Characterization of Glutamate-Gated Chloride Channels in the Pharynx of Wild-Type and Mutant Caenorhabditis elegans Delineates the Role of the Subunit GluCl-a2 in the Function of the Native Receptor

Glutamate-gated chloride (GluCl) channels are the site of action of the anthelmintic ivermectin. Previously, the Xenopus laevis oocyte expression system has been used to characterize GluCl channels cloned from Caenorhabditis elegans. However, information on the native, pharmacologically relevant receptors is lacking. Here, we have used a quantitative pharmacological approach and intracellular recording techniques of C. elegans pharynx to characterize them. The glutamate response was a rapidly desensitizing, reversible, chloride-dependent depolarization (EC50 5 166 mM), only weakly antagonized by picrotoxin. The order of potency of agonists was ibotenate . L-glutamate . kainate 5 quisqualate. Ivermectin potently and irreversibly depolarized the muscle (EC50 5 2.7 nM). No further depolarization was seen with coapplication of maximal glutamate during the maximal ivermectin response, indicating that ivermectin depolarizes the muscle by the same ionic mechanism as glutamate (i.e., chloride). The potency of ivermectin on the pharynx was greater than at any of the GluCl subunits expressed in X. laevis oocytes. This effect of ivermectin was abolished in the mutant avr-15, which lacks a functional GluCl-a2 subunit. However, a chloride-dependent, nondesensitizing response to glutamate persisted. Therefore, the GluCl-a2 subunit confers ivermectin sensitivity and a highaffinity desensitizing glutamate response on the native pharyngeal GluCl receptor. The avermectins are a class of insecticides and anthelmintics that potently activate glutamate-gated chloride (GluCl) channels. The channels are apparently unique to the invertebrate phyla and play vital roles in animal function, thus accounting for the selective toxicity of these drugs. For example, Ivermectin (22,23-dihydroavermectin B1a) paralyzes both the somatic (Kass et al., 1980) and pharyngeal (Geary et al., 1993; Brownlee et al., 1997) musculature of nematodes with exceptional potency. The molecular identity of the target for ivermectin has been pursued in the nonparasitic nematode Caenorhabditis elegans, as a model genetic animal. This has led to the identification of a family of genes that encode subunits for glutamate-gated chloride channels (Cully et al., 1994, 1996; Dent et al., 1997, 2000; Laughton et al., 1997a; Vassilatis et al., 1997). The family consists of at least two classes of subunit, a and b, that may coassemble to form either homomeric or heteromeric ligand-gated chloride channels. The properties of some of these have been determined in the Xenopus laevis oocyte expression system (summarized in Table 1). To date, only two of these genes have been shown to be expressed in C. elegans pharynx and may therefore contribute to the properties of the native channel that regulates feeding. avr-15, encodes for GluCl-a2, which in X. laevis oocytes forms a homomeric chloride-channel, gated by glutamate and high concentrations of ivermectin (10 mM). ad1051 is proposed to be a null allele of avr-15 (Dent et al., 1997). In the same study, behavioral analysis demonstrated that pharynxes dissected from these worms continue to pump in the presence of ivermectin and that the pharyngeal response to iontophoretic application of glutamate was abolished. The GluCl-b subunit is also present in pharynx (Laughton et al., 1997b). In X. laevis oocytes, this also forms a homomeric chloride-channel (Cully et al., 1994). However, in contrast to GluCl-a2, this channel is gated by glutamate, but not by