Effects of lidocaine and DCJW on the recovery from fast inactivation of Na⁺ currents.

<p>The protocol was the same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067290#pone-0067290-g006" target="_blank">Figure 6D</a>. A. Time course of recovery from fast inactivation in the presence of 2 mM lidocaine B. Histogram bars summarizing the effects of 2 mM lidocaine on the rates of recovery from fast inactivation at 20 ms. Data were deduced from the curves shown in A (one-way ANOVA: F_(3,19) = 7.71, p<0.0021, post-hoc Tukey test). C. Time course of recovery from fast inactivation in presence of 2 µM DCJW.</p

Semi-quantitative RT-PCR analysis of PaTEH1 expression in various <i>P. americana</i> tissues.

<p>RT-PCR was performed using 5 µg of mRNA extracted from head, thoracic ganglia, nerve cord, muscles, gut and mushroom-shaped accessory gland. Actin (Genbank accession number AY116670) was used as internal quantitative control.</p

Parameters of voltage-dependence of activation and fast and slow steady-state inactivation for DmNa_v1.1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B or PaTEH1Δ(270-280) in <i>Xenopus</i> oocytes in the presence and absence of lidocaine and DCJW.

<p>Values are mean ± SEM derived from the number of individual experiments, each performed with a different oocyte. The number of recorded oocytes is indicated in parenthesis.</p

Modulation of Na⁺ current densities by different TEH1 auxiliary subunit subtypes.

<p>A. Family of Na⁺ currents measured at test potentials of −70 mV to 40 mV from a holding potential of −100 mV for DmNa_v1-1 alone (α) or co-expressed with DmTEH1 (α+DmTEH1), PaTEH1A (α+PaTEH1A), PaTEH1B (α+PaTEH1B) or PaTEH1Δ(270-280) (α+PaTEH1Δ(270-280)) injected with about 35 ng, 8 ng, 2 ng, 3 ng and 2 ng of RNA (α∶β, 1∶1), respectively. The protocol of depolarizing voltage-clamp steps is shown. B. Na⁺ current density per ng of injected RNA after 3-days incubation, except for DmNa_v1-1 (10 days after injection). Results are expressed in µA per nF per ng of injected RNA (One-way ANOVA: F_(4,55) = 15.11, p<0.0001 post hoc Tukey test). The number of tested oocytes is indicated in the bar histogram.</p

Organization of genomic region and mRNA and encoding TEH1-like subunit of <i>P. americana</i>.

<p>A. The genomic region of <i>Pateh1</i> consists in two exons interrupted by a short intron. The intron sequence is in lowercase letters and splice donor site, branch site and splice acceptor site are underlined. Sizes of exons and intron are indicated in parentheses. *: stop codon. B. Exclusion or retention of the intron led to PaTEH1A and PaTEH1B variants with different C-terminal ends. The 94 bp intron sequence contains a short new coding sequence followed by an in-frame stop codon. This generates a second protein (PaTEH1B) with a novel C-terminal end.</p

Modulation of Na_v channels expression by intron retention mechanism of <i>teh1</i>-<i>like</i> gene of <i>P. americana</i>.

<p>Immunofluorescent confocal images of representative oocytes injected with water (control) or with 3 ng of DmNa_v1.1/PaTEH1A (α+PaTEH1A) or PaTEH1B (α+PaTEH1B) RNAs. A. Immunostaining representative snapshot with an anti Pan-Na_v (Alexa®488) antibody and associated wireframe plots drawn with ImajeJ software (version 1.4.3.67). B. Immunostaining representative snapshot with an RGS-His (Alexa®546) antibody and associated wireframe plots drawn with ImajeJ software. C. Relative fluorescence was measured using the ImajeJ software (version 1.4.3.67). For each measurement, an identical region of interest of 5002.2 µm² (292×36 pixels) was defined. Mean relative fluorescence intensity values measured with Alexa®488 (white) and Alexa®546 (grey) are mean ± SEM for 6 oocytes. PaTEH1A shows a stronger expression of the α-subunit than PaTEH1B in oocytes (One-way ANOVA: Alexa®488: F_(2,16) = 24.86, p<0.0001 and Alexa®546: F_(2,15) = 39.81, p<0.0001, post hoc Tukey test). a.u: arbitrary unit.</p

Primary structure analysis of TEH1-like auxiliary subunits of <i>P. americana</i>.

<p>A. Clustal W alignment of PaTEH1A (GenBank accession number KC206367), PaTEH1B (accession number KC206368) and DmTEH1 (accession number NP_649959). Transmembrane segments (M1 and M2) are indicated with bold line above the sequences. Conserved N-glycosylation sites are indicated by closed inverted triangles (▾) and O-glycosylation sites of PaTEH1s subunits are indicated by asterisk (*) (<a href="http://www.cbs.dtu.dk" target="_blank">www.cbs.dtu.dk</a>). Gaps are indicated by dashes. The undecapeptide (VALLDCEEDRT) and the decapeptide (YVPLSVHDTR) at the C-terminal ends of PaTEH1 variants are boxed. B. Hydrophobicity profile and deduced topological organization of PaTEH1A and PaTEH1B. Hydrophobicity analysis was performed using the algorithm of Kyte and Doolittle (1982). The amino acid residue position is plotted along the x-axis and the calculated mean hydrophobicity is plotted along the y-axis. Regions above the line are hydrophobic. The two putative membrane-spanning segments are indicated as M1 and M2.</p

Biophysical properties of Na⁺ currents elicited by co-expression of DmNa_v1-1 with DmTEH1 or PaTEH1-like subunits.

<p>A. Voltage-dependence of activation (One-way-ANOVA: F_(3,65) = 6.67, p<0.0005, post-hoc Tukey test). G represents the conductance. B. Voltage dependence of fast steady-state inactivation (One-way-ANOVA: F_(3,75) = 6.84, p = 0.0009, post-hoc Tukey test). C. Voltage-dependence of slow steady-state inactivation (One-way-ANOVA: F_(3,23) = 4.49, p = 0.014, post-hoc Tukey test). D. Recovery from fast inactivation (One-way-ANOVA: F_(3,62) = 2.104, p = 0.1098, post-hoc Tukey test). Na⁺ current amplitudes (I) were measured using the pulse protocols described in the Materials and Methods section and were normalized to the largest current amplitude (Imax). Values are mean ± SEM. The number of individual experiments, each performed with a different oocyte, is indicated in parentheses. Standard protocols are shown in insets.</p

Sequences of the oligonucleotides used in PCR and their corresponding region.

<p>Designation of oligonucleotide mixtures: S = G+C; Y = C+T; M = A+C.</p><p>UTR, untranslated region; ORF, open-reading frame.</p><p>Restriction enzyme recognition sequences are underlined and correspond to <i>Xma</i>I site (CCCGGG) and <i>Xba</i>I site (TCTAGA).</p

Susceptibility of AbDhn-deficient mutants to temperature stress.

<p>Calibrated water suspensions of conidia from the wild-type (WT) strain <i>Abra43</i> and AbDhn-deficient mutants were left for 10 h at various temperatures (−20°C, +4°C, +20°C, +40°C). Conidia were then used to inoculate microplate wells and nephelometric growth curves were established over a 33 h period. ΔLag time was calculated as the difference between the lag time at the tested temperature and the lag time at 20°C and was used as a parameter to estimate the effect of the treatment on spore viability. Error bars indicate standard deviations and asterisks indicate values that are significantly (<i>P</i><0.01) higher than that of the wild-type. Each genotype was analysed in triplicate and the experiments were repeated twice times per growth condition.</p