Conservation of residues involved in drug binding in <i>Caligus</i> and <i>C. elegans</i> GluClα.

<p>Residues involved in hydrogen-bonding (green) and van der Waals (grey) interactions of ivermectin or emamectin with CrGluClα (CrGluClα-IVM, CrGluClα-EMA, see text) are compared with those identified in the CeGluClα-ivermectin complex (CeGluClα-IVM) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004402#ppat.1004402-Hibbs1" target="_blank">[16]</a>. Only those interactions existing during >70 ns of the 140 ns MD runs are shown. Residues highlighted in red are those apparently involved in creating a hydrophobic seal in the channel pore in the absence of agonists <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004402#ppat.1004402-Cheng1" target="_blank">[31]</a>.</p

Morphology and body weights of <i>Kcnj13</i> null mutant mice.

<p>a. Analysis of Kir7.1 expression in WT, heterozygous and null mutant mice; cyclophilin A (Cyc1) is used as constitutively expressed control gene. b. Gross morphology of WT, and heterozygous and homozygous <i>Kcnj13</i> null mutant newborn pups. c. Body weight vs. embryonic stage for WT (circles), <i>Kcnj13</i>^<i>+/-</i> (triangles), and <i>Kcnj13</i>^-/- (squares) embryos. Results are expressed as mean ± S.E.M. of the following numbers of embryos: 12.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 9, <i>Kcnj13</i>^-/- 4; 13.5 dpc: WT 9, <i>Kcnj13</i>^<i>+/-</i> 14, <i>Kcnj13</i>^-/- 2; n 14.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 11, <i>Kcnj13</i>^-/- 6; 15.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 20, <i>Kcnj13</i>^-/- 14; 16.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 4; 17.5 dpc:WT 5, <i>Kcnj13</i>^<i>+/-</i> 12, <i>Kcnj13</i>^-/- 6; 18.5 dpc: WT 4, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 6; P0: WT 7, <i>Kcnj13</i>^<i>+/-</i> 15, <i>Kcnj13</i>^-/- 10. * p< 0.001; ** p <0.05 for the differences between <i>Kcnj13</i>^-/- and <i>Kcnj13</i>^<i>+/+</i> data (ANOVA).</p

A. Channel pore radius along the z-axis in the receptor alone (black) and in the CrGluClα-emamectin (green) and CrGluClα-ivermectin (red) systems.

<p>A length, mainly intramembrane, of the pore is shown. The discontinuous lines show the pore segment lined by M2 α-helices. Values have been taken at the end of MD trajectories. B and C. Lateral views of the pore at time zero (C) and at the end (B) of the MD trajectory for CrGluClα in absence of drugs. Only four M2 helices are shown with the fifth removed for clarity. Residues P288, A292 and L299 are shown in licorice. Water occupancy is shown as licorice and surface.</p

Basolateral expression of Kir7.1 channel in the epithelium of the airways.

<p>Immunohistochemical detection of Kir7.1 channel in trachea (left) and bronchiole (right) in adult <i>Kcnj13</i>^+/+, newborn <i>Kcnj13</i>^+/+ or newborn <i>Kcnj13</i>^-/- mice. Tissue sections were treated with anti-Kir7.1 antibody (1:15,000). Kir7.1 expression was restricted to the basolateral membrane of airway epithelium in adult and newborn <i>Kcnj13</i>^+/+ mice. Staining in <i>Kcnj13</i>^-/- tissues shows complete absence of specific immunoreactive signal. Nuclei were counterstained with Fast Red. Scale bar represents 50 μm.</p

Pulmonary abnormalities in embryonic lungs from <i>Kcnj13</i>^-/- mice.

<p>a. Hematoxylin and eosin stained lung sections taken at various gestational stages as indicated. Morphological differences in KO lungs were observed at E18.5 and P0. Null mutant mice show a lower air space and thicker walls at lung terminal sacs compared to WT and heterozygous mice. No differences were visible between <i>Kcnj13</i>^+/+ and <i>Kcnj13</i>^+/- genotypes. Scale bars represent 100 μm. b. Morphometric analysis of terminal sac spaces in lungs at various gestational stages. Significant reduction in spaces was observed in Kir7.1 deficient mice from E18.5 onwards. Results are expressed as mean ± S.E.M, # p<0.05 and * p<0.01 for the difference with WT by ANOVA. c. Graphical representation of newborn lung flotation test. Grey sections of columns correspond to percent of floating lungs, with black being the percent sinking lungs.</p

Variations in deduced CrGluClα primary structure.

<p>The rooted phylogenetic tree was done by using the UPGMA method after aligning twelve representative clones with CLUSTALW software (<a href="http://www.genome.jp/tools/clustalw" target="_blank">http://www.genome.jp/tools/clustalw</a>). The positions of amino acid changes (V27I, D73G, R387K and L411Q), or deletions (isoleucine 20 (I or Δ), and alanine 376 plus serine 377 (AS or Δ)) are shown. The clone name is composed by CrGluClα, source and Arabic number. Source: Valdivia (Vald), Darwin (Dw) and Errázuriz (Err). Arabic number is the number assigned during the cloning process.</p

Survival of <i>Kcnj13</i>^- mutant mice.

<p>Survival of <i>Kcnj13</i>^- mutant mice.</p

Effect of ivermectin or emamectin upon CrGluClα-T318A-dependent currents.

<p>A and B. Effect of increasing concentrations of emamectin and ivermectin on WT CrGluClα-dependent currents. The currents were first activated by addition of 50 µM glutamate and during continuous exposure to glutamate increasing concentrations of emamemctin or ivermectin were added. Reduction of [Cl⁻] to 7.6 mM or addition of 100 mM PTX are also indicated. C and D. Similar experiments performed using the CrGluClα-T318A mutant. E and F. Concentration dependence of the effects of avermectins on WT and T318A mutant CrGluClα receptors. Normalised average responses to emamectin and ivermectin are shown (means±SEM, n = 6 for all experiments shown). Fits of Hill decay functions to the average values are shown by the solid lines.</p

Energy and affinity values for the best pose of ivermectin and emamectin in docking assays with CrGluClα molecular model.

<p>Energy and affinity values for the best pose of ivermectin and emamectin in docking assays with CrGluClα molecular model.</p

Irreversible activation by emamectin of CrGluClα receptor expressed in <i>Xenopus</i> oocytes.

<p>A. Current trace obtained at 60 mV during bath application of emamectin at concentrations going from 5 nM to 10 µM during the times shown in the boxes. The effect of picrotoxin addition at 100 µM is also shown immediately after removal of emamectin. Wash out of picrotoxin in the absence of emamectin returns current to the levels attained in the presence of high concentration of emamectin. B: Current-voltage relations taken from voltage-ramps applied during experiment in A. Control denotes current prior to emamectin addition. C. Dose-response relationship of the emamectin-sensitive currents. Data are normalized (mean ± SEM) to the maximal effect of emamectin and originate in seven separate experiments. The line is a Hill equation fitted simultaneously to measurements taken at −80 and 60 mV giving a value for EC₅₀ of 202±21 nM and n_H 1.1±0.11.</p