Kulke D et al. (2014), Characterization of the Ca2+-gated and voltage-...

XB-ART-50423

PLoS Negl Trop Dis 2014 Dec 18;812:e3401. doi: 10.1371/journal.pntd.0003401.

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Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

Kulke D , von Samson-Himmelstjerna G , Miltsch SM , Wolstenholme AJ , Jex AR , Gasser RB , Ballesteros C , Geary TG , Keiser J , Townson S , Harder A , Krücken J .

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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.

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Species referenced: Xenopus laevis
Genes referenced: acan aga kcnma1 lat tirap

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	Figure 2. Frequencies of splice variants in terms of fragments per kilobase of exons per million fragments mapped (FKPM).Libraries were compared according to developmental stage (A) and (C) as well as different sexes or tissues (B and D). Splice variants were grouped according to the encoded protein, i.e. frequencies of splice variants encoding the same protein and differing in sequence downstream of the stop codon were added together. A) and B) show expression patterns of Tsuslo-1.1 and C) and D) indicate patterns for Tsuslo-1.2 splice variants. L1, L2, L3, L4, first, second, third, fourth stage larvae; Mal., males, Mal. post., posterior part of males; Fem., Females; Fem. post., posterior part of females; Stich., stichosome.
	Figure 3. Phylogenetic analysis of nematode Slo-1 channels.A) Phylogram obtained by maximum likelihood analysis from full-length Slo-1 channels. Slo-1 protein sequences from the nematode species Caenorhabditis elegans (Cel), Caenorhabditis briggsae (Cbr), Caenorhabditis remanei (Cre), Pristionchus pacificus (Pca), Haemonchus contortus (Hco), Cooperia oncophora (Con), Ancylostoma caninum (Acan), Onchocerca gutturosa (Ogu), Brugia malayi (Bma), Dirofilaria immitis (Dim), Toxocara canis (Tca), Parascaris equorum (Peq), Ascaris suum (Asu), Meloidogyne incognita (Min), Strongyloides ratti (Sra), Trichuris muris (Tmu) and Trichuris suis (Tsu) were aligned together with orthologs from Drosophila melanogaster (Dme), Anopheles gambiae (Aga), Pediculus humanus corporis (Phu), Daphnia pulex (Dpu), Aplysia callifornica (Acal), Gallus gallus (Gal), Bos taurus (Bta) and Homo sapiens (Hsa), which were used as outgroup, using ClustalX2. For B. malayi only the experimentally identified splice variant Slo-1f and for C. elegans only the variants Slo-1a-c were included. The JTT model of amino acid substitutions was used and PhyML was set to optimize the number of invariable sites while amino acid frequencies were based on the model. The number of Γ distributed substitution rate categories was set to 16 and PhyML optimized the Γ shape parameter. Support for individual nodes was calculated using the Shimodaira-Hasegawa modification and a Bayesian transformation of the approximate likelihood ratio test and results are shown close to the nodes before and after the slash, respectively. For those cases where support values were not shown next to the node they are shown on the right and refer to the most terminal node on the same vertical position. The scale bar represents 0.2 substitutions per site. C, Crustacea; G, Gastropoda; Vert, Vertebrata; Arthropod, Arthropoda, M, Mollusca; L, Lophotrophora; Deut, Deuerostomia. B) Phylogenetic tree calculated on an alignment of the conserved alternative exons from all Ecdysozoa included in the tree in A). In addition, four alternative exons identified in BmaSlo-1h and in the genome sequences of Onchocerca volvulus (OvoSlo-1 and OvoSlo-1 alt. exon) and A. suum (AsuSlo-1 alt. exon) were included. Parameters were identical to those used to calculate the tree from full-length sequences.
	Figure 4. Currents observed in voltage-clamp experiments.Representative currents determined in oocytes injected with water (A), Celslo-1a cRNA in the absence of emodepside (B) and Celslo-1a cRNA in the presence of 10 µM emodepside (C).
	Figure 5. Determination of emodepside effects on current voltage curves in Xenopus laevis oocytes injected with Celslo-1a cRNA.A) Current voltage curves (IVCs) (means ± SEM) were recorded in water-injected oocytes without addition of drugs (basal), after addition of the vehicle (0.1% DMSO, 0.003% Pluronic F-68) and after addition of 10 µM emodepside (emo) (n = 10). B) Currents obtained from oocytes injected with water (n = 10) or Celslo-1a cRNA in the absence of any drug (basal, n = 8) or in the presence of vehicle (0.1% DMSO, 0.003% Pluronic F-68, n = 8). The asterisk highlights a significant difference of both curves obtained from Celslo-1a cRNA injected groups of oocytes to the water-injected oocytes. C) Currents were recorded in Celslo-1a injected oocytes at the presence of vehicle (0.1% DMSO, 0.003% Pluronic F-68), or emodepside (1 µM or 10 µM emo) (n = 10). For 10 µM emodepside the upper asterisks symbolize the comparison to 1 µM emodepside and the lower the comparison to the vehicle control. The asterisks at the 1 µM emodepside curve indicate significant differences to the control. D) Oocytes injected with Celslo-1a were incubated with 1 µM (n = 6) or 10 µM (n = 7) emodepside (emo) before recording the IVC curves. Then, oocytes were perfused with normal frog ringer for 5 or 10 min, respectively, before a second IVC was recorded from the same oocytes (n = 7). E) Oocytes were preincubated in the absence of drugs (basal) or with 1 µM verruculogen (verruc) before IVCs were recorded. Then, oocytes were perfused for 2 min before 1 µM emodepside (emo) was added (n = 8). F) Oocytes injected with water (n = 10) or with Celslo-1 cRNA (n = 6) were incubated in the absence of drugs (basal) or with 1 µM emodepside before IVCs were recorded. After perfusion with normal frog ringer for 2 min, verruculogen (verruc) was added for 2 min followed by recording of IVCs., p<0.05; , p<0.01; **, p<0.001.
	Figure 1. Estimation of splice variant abundances encoding truncated or full-length versions of Trichuris muris Slo-1.1. A) Primers flanking the introns retained in the cDNAs encoding the truncated proteins (TmuSlo-1.1c and TmuSlo-1.1d) were used in RT-PCR (lanes 1, 2, 5, and 6) and genomic PCR (lanes 4 and 8). Template was either derived from the Monheim (lanes 1–4) or the Edinburgh Zoo (lanes 5–8) isolate. Lanes 3 and 7 show no reverse transcription controls. M, 100 bp ladder. B) Representative samples separated on the Bioanalyzer. The upper panel shows a genomic PCR product, the middle panel the control without reverse transcription and the bottom panel the RT-PCR.

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