SLO-1-Channels of Parasitic Nematodes Reconstitute Locomotor Behaviour and Emodepside Sensitivity in Caenorhabditis elegans slo-1 Loss of Function Mutants

Abstract

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5′ flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.

Ion Channels and Drug Transporters as Targets for Anthelmintics

“…slo-1 gain-of-function mutations phenocopy the effects of emodepside and slo-1 null mutants are rescued by transformation with wild type slo-1 [77]. Orthologues of slo-1 from parasitic nematodes also restore emodepside sensitivity to the C. elegans loss-of-function mutants [78]. In contrast, a human orthologue of slo-1 does not confer emodepside sensitivity to these mutants, though it does rescue some of the behavioral deficiencies associated with the absence of the channel [79].…”

“…The current view appears to be that emodepside can act either directly or indirectly on SLO-1 channels. In the pharynx, emodepside apparently acts upstream of the SLO-1 channel, likely via the latrophilin receptor, to modulate SLO-1 activity, perhaps via phosphorylation, while the latrophilin receptor is not involved in emodepside activity on body wall musculature [78]. …”

Neurobiology of plant parasitic nematodes

“…Given that J2 stage plant parasitic nematodes have to locate their host plant using sensory mechanisms, it will likely be helpful to use this information as a platform to inform functional studies on these pests. There is a strong precedent for this 'model hopping' approach in veterinary medicine where it has made a major contribution to understanding the mode of action of novel anthelmintics and highlighting new anthelmintic targets (Cully et al, 1994;Kaminsky et al 2008;Welz et al 2011). Thus, there is the promise that similar approaches may yield insight into plant parasitic nematodes and their control.…”

Ion channels and receptor as targets for the control of parasitic nematodes

“…To date, only one anthelmintic, emodepside, has been shown to require a voltage-gated ion channel for its activity (Guest et al, 2007;Welz et al, 2011). Emodepside inhibits locomotion, feeding, egg-laying and slows development of C. elegans and parasitic worms Willson et al, 2003;Bull et al, 2007;Welz et al, 2011), and these multiple effects may be due to activation of more than one target.…”

“…Emodepside inhibits locomotion, feeding, egg-laying and slows development of C. elegans and parasitic worms Willson et al, 2003;Bull et al, 2007;Welz et al, 2011), and these multiple effects may be due to activation of more than one target. Following a screen to identify C. elegans mutants resistant to emodepside, it was found that worms with loss-of-function mutations in slo-1, a gene that encodes a voltage-and calcium-activated potassium, or BK, channel (Atkinson et al, 1991), are resistant to emodepside inhibition of locomotion and pharyngeal pumping whereas gain-of-function mutants in the same gene phenocopy the action of the drug (Guest et al, 2007).…”