 2, 2004 Originally published In Press as doi:10.1074/jbc.M404370200 on July 27, 2004

J. Biol. Chem., Vol. 279, Issue 41, 42476-42483, October 8, 2004
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The Caenorhabditis elegans unc-63 Gene Encodes a Levamisole-sensitive Nicotinic Acetylcholine Receptor {alpha} Subunit*

Emmanuel Culetto{ddagger}§, Howard A. Baylis§||, Janet E. Richmond**, Andrew K. Jones{ddagger}, John T. Fleming§{ddagger}{ddagger}, Michael D. Squire§§§, James A. Lewis§¶¶, and David B. Sattelle{ddagger}§||||

From the {ddagger}Medical Research Council Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom, ||Department of Zoology and §The Babraham Institute Laboratory of Molecular Signalling, University of Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom, and **Department of Biology, University of Illinois, Chicago, Illinois 60607

Received for publication, April 20, 2004 , and in revised form, July 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The anthelmintic drug levamisole causes hypercontraction of body wall muscles and lethality in nematode worms. In the nematode Caenorhabditis elegans, a genetic screen for levamisole resistance has identified 12 genes, three of which (unc-38, unc-29, and lev-1) encode nicotinic acetylcholine receptor (nAChR) subunits. Here we describe the molecular and functional characterization of another levamisole-resistant gene, unc-63, encoding a nAChR {alpha} subunit with a predicted amino acid sequence most similar to that of UNC-38. Like UNC-38 and UNC-29, UNC-63 is expressed in body wall muscles. In addition, UNC-63 is expressed in vulval muscles and neurons. We also show that LEV-1 is expressed in body wall muscle, thus overlapping the cellular localization of UNC-63, UNC-38, and UNC-29 and suggesting possible association in vivo. This is supported by electrophysiological studies on body wall muscle, which demonstrate that a levamisole-sensitive nAChR present at the C. elegans neuromuscular junction requires both UNC-63 and LEV-1 subunits. Thus, at least four subunits, two {alpha} types (UNC-38 and UNC-63) and two non-{alpha} types (UNC-29 and LEV-1), can contribute to levamisole-sensitive muscle nAChRs in nematodes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nicotinic acetylcholine receptors (nAChRs)1 are Cys-loop ligand-gated ion channels formed by five polypeptide subunits (1). Typical features of Cys-loop ligand-gated ion channels include an N-terminal extracellular domain where ligand binding occurs and the Cys-loop, which is two disulfide-bonded cysteines separated by 13 residues and four transmembrane regions (M1–4), the second of which contains many channel-lining residues (2). The nAChR subunits possessing two adjacent cysteines in the acetylcholine (ACh) binding site are referred to as {alpha} subunits, whereas those with no such motif are referred to as non-{alpha} subunits ({beta}, {gamma}, {delta}, and {epsilon}). Birds and mammals possess 17 subunits characterized as either "muscle" ({alpha}1, {beta}1, {gamma}, {delta}, and {epsilon}) or "neuronal" ({alpha}2–10, {beta}2–4) subtypes (3, 4). Recent analysis of the pufferfish genome has revealed a larger nAChR gene family consisting of 28 subunits that probably arose through genome duplication (5). One of the most extensive and diverse nAChR gene families currently known is that of the nematode Caenorhabditis elegans which consists of at least 27 subunits (6).

Early investigations identified C. elegans mutants resistant to the anthelmintic drug levamisole, which causes paralysis of nematode body wall muscles (7–9). Among the 12 levamisole-resistant loci, it has been shown that lev-1 and unc-29 both encode non-{alpha} nAChR subunits, whereas unc-38 encodes a nAChR {alpha} subunit (10). Expression in Xenopus laevis oocytes of combinations of these subunits that include UNC-38 resulted in small amplitude, dose-dependent inward currents in response to ACh and levamisole that were suppressed by several nAChR antagonists (10). More recently, electrophysiological studies have shown that UNC-29 and UNC-38 are essential components of the native levamisole-sensitive nAChR at the nematode neuromuscular junction (11).

Four other genes mediating levamisole resistance have been identified. These are: unc-22, the product of which is known as twitchin (12); unc-50, which encodes for a novel transmembrane protein (13); unc-68, which encodes for a ryanodine receptor (14); and lev-11, which encodes for tropomyosin (15). The characterization of the remaining levamisole-resistant loci (unc-63, unc-74, lev-8, lev-9, and lev-10) have yet to be reported. Here we describe the cloning of a new nAChR {alpha} subunit and show that it corresponds to the unc-63 locus (7, 8). Green fluorescent protein (GFP) reporter constructs reveal that UNC-63 is expressed in body wall muscles as well as neurons. Using electrophysiology on muscle preparations, we also show that unc-63 is necessary for the function of the levamisole-sensitive nAChR at the neuromuscular junction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
C. elegans Strains and General Methods—The handling of C. elegans was performed as described by Sulston and Hodgkin (16). The following strains were used: N2 wild type C. elegans (Bristol variety), ZZ13 unc-63(x13) I, ZZ26 unc-63(x26) I, DH404 unc-63(b404), ZZ37 unc-63(x37) I, ZZ1004 unc-63(x18) dpy-5(e61) I, and CB211 lev-1(e211) IV. Tests for Levamisole Sensitivity and Locomotor Function—The sensitivity to levamisole was assessed on nematode growth media plates containing 1 mM levamisole. Locomotion was assessed by transferring adult hermaphrodites to nematode growth media plates without levamisole and after 1 h, counting the number of body bends/min.

DNA Extraction and Sequencing—Cloning was carried out using standard methods (17). The C. elegans genomic DNA extraction protocol was obtained from www.dartmouth.edu/artsci/bio/ambros/protocols/worm_protocols.html. Plasmid DNA for micro-injection into nematodes was purified using a Qiagen plasmid mini kit (Qiagen). Sequencing was performed at the Babraham Institute or at the Oxford University Biochemistry Department Sequencing Facility.

Cloning jtf#38 cDNA—A mixed stage N2 cDNA library in {lambda}gt10 (provided by S. Kim) was hybridized with an unc-38 probe at 50 °C as described previously (10). The final washes were carried out at moderate stringency (65 °C in 2x SSC, 0.1% SDS). Sequence analysis revealed that one of the positive clones, jtf#38, contained a partial cDNA homologous to nAChR subunits. The 5' and 3' ends of the jtf#38 coding region was determined using the 5'/3' RACE kit (Roche Diagnostics).

Sequence Analysis—Sequence alignment and analysis were performed using ClustalX. The BLAST alignment tool (18) was used to search the genome data base.

Genetic Localization of the jtf#38 Gene—The jtf#38 cDNA was used to probe an ordered grid of yeast artificial chromosomes (YACs) following the protocol described by Coulson et al. (19).

Sequencing of Mutant Alleles—Reverse transcriptase-PCR was performed to amplify unc-63 and lev-1 from mutant alleles, and the PCR products were sequenced. After a putative mutation was found in the cDNA, the corresponding genomic fragment from the mutants was amplified using single worm PCR (20) and sequenced.

Germ Line Transformation—Germ line transformation was performed according to the method of Mello et al. (21).

Mutant Rescue Experiment—We were unable to make a GFP construct consisting of the entire unc-63 coding region for both the rescue of unc-63 mutants and localization of the UNC-63 subunit. Thus, for mutant rescue studies, we used a construct (punc-63.1) that was generated by PCR on wild type genomic DNA using the Expand Long Template system (Roche Applied Science) and the primers 5'-TATTTGGCGGCCGCTCTGTGACTGCCTATGG-3' and 5'-GGAAGAGGTACCATGGCAGAACACGTGATG-3' (engineered restriction sites are underlined). The PCR product was cloned into the pGEM-T plasmid (Promega), and the clones were subsequently checked using restriction analysis and sequencing. The punc-63.1 construct contained a 12.5-kb insert comprising 4.5 kb of 5' upstream region, all of the genomic coding region, and 1 kb of downstream 3' sequence. Germ line transformation was performed by co-injecting the test DNA (80 µg ml–1) and the marker plasmid pPD93.65 (100 µg ml–1). Injection of pPD93.65, which contains the GFP gene under the control of the myosin heavy chain unc-54 gene promoter, resulted in GFP expression in all body wall muscle cells. Transgenic animals with GFP fluorescence in body wall muscle cells were used for further studies on the phenotype of rescued worms. Three stable lines were obtained.

Generation of UNC-63::GFP and LEV-1::GFP Constructs—As indicated in Fig. 4A, the UNC-63::GFP fusion construct was made using the expression vector pPD95.70 and a PCR product containing 4.5 kb upstream of the unc-63 start codon to part of exon 7 (the encoding part of the large cytoplasmic loop). The primers used to amplify the unc-63 genomic fragment were: 5'-TTTTGGCCCGGGATGTGTTGTTGGGGATCG-3' and 5'-TATTTGGCATGCTCTGTGACTGCCTATGG-3'. For the LEV-1::GFP construct, 9 kb of genomic DNA, including 4 kb upstream of the lev-1 start codon, was amplified using the primers 5'-AGCTCCTCTTCCGGCCACTCG-3' and 5'-TTCAGAAAATACCAAGAACTGTGTCGTTGG-3' and then cloned into vector pPD95.79. The GFP was fused in-frame with the C terminus of LEV-1 (Fig. 4A), removing the two C-terminal amino acids of LEV-1. The UNC-63::GFP fusion construct (80 µg ml–1) was co-injected with plasmid pRF4 (100 µg ml–1) into wild type animals. Transgenic animals were selected by their roller phenotype (21). The LEV-1::GFP fusion construct (50–80 µgml–1) was injected into the lev-1(e211) mutant. Transgenic worms with GFP fluorescence were selected, and the animals were viewed by fluorescence microscopy on a Leica confocal system.



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  		FIG. 4.
Expression pattern of the UNC-63::GFP and the LEV-1::GFP constructs. A, structure of the injected GFP constructs. B, composite image of L3/L4 transgenic larvae stably transformed with UNC-63::GFP. The GFP signal is observed in a large number of neurons in the lumbar ganglia (lg), pre-anal ganglia (pag), head ganglia (hg), and ventral nerve cord (arrows indicate motor neuron cell bodies). Scale bar = 50 µm. C, UNC-63::GFP signal is shown in a subset of neurons in the anal ganglia. No expression is observed either in the sphincter muscle cell or in the anal depressor muscle. Scale bar = 5 µm. D, dorsal view of a young adult animal. All the body wall muscle cells, visible as large trapezoidal cells (example indicated by arrow), express UNC-63::GFP. A dark strip separates the left and right quadrants. Scale bar = 50 µm. E, four neurons (see arrows) of the posterior lateral ganglion express UNC-63::GFP. Scale bar = 5 µm. F, the UNC-63::GFP signal is expressed in some of the vulval muscle cells indicated by arrows. Scale bar = 10 µm. G, the lev-1::GFP construct was injected into the lev-1 mutant (allele e211). GFP expression is observed from embryo to the adult stage, where signal is found in a subset of neurons in the ventral nerve cord, indicated by arrows. H, LEV-1::GFP expression in body wall muscles. Scale bar = 50 µm.

 
Electrophysiology—Whole-cell, voltage clamp recordings were obtained from the ventral medial body wall muscles of dissected C. elegans adults, as described previously (11). An EPC-9 patch clamp amplifier (HEKA, Lambrecht, Germany) was deployed to record currents, which were digitized at 2.9 kHz using an IT6 interface (Instrutech, Great Neck, New York). Data acquisition was controlled by Pulsefit software (HEKA) run on a Power Mac 6500/255 computer. Recordings were made at a holding potential of –60 mV. Extracellular saline of the following composition (in mM) was used: 150 NaCl, 5 KCl, 5 CaCl2, 4 MgCl2, 10 glucose, 5 sucrose, 15 HEPES (pH 7.3, ~330 milliosmoles). The patch pipette was filled with the following solution (in mM): 120 KCl, 20 KOH, 4 MgCl2, 5 TES, 0.25 CaCl2, 4 NaATP, 36 sucrose, 5 EGTA (pH 7.2, ~315 milliosmolar). Subsequent analysis and graphing were carried out using Pulsefit (HEKA) and Igor Pro (Wavemetrics, Lake Oswego, Oregon). Statistical analysis was performed using the unpaired t test (for ACh responses) or the Mann Whitney test (for levamisole responses and dihydro-{beta}-erythroidine block). All statistically derived values are given as mean ± S.E.

Chemicals—Unless otherwise indicated all chemicals were obtained from Sigma (UK).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning a Novel C. elegans nAChR Subunit—We employed a cross-hybridization strategy to identify new nAChRs in C. elegans. A C. elegans cDNA phage library was screened at low stringency using the unc-38 and unc-29 cDNAs as probes. Several positive clones were obtained, including one which hybridized specifically at moderate stringency to the unc-38 cDNA probe. The clone was a partial cDNA, in which the amino acid sequence showed significant identity with previously characterized nAChR subunits. We utilized RACE PCR to complete the cDNA sequence, and the RNA splice leader SL1 (22) was observed at the 5' end. The full-length cDNA clone was designated jtf#38 (GenBankTM accession number AAK83056 [GenBank] .

Location of the jtf#38 Clone on the C. elegans Physical and Genetic Maps—We mapped the location of jtf#38 to YACs Y55F5 and Y72D6 on the physical map by hybridizing the cloned cDNA to a YAC grid (Fig. 1A). Y55F5 and Y72D6 are located near the center of chromosome I. During the course of this study, the genomic sequence of the region containing jtf#38 was completed by the C. elegans sequencing consortium (23). We therefore compared the genomic region and the jtf#38 cDNA sequences and found that jtf#38 corresponds to the predicted gene Y110A7A.3, which encodes a putative nAChR subunit. The gene is composed of 10 exons spanning 7.5 kb (Fig. 1B). Because Y110A7A.3 lies close to the levamisole-resistant locus unc-63, we investigated whether Y110A7A.3 corresponds to unc-63.



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  		FIG. 1.
Cloning of unc-63. A, chromosomal localization of the C. elegans jtf#38 cDNA. Jtf#38 was mapped on a YAC grid to YAC Y55F5 and Y72D6, spanning the unc-63 locus on chromosome I. B, the genomic organization of the unc-63 gene is shown with location of identified changes found in unc-63 mutant alleles.

 
Y110A7A.3 Is the unc-63 Locus—To test whether Y110A7A.3 corresponds to unc-63, we first sequenced Y110A7A.3 genomic DNA in five unc-63 mutant alleles. All mutant alleles were isolated using ethyl methane-sulfonate mutagenesis (7). The unc-63 mutants exhibited a range of phenotypes from strongly uncoordinated locomotion with strong levamisole resistance (alleles x37, x13, x18) to almost wild type locomotion with mild levamisole resistance (alleles b404, x26). We found one missense mutation (x26), one nonsense mutation (x13), one deletion (b404), and two mutations (x37 and x18) that disrupt splice junction consensus sites (Fig. 1B). These results provide strong evidence that unc-63 corresponds to the nAChR subunit encoded by Y110A7A.3.

In addition, we rescued the unc-63 uncoordinated phenotype by injecting the construct punc-63.1 into the syncitial gonad of young adult unc-63(x37) animals. Three independently transformed lines carrying the extrachromosomal array of unc-63 (LM200, unc-63(x37);Exunc-63) were obtained, each of which showed restored sensitivity to levamisole and phenotypically wild type locomotion. We analyzed the phenotype of one rescued mutant by comparing the speed of locomotion of strains N2 (wild type), unc-63(x37);Exunc-63, and unc-63(x37). The locomotion characteristics were as follows (bends/min ± S.D., n = number of animals): wild type (24 ± 2, n = 30), unc-63(x37);Exunc-63 (21 ± 2, n = 49), and unc-63(x37) (10 ± 1, n = 20). Together with the sequences of five unc-63 mutant alleles, these results show that the jtf#38 cDNA clone we isolated corresponds to the unc-63 gene.

Features of the UNC-63 Polypeptide—As shown in Fig. 2, UNC-63 consists of 502 amino acids and possesses motifs common to Cys-loop ligand-gated ion channels, including an N-terminal signal peptide of 23 amino acids (24), 4 transmembrane regions, and the Cys-loop (1). Also present are conserved stretches of amino acids in loops A–F, which are involved in ligand binding. In loop C, there are two adjacent cysteines, defining UNC-63 as a nAChR {alpha} subunit. Using the GCG MOTIFS program, we identified one putative glycosylation site at position Asn-136 and potential phosphorylation sites within the large M3–M4 intracellular loop (Fig. 2). A phylogenetic tree of C. elegans nAChR subunits based on derived sequence identity is shown in Fig. 3. As indicated in the tree, UNC-63 is most similar to UNC-38, sharing 49% identity. However, it is interesting to note that in loop C of UNC-63, the typical YXCC motif is present rather than the unusual YXXCC motif found in loop C of UNC-38 (6).



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  		FIG. 2.
Deduced amino acid sequence of UNC-63. The protein alignment, also including UNC-38, LEV-1, UNC-29, and the human muscle {alpha} nAChR subunit, was constructed using the ClustalX algorithm (31) and is shown using the GeneDoc program (www.psc.edu./biomed/genedoc). Amino acids are numbered beginning at the first methionine. The amino acids of loops A–F contributing to the ACh binding domain are indicated as well as the four transmembrane regions (M1–M4). The potential N-glycosylation site of UNC-63 is indicated by #, and putative phosphorylation sites within the large intracellular loop of UNC-63 are shown by *, which include calmodulin-dependent protein kinase (CaMKII) sites (Thr-352, Thr-396, and Ser-432) and a protein kinase C site (Ser-418).

 



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  		FIG. 3.
Phylogenetic tree showing the relationship of UNC-63 to other members of the C. elegans nAChR subunit family. The tree was constructed using ClustalX (31) and displayed using the TreeView application (32). Numbers at each fork show bootstrap values with 1000 replicates, and the scale bar represents substitutions/site. The C. elegans ionotropic GABA receptor subunit UNC-49B (33) was chosen as the out-group. The C. elegans nAChR subunits are classified in five major subgroups: DEG-3-like, ACR-16-like, UNC-38-like, ACR-8-like, and UNC-29-like (34). UNC-38, UNC-63, UNC-29, and LEV-1 are highlighted in bold.

 
The unc-63 Gene Is Expressed in Both Muscle Cells and Neurons—To determine the tissue-specific localization of unc-63, we injected N2 animals with a genomic fragment of unc-63 fused to DNA encoding GFP (25), as shown in Fig. 4A. In transgenic animals expressing the UNC-63::GFP fusion protein fluorescence was observed in all body wall muscles and in vulval muscles (Fig. 4). We also detected expression in many cells of the nervous system, including motor neurons in the ventral nerve cord (AS, DA, DB, which innervate dorsal muscles; VB, VD and DB, which innervate ventral muscles; VC which innervates vulval and ventral muscles) and neurons in the head, posterior lateral, pre-anal, and lumbar ganglia. The expression of unc-63 in both body wall muscles and motor neurons is consistent with locomotion defects observed for unc-63 mutants.

LEV-1 Is a Possible Partner for the UNC-63 {alpha} Subunit in a Native nAChR—The subunits UNC-38, UNC-29, and LEV-1 are also associated with levamisole resistance in C. elegans. Both UNC-29 (10) and UNC-38 (11) are expressed in the body wall muscles of C. elegans, overlapping at least in part with the UNC-63 expression pattern. This raises the possibility that UNC-63, UNC-38, and UNC-29 subunits may co-assemble to form a native levamisole-sensitive receptor. To test whether LEV-1 might also be a component of this body wall muscle receptor, we determined the expression pattern of LEV-1. We made a gene fusion construct (lev-1::GFP) with 4 kb of the lev-1 5' genomic sequence and the entire lev-1 genomic coding sequence fused at the C terminus to GFP (Fig. 4A). The transgene was then injected into the recessive lev-1(e211) mutant. The lev-1(e211) allele contains a missense mutation (G461E) located in the M4 region and exhibits normal locomotion in the absence of levamisole, becoming uncoordinated (but not hypercontracted as with wild type) in the presence of 1 mM levamisole. Injected lev-1::GFP restored levamisole sensitivity to the lev-1 mutant, demonstrating that the transgene rescued lev-1(e211), and as shown in Fig. 4, G and H, GFP expression was observed in all body wall muscle cells and in a subset of motor neurons in the ventral nerve cord. Thus LEV-1 has overlapping expression with UNC-63 as well as UNC-38 and UNC-29.

UNC-63 and LEV-1 Are Part of the Levamisole-sensitive Acetylcholine Receptor—Two pharmacological classes of acetylcholine receptors function on muscles at C. elegans neuromuscular junctions. One is specifically activated by levamisole, whereas the other is activated by nicotine and inhibited by dihydro-{beta}-erythroidine (11). The levamisole-sensitive current recorded from body wall muscle requires the function of unc-38 and unc-29 (11). We wanted to test whether unc-63 and lev-1 are also required for functional levamisole-sensitive muscle nAChRs. Therefore the electrophysiological properties of the C. elegans body wall muscle nAChRs were examined in wild type, unc-63, and lev-1 mutant animals. As observed previously (11), the muscles of wild type worms responded to 100 µM ACh by generating inward currents with rapid onset and decay, and the same cells responded to 100 µM levamisole with similar currents but with slower onset and decay. As shown in Fig. 5A, the response to 100 µM levamisole was almost completely abolished for unc-63(x37) and dramatically reduced to only 14% of that observed in the wild type in lev-1(e211). We then tested whether the second nAChR type was still present in both mutants by pressure application of ACh onto voltage-clamped body wall muscle cells of unc-63(x37) and lev-1(e211) mutants (Fig. 5B). In both mutants, ACh elicited a robust inward current, although the responses were smaller than the wild type. This reduction in response to ACh is consistent with the loss of levamisole receptor contribution to the total ACh response in unc-63(x37) and lev-1(e211). We also measured the effects of dihydro-{beta}-erythroidine on the ACh-elicited responses (Fig. 5B). In wild type worms, the inward current was blocked by 84%, whereas in lev-1(e211), there was an 89% block, and an almost complete block of 95% was observed in unc-63(x37), consistent with loss of the second muscle nAChR type. Overall, these data suggest that levamisole-sensitive receptors in body wall muscle require the functional expression of UNC-63 and LEV-1 in addition to UNC-29 and UNC-38.



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  		FIG. 5.
unc-63(x37) and lev-1(e211) mutants showed reduced amplitude levamisole-induced currents in body wall muscles of C. elegans. A, body wall muscles produce an inward current in response to a 100-ms pressure-ejected pulse of levamisole. The levamisole response is almost completely abolished in the unc-63 mutant and is largely reduced in the lev-1 mutant (wild type (WT), 279 ± 28 pA, n = 5; unc-63(x37), 2.7 ± 5.8 pA, n = 3; lev-1(e211), 39 ± 7.4 pA, n = 4) as shown in typical traces and a histogram with averaged amplitudes. B, pressure ejection of ACh (100-ms pulses) produces a robust inward current in wild type body wall muscles. This current represents the activation of both the levamisole-sensitive current and the levamisole-insensitive current. The ACh response was significantly reduced in unc-63 and lev-1 mutants as reflected in the example traces and histograms (wild type (WT), 1963 ± 156 pA, n = 7; unc-63(x37), 1356 ± 142 pA, n = 7; lev-1(e211), 1559 ± 156 pA, n = 8). Blocking the levamisole-insensitive current with dihydro-{beta}-erythroidine (DH{beta}E) almost completely abolished the ACh response in unc-63 mutants and markedly reduced the response in lev-1 mutants and wild type worms (wild type, 319 ± 45 pA, n = 9; unc-63(x37), 66 ± 19 pA, n = 6; lev-1(e211), 154 ± 48 pA, n = 6). Values in all histograms which are significantly different from wild type are marked with an asterisk.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Levamisole is a potent anthelmintic drug, which is used to eradicate nematode infestations in a number of domestic animals (26). Application of levamisole to wild type C. elegans causes muscle hypercontraction, paralysis, and ultimately death, although the exact cause of death is unknown. Through the analysis of levamisole-resistant mutants, we can begin to understand the actions of levamisole by identifying targets within the worm that are required for levamisole sensitivity. Previous studies suggest that the molecular targets of levamisole include nAChRs at the C. elegans neuromuscular junction (8, 9, 11). Application of levamisole directly onto voltage-clamped C. elegans body wall muscles produces inward currents that are abolished in mutants encoding the nAChR subunits UNC-38 and UNC-29 (11). This observation is consistent with the muscle expression of these two subunits and can explain the observed muscle hypercontraction of worms exposed to levamisole.

Here we report the identification of a novel nAChR subunit in C. elegans, which shows greatest sequence similarity with UNC-38 and physically maps in the vicinity of unc-63, one of 12 loci identified in genetic screens for mutants that confer levamisole resistance (7). Sequence analysis of five unc-63 alleles revealed mutations in the predicted open reading frame of the nAChR clone, indicating that unc-63 encodes the novel nAChR. This was confirmed by demonstrating that the strongly levamisole-resistant allele unc-63(x37) could be behaviorally rescued by expressing an extrachromosomal array of the wild type nAChR cDNA clone. Using an unc-63::GFP fusion construct, we showed that UNC-63 is present in muscles. Patch clamp electrophysiology was deployed to show that muscle responses to direct applications of levamisole are virtually eliminated in the unc-63(x37) allele, indicating that UNC-63 is an essential component of a levamisole receptor.

We have also demonstrated that LEV-1, in addition to UNC-63, UNC-38, and UNC-29, is expressed in the body wall muscles of C. elegans. The lev-1(e211) mutant worms are resistant to levamisole, although their locomotion appears normal. Electrophysiological analysis of the lev-1(e211) mutant animals indicated that 86% of the levamisole-sensitive current was abolished (Fig. 5A). Because lev-1(e211) produces a missense mutation, the residual levamisole response may reflect only a partial loss of function of the LEV-1 subunit or the presence of a poison subunit that reduces the efficacy of the heteromeric receptor. Alternatively, the LEV-1 subunit may be less essential, possibly being replaced by other non-{alpha} subunits such as UNC-29.

We also show that UNC-63 and LEV-1 subunits, similar to UNC-29 and UNC-38, are expressed in a subset of C. elegans neurons. This suggests that these subunits participate in neuronal excitability, and these may represent additional targets for levamisole. Other nAChR subunits have overlapping expression with UNC-63 in several identified neurons such as ACR-5 (DB, VB) (27) and ACR-2 (DA, DB, VA, VB) (28). The composition of the nAChR receptors in these neuronal locations and their potential roles in levamisole sensitivity will require further analysis.

Thus, we have identified a fourth nAChR subunit that is a constituent of nematode levamisole-sensitive receptors, and we have established that LEV-1 contributes to the normal functioning of this receptor. It remains to be determined whether there is either a muscle levamisole-sensitive nAChR composed of UNC-38, UNC-63, UNC-29, and LEV-1 or perhaps more than one receptor, each made from various combinations of the four subunits. It is clear, however, that UNC-38, UNC-63, and UNC-29 are required subunits of all functional levamisole receptors on the medial body muscles. It is worth noting that, in terms of sequence identity, UNC-38 and UNC-63 are most closely related to the {alpha} subunit ACR-6, whereas UNC-29 and LEV-1 are most similar to the non-{alpha} subunits ACR-2 and ACR-3 (Fig. 3). Although ACR-6 has yet to be characterized, we know that ACR-2 or ACR-3 coexpress with UNC-38 in Xenopus oocytes to form functional ion channels upon which levamisole acts as an agonist (29, 30). Indeed, ACR-2 was shown to be expressed in a number of ventral cholinergic motor neurons (28) overlapping with the neuronal expression of UNC-63. It would thus be of interest to determine the expression patterns of ACR-3 and ACR-6 as a first step in evaluating their potential as further components of neuronal levamisole-sensitive nAChRs. Studies of these subunits as well as other levamisole-resistant loci may prove instructive in understanding the molecular mechanisms of drug action and developing improved parasite control agents as well as investigating mechanisms of drug resistance.


   FOOTNOTES
 
* This work was supported by grants from the Medical Research Council (to E. C., H. A. B., and D. B. S.) and the Biotechnology and Biosciences Research Council (to E. C. and D. B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AAK83056 [GenBank] /I> Back

Present address: Laboratoire de Génétique Moléculaire, Institut de Génétique et Microbiologie, Unité Mixte de Recherche 8621, Université Paris XI, Bat. 400, 91405 Orsay Cedex, France. Back

{ddagger}{ddagger} Present address: Dept. of Pediatric Haematology and Oncology, Massachusetts General Hospital, Boston, MA 02114. Back

§§ Present address: Dept. of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK. Back

¶¶ Present address: Occupational and Safety Programs, University of Texas, San Antonio, TX 78249. Back

|||| To whom correspondence should be addressed. Tel.: 44-1865-272-145; Fax: 44-1865-282-651; E-mail: david.sattelle{at}anat.ox.ac.uk.

1 The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; GFP, green fluorescent protein; YAC, yeast artificial chromosome; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; RACE, rapid amplification of cDNA ends. Back


   ACKNOWLEDGMENTS
 
We thank Dr. A. Fire for providing pPD plasmids and Dr. A. Coulson (Sanger Centre, Hinxton, UK) for providing the YAC grid. We also thank B. Esmaeili, E. B. Maryon, and D. E. Featherstone for critically reading the manuscript.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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