Jospin M , Qi YB , Stawicki TM , Boulin T , Schuske KR , Horvitz HR , Bessereau JL , Jorgensen EM , Jin Y .

GM024663 NIGMS NIH HHS , NS034307 NINDS NIH HHS , NS035546 NINDS NIH HHS , Howard Hughes Medical Institute , R01 NS035546 NINDS NIH HHS , HHMI_JIN_Y Howard Hughes Medical Institute , R01 GM024663 NIGMS NIH HHS , R37 NS034307 NINDS NIH HHS , R01 NS034307 NINDS NIH HHS , R37 GM024663 NIGMS NIH HHS

	Figure 1. acr-2 locus.(A) Genetic position of acr-2 on the left arm of the X chromosome. (B) Representative cosmids and subclones used in germline transformation rescue of acr-2(n2420gf). Rescue of spontaneous convulsion is indicated by a plus sign (+); and no rescue by a minus sign (−). Canonical cosmids F38B6 and K11G12 are shown in parentheses. (C) ACR-2 is a member of the UNC-29 class non–α-subunits of the acetylcholine receptor family [14]. (D) The n2420 mutation causes a valine-to-methionine change in the 13′ position of the pore-forming second transmembrane domain (TM2, residues are colored in teal). The amino acid at the 13′ position is oriented toward the pore; orange mark residues lining the pore [20].
	Figure 2. Mutant phenotypes and pharmacological analysis of acr-2(n2420gf) mutants.(A) Suppression of the convulsions of acr-2(n2420gf) mutants by exogenous mecamylamine (100 µM). The effects of mecamylamine are reversible. (B) acr-2(n2420gf) mutants are hypersensitive, and acr-2(lf) mutants are moderately resistant to aldicarb. The graph shows the time course of the response of adult hermaphrodites to 0.5 mM aldicarb, from three trials with ten animals per genotype per trial. *p<0.001 between wild type and mutants (two-way ANOVA). (C) acr-2(n2420gf) mutants are hypersensitive to levamisole. The graph shows the time course of the response of 1-d-old hermaphrodites to 1 mM levamisole, from three trials with ten animals per genotype per trial. *p<0.001 between wild type and acr-2(n2420gf) (two-way ANOVA). (D) Quantification of convulsion rates of 1-d-old hermaphrodites of various genotypes. (E) Developmental onset of the convulsion phenotype of acr-2(n2420gf) animals occurs during the L3 stage. OA, 2-d-old adult; YA, 1-d-old adult.
	Figure 3. acr-2 is expressed and functions in the cholinergic ventral cord motor neurons.(A) Expression pattern from a 3.5-kb acr-2 promoter-driven Pacr-2-GFP transgene (juIs14) in an L1 (upper image) and an L4 larva. Middle image is a side view, and the bottom image is a ventral view of an L4 larva. The number and positions of the cells and the sides of the commissures indicate that they are embryonically born DA and DB and postembryonically born VA and VB neurons. Expression is also seen in DVC in the tail (arrow, middle panel). Scale bars indicate 10 µm. (B). The top image shows nonoverlapping expression of Pacr-2-GFP (juIs14) (green) and Pttr-39-mcherry (juIs223) (red) marking the DD and VD GABAergic neurons. The bottom images show nonoverlapping expression of the 1.8-kb promoter-driven Pacr-2-mcherry transgene (juEx2045) (red) and those of Pglr-1-GFP (nuIs1) (green) or Pnmr-1-GFP (akIs3) (green), which label the command interneurons in the head (left panels) and tail (right panels) ganglia. Transcriptional activity of the acr-2 1.8-kb promoter is not seen in any head neurons. Scale bars indicate 10 µm. (C) Quantification of cell-type–specific transgenic rescue of the convulsion defects in acr-2(n2420gf) animals. Pacr-2::acr-2 contains the full-length genomic coding sequence driven by the 3.5-kb-long promoter. Pacr-2::acr-2(mini) contains cDNA that replaced genomic DNA from exon 2, driven by the 1.8-kb promoter. Punc-25::acr-2 contains the full-length genomic coding sequences driven by the 1.4-kb unc-25 promoter. n = 10, 10, 5, 10, and 8 for N2, acr-2(n2420gf), acr-2(n2420gf);Pacr-2-acr-2(oxEx707), acr-2(n2420gf); Pacr-2-acr-2(mini) (juEx2336), and acr-2(n2420gf);Punc-25-acr-2(juEx32), respectively.
	Figure 4. Acetylcholine neurotransmission is reduced in acr-2 null mutants.(A) acr-2 gene structure with the intragenic mutations identified from the acr-2(n2420gf) suppressor screen. acr-2(n2595 n2420) contains a nonsense mutation at the codon for tryptophan 175 in addition to the original gain-of-function mutation and is used as the null mutant in this figure. ok1887 removes the first three exons and inserts 420 bp of unrelated DNA. Details of the nucleotide and amino acid changes are shown in Table S1 and Figure S1. Boxes indicate exons; lines, introns; M, transmembrane domain; an asterisk (*) indicates mutations at the splice junctions; X indicates stop codon mutations. (B) Average speed of wild-type (3.9 mm/min±0.3 SEM, n = 16), acr-2(n2595 n2420) (2.8 mm/min±0.1 SEM, n = 16), and acr-2(n2595 n2420) worms carrying a Pacr-2::acr-2 construct (4.0 mm/min±0.4 SEM, n = 16). An ANOVA test followed by a Dunn's post test was used to analyze the data; *p<0.05. (C) Acetylcholine mini frequencies recorded in 2 mM external CaCl2 from the wild type (21.9 events/s±3.2 SEM, n = 5) and acr-2(n2595 n2420) (12.9 events/s±1.6 SEM, n = 9) are significantly different (*p = 0.015). GABA mini frequencies recorded in 2 mM external CaCl2 from the wild type (14.9 events/s±5.4 SEM, n = 5) and acr-2(n2595 n2420) (10.4 events/s±2.2 SEM, n = 9) are not significantly different (p = 0.38). Data were analyzed using a two-tailed unpaired t-test. (D) Representative traces of minis recorded at two holding potentials, −60 and −10 mV, on body muscle cells from wild-type and acr-2(n2595 n2420) worms in 2 mM external CaCl2.
	Figure 5. acr-2(n2420gf) mutants exhibit reduced GABA neurotransmission.(A) Representative traces (upper panel) and frequencies (lower panel) of endogenous postsynaptic currents recorded at two holding potentials, −60 and −10 mV, from wild-type and acr-2(n2420gf) worms in 0.5 mM external CaCl2. The frequency of miniature postsynaptic currents from cholinergic neurons is increased in the gain-of-function mutant (wild type: 12.1 events/s±1.1 SEM, n = 8; acr-2(n2420gf): 18.3 events/s±2.2 SEM, n = 9; *p = 0.0307). The frequency of miniature currents induced by GABAergic motor neurons was only slightly reduced in the gain-of-function mutant (wild type: 9.2 events/s±2.3 SEM, n = 8; acr-2(n2420gf): 7.2 events/s±2.1 SEM, n = 9; p = 0.5342). Data were analyzed using a two-tailed unpaired t-test. (B) Representative traces (upper panel) and frequencies (lower panel) of endogenous postsynaptic currents recorded at two holding potentials, −60 and −10 mV, from wild-type and acr-2(n2420gf) worms in 2 mM external CaCl2. Acetylcholine currents frequencies recorded from the wild type (18.9 events/s±2.3 SEM, n = 10) and acr-2(n2420gf) (18.7 events/s±2.6 SEM, n = 14) are not significantly different (p = 0.9555). GABA currents recorded from the wild type (9.9 events/s±1.7 SEM, n = 10) and acr-2(n2420gf) (1.2 events/s±0.6 SEM, n = 14) are significantly different (*p = 0.0007). Data were analyzed using a two-tailed unpaired t-test with Welch's correction. (C) Representative traces (upper panel) and frequencies (lower panel) of endogenous postsynaptic currents recorded at two holding potentials, −60 and −10 mV, from wild-type and acr-2(n2420gf) worms in 5 mM external CaCl2. Acetylcholine currents frequencies recorded from the wild type (24.9 events/s±3.4 SEM, n = 8) and acr-2(n2420gf) (7.6 events/s±2.7 SEM, n = 7) are significantly different (*p = 0.0017). GABA currents recorded from the wild type (13.4 events/s±2.0 SEM, n = 8) and acr-2(n2420gf) (2.3 events/s±1.0 SEM, n = 7) are significantly different (**p = 0.0004). Data were analyzed using a two-tailed unpaired t-test.
	Figure 6. Mutations in acetylcholine receptor subunits suppress convulsions of acr-2(n2420gf) mutants.(A) acr-12 gene structure with the loss-of-function mutations identified as extragenic suppressors of acr-2(n2420gf). ok367 has a 1,368-bp deletion and is a null allele of acr-12. Boxes indicate exons; lines, introns; TM, transmembrane. (B) The convulsions of acr-2(n2420gf) mutants are suppressed by loss-of-function mutations in unc-63, unc-38, unc-50, or unc-74, but not in unc-29, lev-1, and several other acr genes tested. The alleles used were unc-63(e384), unc-38(e284), unc-50(n2623), unc-74(n2614), unc-29(x29), lev-1(e211), acr-16(ok789), acr-5(ok180), lev-8(ok1519), ric-3(hm9), and lev-10(x17), all of which are null mutations. Red columns represent ancillary proteins, and green columns represent acetylcholine receptor subunits. A minimum of eight to ten animals per genotype were scored. (C) Cell-type–specific transgenic expression of acr-12 and unc-63 shows that both are required in the ventral cord cholinergic motor neurons to suppress acr-2(n2420gf). The alleles used were unc-63(e384) and acr-12(ok367). Pmyo-3 promoter drives expression in all body muscles, and Prab-3 drives expression in all neurons [49]. The Pacr-2 promoter contains 1.8 kb of acr-2 upstream sequences driving expression in A and B neurons (Figure 3A), and the Punc-25 promoter drives expression in the four RME and 19 D neurons [38]. The colors of the columns represent grouping of genotypes. A minimum of eight to ten animals per genotype were scored.
	Figure 7. Composition of the ACR-2R receptor reconstituted in Xenopus oocytes.(A) The ACR-2R receptor shows high sensitivity to acetylcholine and is insensitive to levamisole or choline. Mecamylamine completely blocks the current. The ACR-2R receptor includes all five subunits. (B) The ACR-2(V13'M)R receptor shows high sensitivity to acetylcholine and an increased sensitivity to DMPP, and weakly responds to levamisole and choline. The ACR-2(V13'M)R receptor includes all five subunits. (C) Quantification of the relative efficacies of cholinergic agonists compared to acetylcholine. Numbers above the bars represent the numbers of oocytes recorded for each condition. Example of traces are shown in (A and B). Replacement of ACR-2(+) by ACR-2(n2420) increased agonist efficacy for DMPP, choline, and levamisole, whereas nicotine efficacy was unchanged. ACh: 100 µM acetylcholine; Nic: 100 µM nicotine; DMPP: 100 µM DMPP; Cho: 1 mM choline; and Lev: 100 µM levamisole. (D) Inclusion of ACR-3 greatly increases the current of the ACR-2R. cRNAs for acr-12, ric-3, unc-38, unc-50, unc-63, and unc-74 were coinjected for each condition. Average peak current for acr-2 + acr-3 coinjection was 111±68 nA (n = 34). (E) Dose–response curves for acetylcholine action on the ACR-2 and ACR-2(V13'M) receptors show that EC50 is comparable. All recordings were made with 1 mM external CaCl2. Error bars are standard errors of the mean. Asterisks indicate that data are significantly different at p<0.05 (*) or p<0.01 (**). ACh: acetylcholine; Nic: nicotine; DMPP: 1,1-dimethyl-4-phenylpiperazinium; Cho: choline; Lev: levamisole; Mec: mecamylamine.

Albuquerque, Mammalian nicotinic acetylcholine receptors: from structure to function. 2009, Pubmed

Albuquerque, Mammalian nicotinic acetylcholine receptors: from structure to function. 2009, Pubmed
Bamber, The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor. 1999, Pubmed , Xenbase
Baylis, ACR-3, a Caenorhabditis elegans nicotinic acetylcholine receptor subunit. Molecular cloning and functional expression. 1997, Pubmed , Xenbase
Bertrand, Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic receptor. 1993, Pubmed , Xenbase
Boulin, Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor. 2008, Pubmed , Xenbase
Brenner, The genetics of Caenorhabditis elegans. 1974, Pubmed
Brown, Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study. 2006, Pubmed
Charnet, An open-channel blocker interacts with adjacent turns of alpha-helices in the nicotinic acetylcholine receptor. 1990, Pubmed
Cinar, Expression profiling of GABAergic motor neurons in Caenorhabditis elegans. 2005, Pubmed
Coulson, Toward a physical map of the genome of the nematode Caenorhabditis elegans. 1986, Pubmed
Culetto, The Caenorhabditis elegans unc-63 gene encodes a levamisole-sensitive nicotinic acetylcholine receptor alpha subunit. 2004, Pubmed
Davis, Rapid single nucleotide polymorphism mapping in C. elegans. 2005, Pubmed
Drisdel, Neuronal alpha-bungarotoxin receptors are alpha7 subunit homomers. 2000, Pubmed
Eimer, Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50. 2007, Pubmed
Engel, New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome. 1996, Pubmed
Fleming, Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits. 1997, Pubmed , Xenbase
Galzi, The multiple phenotypes of allosteric receptor mutants. 1996, Pubmed
Galzi, Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. 1992, Pubmed , Xenbase
Gottschalk, Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. 2005, Pubmed
Halevi, The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors. 2002, Pubmed , Xenbase
Hansen, Galanthamine and non-competitive inhibitor binding to ACh-binding protein: evidence for a binding site on non-alpha-subunit interfaces of heteromeric neuronal nicotinic receptors. 2007, Pubmed
Hille, Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH. 1975, Pubmed
Jin, The Caenorhabditis elegans gene unc-25 encodes glutamic acid decarboxylase and is required for synaptic transmission but not synaptic development. 1999, Pubmed
Jones, The nicotinic acetylcholine receptor gene family of the nematode Caenorhabditis elegans: an update on nomenclature. 2007, Pubmed
Jones, Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans. 2004, Pubmed
Jospin, The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans. 2002, Pubmed
Karlin, Emerging structure of the nicotinic acetylcholine receptors. 2002, Pubmed
Keramidas, Ligand-gated ion channels: mechanisms underlying ion selectivity. 2004, Pubmed
Lee, Nicotinic receptor interloop proline anchors beta1-beta2 and Cys loops in coupling agonist binding to channel gating. 2008, Pubmed
Lewis, Levamisole-resistant mutants of the nematode Caenorhabditis elegans appear to lack pharmacological acetylcholine receptors. 1980, Pubmed
Mann, The multifaceted role of inhibition in epilepsy: seizure-genesis through excessive GABAergic inhibition in autosomal dominant nocturnal frontal lobe epilepsy. 2008, Pubmed
McIntire, Genes required for GABA function in Caenorhabditis elegans. 1993, Pubmed
Mello, Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. 1991, Pubmed
Rand, Acetylcholine. 2007, Pubmed
Richmond, One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. 1999, Pubmed
Ruaud, Activation of nicotinic receptors uncouples a developmental timer from the molting timer in C. elegans. 2006, Pubmed
Schuske, The GABA nervous system in C. elegans. 2004, Pubmed
Squire, Molecular cloning and functional co-expression of a Caenorhabditis elegans nicotinic acetylcholine receptor subunit (acr-2). 1995, Pubmed , Xenbase
Stitzel, Naturally occurring genetic variability in the nicotinic acetylcholine receptor alpha4 and alpha7 subunit genes and phenotypic diversity in humans and mice. 2008, Pubmed
Towers, The Caenorhabditis elegans lev-8 gene encodes a novel type of nicotinic acetylcholine receptor alpha subunit. 2005, Pubmed
Treinin, A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans. 1995, Pubmed
Unwin, Refined structure of the nicotinic acetylcholine receptor at 4A resolution. 2005, Pubmed
Varanda, The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist. 1985, Pubmed
Vashlishan, An RNAi screen identifies genes that regulate GABA synapses. 2008, Pubmed
Wang, SLO-1 potassium channels control quantal content of neurotransmitter release at the C. elegans neuromuscular junction. 2001, Pubmed , Xenbase
White, The structure of the nervous system of the nematode Caenorhabditis elegans. 1986, Pubmed
White, The structure of the ventral nerve cord of Caenorhabditis elegans. 1976, Pubmed
White, The sensory circuitry for sexual attraction in C. elegans males. 2007, Pubmed
Wicks, Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. 2001, Pubmed