Characterization of Glutamate-Gated Chloride Channels in the Pharynx of Wild-Type and MutantCaenorhabditis elegansDelineates the Role of the Subunit GluCl-α2 in the Function of the Native Receptor

Abstract

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 (EC(50) = 166 microM), only weakly antagonized by picrotoxin. The order of potency of agonists was ibotenate > L-glutamate > kainate = quisqualate. Ivermectin potently and irreversibly depolarized the muscle (EC(50) = 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-alpha2 subunit. However, a chloride-dependent, nondesensitizing response to glutamate persisted. Therefore, the GluCl-alpha2 subunit confers ivermectin sensitivity and a high-affinity desensitizing glutamate response on the native pharyngeal GluCl receptor.

The actions of chloride channel blockers, barbiturates and a benzodiazepine on Caenorhabditis elegans glutamate- and ivermectin-gated chloride channel subunits expressed in Xenopus oocytes

“…Flunitrazepam-binding sites have been identified in the central nervous system of insects but interestingly the most potent ligand at displacing this binding, Ro5-4864, is selective for the vertebrate peripheral benzodiazepine-binding site (Lummis 1990). However, in the present study, the benzodiazepine, flurazepam, failed to modulate C. elegans GluCla1b subunits expressed in oocytes or the native GluCl receptors in the pharynx (Pemberton et al 2001). It may be that the glutamate-gated chloride channels in nematodes do not possess benzodiazepine modulatory binding sites.…”

“…Amobarbital which was potent against glutamate on the heteromer in this study was inactive against the native glutamate-gated chloride channel in the pharynx of C. elegans (Pemberton et al 2001), perhaps reflecting a different subunit stoichiometry in this tissue e.g., the presence of an avr-15 subunit rather than glc-1. In contrast, pentobarbital, 100 lM, enhanced both the amplitude and duration of the GABA responses of cultured locust neurones by up to 70% (Lees et al 1987).…”

“…Etter et al concluded that picrotoxin blocked GluCl channels by binding to a site accessible when the channel was open. Picrotoxin weakly antagonised the action of glutamate on the native receptor in the pharynx of C. elegans, for example, 100 lM picrotoxin only reduced the response to 50 lM glutamate by 25% (Pemberton et al 2001). Using the heteromer expressed in Xenopus oocytes, 50 lM picrotoxin reduced the response to 300 lM glutamate by 60% (Etter et al 1999).…”

“…Other chloride channels are resistant to both NPPB and DNDS, e.g., the cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium (Copello et al 1993). Another arylaminobenzoate, flufenamic acid, 200 lM, reduced the response to 1 lM ivermectin on GluCla1b subunits expressed in oocytes by 60% (Cully et al 1994) but had no effect when applied at 100 lM on the response to 50 lM glutamate on native glutamate-gated chloride channels in C. elegans pharynx (Pemberton et al 2001).…”

Comparative pharmacology and computational modelling yield insights into allosteric modulation of human α7 nicotinic acetylcholine receptors

“…In addition to nematode acetylcholine receptors, these targets also include P2X receptors of Schistosoma [32] and of mouse ( [33]) and histamine-gated chloride channels of Drosophila [34], as well as nematode [35][36][37][38] and insect [39,40] glutamate-gated chloride channels, GABA receptors from nematodes to vertebrates [41][42][43][44] and human glycine receptors [45]. There is also evidence for an action of IVM on an intracellular ligand-gated ion channel, the ryanodine receptor [46].…”