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 ...

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Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

Delayed-rectifier K؉ currents (I DR ) in pancreatic ␤-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltagegated K ؉ channel, K V 2.1, is expressed in ␤-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I DR in rodent ␤-cells. A novel peptidyl inhibitor of K V 2.1/K V 2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration ϳ1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to ␤-cell physiology. In mouse ␤-cells, GxTX-1 inhibits 90% of I DR and, as for K V 2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the ␤-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the ␤-cell I DR , which may provide advantages over currently used therapies for the treatment of type 2 diabetes.

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc 1 , is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc 1 head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamategated chloride channel subunit DmGluCl␣. To examine the effect of this mutation on the biophysical properties of DmGluCl␣ channels, it was introduced into a recombinant DmGluCl␣, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluCl␣ channels. However, mutant channels were Ϸ10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluCl␣ channels.