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Figure 2. Organization of genomic region and mRNA and encoding TEH1-like subunit of P. americana.A. The genomic region of Pateh1 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.
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Figure 3. Semi-quantitative RT-PCR analysis of PaTEH1 expression in various P. americana tissues.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.
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Figure 4. Modulation of Na+ current densities by different TEH1 auxiliary subunit subtypes.A. Family of Na+ currents measured at test potentials of −70 mV to 40 mV from a holding potential of −100 mV for DmNav1-1 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 DmNav1-1 (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.
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Figure 5. Modulation of Nav channels expression by intron retention mechanism of teh1-like gene of P. americana.Immunofluorescent confocal images of representative oocytes injected with water (control) or with 3 ng of DmNav1.1/PaTEH1A (α+PaTEH1A) or PaTEH1B (α+PaTEH1B) RNAs. A. Immunostaining representative snapshot with an anti Pan-Nav (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.
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Figure 6. Biophysical properties of Na+ currents elicited by co-expression of DmNav1-1 with DmTEH1 or PaTEH1-like subunits.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.
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Figure 7. TEH1 isoforms-mediated changes in the sensitivity of DmNav1-1 to Na+ channel inhibitors.A. Na+ current traces obtained by a 20 ms depolarizing pulse to −10 mV from a holding potential of −100 mV, in the absence (control, grey trace) and in the presence of 2 mM lidocaine at 10 min (black trace). B. Percentage of tonic inhibition of Na+ current induced by lidocaine (2 mM, 10 min) for DmNav1-1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B and PaTEH1Δ(270-280) (one-way ANOVA: F(3,18) = 4.85, p<0.0121, post-hoc Tukey test). C. Na+ current traces obtained by a 20 ms depolarizing pulse to −10 mV from a holding potential of −100 mV, in the absence (control, grey trace) and in the presence of 2 µM DCJW at 30 min (black trace). D. Percentage of inhibition of Na+ current in response to DCJW (2 µM, 30 min) for DmNav1-1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B and PaTEH1Δ(270-280) (one-way ANOVA: F(3,22) = 13.22, p<0.0001, post-hoc Tukey test). Values are mean ± SEM. a.u: arbitrary unit. The number of tested oocytes is indicated in the bar histogram.
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Figure 8. Effects of lidocaine and DCJW on gating properties of Na+ currents.Electrophysiological properties of DmNav1-1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B and PaTEH1Δ(270-280) in the absence (white) and the presence of 2 mM lidocaine (black) and 2 µM DCJW (grey). A. Mean data with SEM for V1/2 of activation. B. Mean data with SEM for V1/2 of fast steady-state inactivation. C. Mean data with SEM for V1/2 of slow steady-state inactivation. D. Summary data for recovery time constant from fast inactivation. Statistical tests were performed using a two-tail unpaired t test (control versus lidocaine and control versus DCJW). The number of tested oocytes is indicated in the bar histogram.
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Figure 9. Effects of lidocaine and DCJW on the recovery from fast inactivation of Na+ currents.The protocol was the same as shown in Figure 6D. 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.
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Figure 1. Primary structure analysis of TEH1-like auxiliary subunits of P. americana.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 (*) (www.cbs.dtu.dk). 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.
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