Characterization of the Ca2+-gated and voltage-dependent k+-channel slo-1 of nematodes and its interaction with emodepside

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

Relative abundance of cDNA amplicons encoding full-length or truncated <i>Trichuris muris</i> Slo-1 channels.

<p>Relative abundance of cDNA amplicons encoding full-length or truncated <i>Trichuris muris</i> Slo-1 channels.</p

Phylogenetic analysis of nematode Slo-1 channels.

<p><b>A</b>) Phylogram obtained by maximum likelihood analysis from full-length Slo-1 channels. Slo-1 protein sequences from the nematode species <i>Caenorhabditis elegans</i> (<i>Cel</i>), <i>Caenorhabditis briggsae</i> (<i>Cbr</i>), <i>Caenorhabditis remanei</i> (<i>Cre</i>), <i>Pristionchus pacificus</i> (<i>Pca</i>), <i>Haemonchus contortus</i> (<i>Hco</i>), <i>Cooperia oncophora</i> (<i>Con</i>), <i>Ancylostoma caninum</i> (<i>Acan</i>), <i>Onchocerca gutturosa</i> (<i>Ogu</i>), <i>Brugia malayi</i> (<i>Bma</i>), <i>Dirofilaria immitis</i> (<i>Dim</i>), <i>Toxocara canis</i> (<i>Tca</i>), <i>Parascaris equorum</i> (<i>Peq</i>), <i>Ascaris suum</i> (<i>Asu</i>), <i>Meloidogyne incognita</i> (<i>Min</i>), <i>Strongyloides ratti</i> (<i>Sra</i>), <i>Trichuris muris</i> (<i>Tmu</i>) and <i>Trichuris suis</i> (<i>Tsu</i>) were aligned together with orthologs from <i>Drosophila melanogaster</i> (<i>Dme</i>), <i>Anopheles gambiae</i> (<i>Aga</i>), <i>Pediculus humanus corporis</i> (<i>Phu</i>), <i>Daphnia pulex (Dpu)</i>, <i>Aplysia callifornica</i> (<i>Acal</i>), <i>Gallus gallus</i> (<i>Gal</i>), <i>Bos taurus</i> (<i>Bta</i>) and <i>Homo sapiens</i> (<i>Hsa</i>), which were used as outgroup, using ClustalX2. For <i>B. malayi</i> only the experimentally identified splice variant Slo-1f and for <i>C. elegans</i> 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>B</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 <i>Bma</i>Slo-1h and in the genome sequences of <i>Onchocerca volvulus</i> (<i>Ovo</i>Slo-1 and <i>Ovo</i>Slo-1 alt. exon) and <i>A. suum</i> (<i>Asu</i>Slo-1 alt. exon) were included. Parameters were identical to those used to calculate the tree from full-length sequences.</p

Estimation of splice variant abundances encoding truncated or full-length versions of <i>Trichuris muris</i> Slo-1.1.

<p><b>A</b>) Primers flanking the introns retained in the cDNAs encoding the truncated proteins (<i>Tmu</i>Slo-1.1c and <i>Tmu</i>Slo-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>B</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.</p

Frequencies of splice variants in terms of fragments per kilobase of exons per million fragments mapped (FKPM).

<p>Libraries were compared according to developmental stage (<b>A</b>) and (<b>C</b>) as well as different sexes or tissues (<b>B</b> and <b>D</b>). 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. <b>A</b>) and <b>B</b>) show expression patterns of <i>Tsu</i>slo-1.1 and <b>C</b>) and <b>D</b>) indicate patterns for <i>Tsu</i>slo-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.</p