Abstract
The prevalence of human and animal helminth infections remains staggeringly high, thus urging the need for concerted efforts towards this area of research. GABA receptors, encoded by the unc-49 gene, mediate body muscle inhibition in Caenorhabditis elegans and parasitic nematodes and are targets of anthelmintic drugs. Thus, the characterization of nematode GABA receptors provides a foundation for rational anti-parasitic drug design. We therefore explored UNC-49 channels from C. elegans muscle cultured cells of the first larval stage at the electrophysiological and behavioral levels. Whole-cell recordings reveal that GABA, muscimol and the anthelmintic piperazine elicit macroscopic currents from UNC-49 receptors that decay in their sustained presence, indicating full desensitization. Single-channel recordings show that all drugs elicit openings of ∼2.5 pA (+100 mV), which appear either as brief isolated events or in short bursts. The comparison of the lowest concentration required for detectable channel opening, the frequency of openings and the amplitude of macroscopic currents suggest that piperazine is the least efficacious of the three drugs. Macroscopic and single-channel GABA-activated currents are profoundly and apparently irreversibly inhibited by ivermectin. To gain further insight into ivermectin action at C. elegans muscle, we analyzed its effect on single-channel activity of the levamisol-sensitive nicotinic receptor (L-AChR), the excitatory receptor involved in neuromuscular transmission. Ivermectin produces a profound inhibition of the frequency of channel opening without significant changes in channel properties. By revealing that ivermectin inhibits C. elegans muscle GABA and L-AChR receptors, our study adds two receptors to the already known ivermectin targets, thus contributing to the elucidation of its pleiotropic effects. Behavioral assays in worms show that ivermectin potentiates piperazine-induced paralysis, thus suggesting that their combination is a good strategy to overcome the increasing resistance of parasites, an issue of global concern for human and animal health.
Highlights
GABA-gated chloride channels, members of the Cys-loop receptor family, play an important inhibitory role in the nervous system of vertebrates [1] and invertebrates [2]
Confocal microscopy observation of cultures obtained from the EG1653 strain, which expresses GFP associated with GABA receptors [16], shows that 78610% of cultured cells with spindle-shaped morphology express GABA receptors, which appear to be homogeneously distributed on the cell membrane (Fig. 1B and C)
To verify that Larva 1 (L1) muscle cells express functional GABA receptors, we measured macroscopic currents elicited by GABA in the whole-cell configuration at 270 mV pipette potential
Summary
GABA-gated chloride channels, members of the Cys-loop receptor family, play an important inhibitory role in the nervous system of vertebrates [1] and invertebrates [2]. Defective GABA neurotransmission causes neurological disorders and GABAA receptors are targets for therapeutically important drugs, including benzodiazepines, barbiturates, and anesthetics [1,3,4]. In adult C. elegans, the GABA receptor has been shown to be composed of UNC-49B and C subunits [9]. The worms become resistant to muscimol, which is a full agonist of vertebrate GABAA receptors. This drug relaxes all body wall muscles and causes lengthening of adult worms [10,11]
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