Date Published: December 18, 2014
Publisher: Public Library of Science
Author(s): Daniel Kulke, Georg von Samson-Himmelstjerna, Sandra M. Miltsch, Adrian J. Wolstenholme, Aaron R. Jex, Robin B. Gasser, Cristina Ballesteros, Timothy G. Geary, Jennifer Keiser, Simon Townson, Achim Harder, Jürgen Krücken, David L. Williams. http://doi.org/10.1371/journal.pntd.0003401
Abstract: The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class.
Partial Text: In Ecdysozoa, large conductance potassium channels (also BK or Maxi-K channels) are encoded by slo-1 genes . Due to their large conductance in the open state, typically exceeding 200 pS, Slo-1 channels are of major importance for repolarization of excitable cells. These channels are tetramers and, in almost every animal species, all subunits are encoded by a single gene – with teleost fish as the only known exception, encoding multiple slo-1 paralogs resulting from whole genome duplications . The opening of Slo-1 channels is controlled by the membrane potential and intracellular free Ca2+-concentrations [Ca2+]i. Depolarization of the membrane as well as very high transient local Ca2+ concentrations are required to open Slo-1 channels . Most other ion channels produce subtypes with altered physiological properties through a combination of different subunits as recently reviewed for acetylcholine and glutamate receptors , . In contrast, several different slo-1 splice variants have been described, e.g. for C. elegans, D. melanogaster, mice and humans, and differential splicing is known to affect channel properties –. In addition, the use of different tissue-specific promoters has been described for D. melanogaster, . Indeed, the number of different splice variants has dramatically increased during evolution, which is demonstrated by the fact that the human slo-1 ortholog kcnma1 encodes 13 alternative exons . In fact, the number of splice variants in Mammalia and Diptera is so high that systematic analysis of the effects of the channel variants generated by alternative splicing on voltage- and Ca2+-sensitivity has only been performed for a few of them. The complexity of Slo-1 channel heterogeneity is further increased by the ability to form heterotetramers  containing different splice variants and the fact that channel responses to depolarization and Ca2+ are modulated by posttranslational modifications such as palmitoylation and phosphorylation at several distinct sites –.
The mode of action of cyclooctadepsipeptides has long been a matter of debate, with ionotropic GABAA receptors , , the latrophilin receptor Lat-1 ,  and the Slo-1 channel - consecutively deemed to be the most likely receptors. Although Slo-1 is generally considered to be the most important target based on experiments using forward and reverse genetics, there has been no direct evidence for emodepside activation of the Slo-1 channel. Responses of excitable muscle/neuronal cells to emodepside develop much slower ,  than usually observed during direct gating of ionotropic receptors, which meant that indirect effects of emodepside on Slo-1 could never be completely excluded . Using an oocyte expression system eliminating all nematode-related confounding factors, the present study for the first time demonstrates direct effects of emodepside on Slo-1 channels. In the presence of emodepside, highly increased currents were observed without depolarization up to a threshold of 0 mV and without any additional stimuli to artificially increase [Ca2+]i levels. These novel findings confirm that Slo-1 is a direct target of emodepside.