The ability of organisms to evolve resistance threatens the effectiveness of every antibiotic drug. We show that in the nematode Caenorhabditis elegans, simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) ␣-type subunits confers high-level resistance to the antiparasitic drug ivermectin. In contrast, mutating any two channel genes confers modest or no resistance. We propose a model in which ivermectin sensitivity in C. elegans is mediated by genes affecting parallel genetic pathways defined by the family of GluCl genes. The sensitivity of these pathways is further modulated by unc-7, unc-9, and the Dyf (dye filling defective) genes, which alter the structure of the nervous system. Our results suggest that the evolution of drug resistance can be slowed by targeting antibiotic drugs to several members of a multigene family. Ivermectin is used to treat numerous parasitic infections of humans, pets, and livestock (1). Treatment with ivermectin is the cornerstone of efforts to eradicate river blindness (onchocerciasis). However, reports of resistance to ivermectin in nematodes are increasingly common (2-4). Ivermectin also kills the nematode Caenorhabditis elegans at therapeutic concentrations, making C. elegans a useful model system in which to examine mechanisms of ivermectin toxicity and resistance. Ivermectin activates glutamate-gated chloride channels (GluCls) that contain ␣-type channel subunits (5-7). In C. elegans, ␣-type subunits are encoded by a family of genes including: glc-1 (encoding GLC-1͞GluCl␣1), avr-15 (encoding AVR-15͞GluCl␣2), and possibly other uncharacterized genes found in the genome sequence (5-8). Severe loss-of-function mutations in glc-1 or avr-15 do not make worms resistant to ivermectin (6, 7), either because GluCls are not physiologically important targets of ivermectin, or because multiple GluCl genes contribute independently to ivermectin sensitivity. To clarify the role of the GluCls in the nematocidal effects of ivermectin, we had screened for ivermectin-resistant mutants (6). Here we analyze the effects of these and other, previously characterized mutations on ivermectin sensitivity. We show that simultaneous mutation of three genes encoding GluCl ␣-type subunits confers high-level resistance to ivermectin. Our results suggest that the ability of ivermectin to target several members of a multigene family may decrease the rate at which resistance evolves. MethodsGenetics. Unless otherwise indicated the mutant alleles used were: avr-14(ad1302), avr-15(ad1051), gcl-1(pk54::Tc1), unc-7(e5), unc-9(e101), osm-1(ad1307), osm-5(ad1308), dyf-11(ad1303), and che-3(ad1306). avr-15(ad1051), glc-1(pk54::Tc1), unc-7(e5), and unc-9(e101) appear to be molecular nulls (refs. 6,7,and 18; T. Starich, personal communication). Ivermectin-resistant mutants were isolated in a screen for ivermectin resistance in an avr-15(ad1051) background by using the mutagen ethyl methanesulfonate as described (6). All strains were outcrossed twice with N2 ...

Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin's effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr-15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr-15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate-gated chloride channel subunits. An avr-15-encoded subunit forms a homomeric channel that is ivermectin-sensitive and glutamate-gated. These results indicate that: (i) an ivermectin-sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate-gated chloride channels in essential excitable cells such as those of the pharynx.

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by D-tubocurarine but not by ␣-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.Fast (ionotropic) cholinergic neurotransmission is generally mediated by nicotinic acetylcholine (ACh) 1 receptors (nAChRs). These are cation-selective channels and hence mediate excitatory neurotransmission. However, electrophysiological evidence of ionotropic, ACh-gated chloride channels in molluscs suggests the existence of fast inhibitory cholinergic neurotransmission as well (1-3). The ACh-gated chloride channels in Aplysia neurons respond to several agonists and antagonists of nAChRs, indicating that, like the nAChRs, they may belong to the superfamily of Cys loop ligand-gated ion channel (LGIC) subunits. Otherwise, little is known about the molecular nature of the receptors that mediate fast inhibitory cholinergic neurotransmission, whether this type of neurotransmission is widespread in the animal kingdom, or how it evolved.The Cys loop LGICs are encoded by a large and diverse gene superfamily. These channels are pentameric and can be homomers or heteromers consisting of as many as four different subunits, each encoded by a different gene (4). Subunits of the Cys loop LGIC superfamily share a topology that consists of a large extracellular ligand-binding domain and four transmembrane domains that form the ion-selective pore (4 -6). In vertebrates, the LGIC superfamily consists of two families of cation-selective channels, the nicotinic ACh receptors and the 5-hydroxytryptamine type 3 receptors, and two families of anion channels, the GABA A receptors and the glycine receptors (7). The repertoire of invertebrate LGICs is larger, including, in addition to homologues of vertebrate channels, histaminegated chloride channels (8, 9), a GABA-gated cation channel (10), a serotonin-gated anion channel (11), several glutamategated anion channels (12-16), and a divergent choline-gated nAChR (17). Thus, the LGIC channel structure appears flexible enough to accommodate diverse ligands and ligand/ion selectivity pairings.Although no genes encoding ACh-gated chloride channels have been previously identified, it is likely that many invertebrate receptors with unusual properties remain to be characterized. The genomes of Caenorhabditis elegans and Drosophila melanogaster reveal numerous predicted Cys loop LGICs that do not obviously belong to any family of known i...

The locomotion of Caenorhabditis elegans consists of forward crawling punctuated by spontaneous reversals. To better understand the important variables that affect locomotion, we have described in detail the locomotory behavior of C. elegans and identified a set of parameters that are sufficient to describe the animal's trajectory. A model of locomotion based on these parameters indicates that reversal frequency plays a central role in locomotion. We found that several variables such as humidity, gravidity, and mechanostimulation influence reversal frequency. Specifically, both gentle and harsh touch can transiently suppress reversal frequency. Thus, reversal behavior is a model for the integration of information from numerous modalities reflecting diverse aspects of the state of an organism.

The subunit stoichiometry of heteromeric glycine-gated channels (GlyRs) determines fundamental properties of these key inhibitory neurotransmitter receptors; however the ratio of α1 to β-subunits per receptor remains controversial. We used single molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.

The genome sequences of Caenorhabditis elegans and Drosophila melanogaster reveal a diversity of cysteine-loop ligand-gated ion channels (Cys-loop LGICs) not found in vertebrates. To better understand the evolution of this gene superfamily, I compared all Cys-loop LGICs from rat, the primitive chordate Ciona intestinalis, Drosophila, and C. elegans. There are two clades of GABA receptor subunits that include both vertebrate and invertebrate orthologues. In addition, I identified nine clades of anion channel subunits found only in invertebrates, including three that are specific to C. elegans and two found only in Drosophila. One well-defined clade of vertebrate cation channel subunits, the alpha 7 nicotinic acetylcholine receptor subunits (nAChR), includes invertebrate orthologues. There are two clades of invertebrate nAChRs, one of alpha-type subunits and one of non-alpha subunits, that are most similar to the two clades of vertebrate neuronal and muscle alpha and non-alpha subunits. There is a large group of divergent C. elegans nAChR-like subunits partially resolved into clades but no orthologues of 5HT3-type serotonin receptors in the invertebrates. The topology of the trees suggests that most of the invertebrate-specific Cys-loop LGIC clades were present in the common ancestor of chordates and ecdysozoa. Many of these disappeared from the chordates. Subsequently, selected subunit genes expanded to form large subfamilies.

The nematode pharynx has a potassium channel with unusual properties, which allows the muscles to repolarize quickly and with the proper delay. Here, the Caenorhabditis elegans exp-2 gene is shown to encode this channel. EXP-2 is a Kv-type (voltage-activated) potassium channel that has inward-rectifying properties resembling those of the structurally dissimilar human ether-à-go-go-related gene (HERG) channel. Null and gain-of-function mutations affect pharyngeal muscle excitability in ways that are consistent with the electrophysiological behavior of the channel, and thereby demonstrate a direct link between the kinetics of this unusual channel and behavior.

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