R1432G mutant alters WT channel localization to the cell membrane in the presence of β₁-subunit.

<p><b>A.</b> Confocal imaging of Na channels in cells co-expressing R1432G mutant and Na_v1.5/WT-FLAG or expressing Na_v1.5/WT-FLAG alone. Immunocytochemistry with anti-FLAG antibodies was performed first on fixed non-permeabilized cells to detect surface FLAG-tagged WT channels (green, left panels). The same cells were then permeabilized to label total mutant and WT Na_v1.5 proteins with anti-Na_v1.5 antibodies (red, middle panels). Light transmission imaging of the HEK293T cells are presented on the right panels. Scale bar, 10 µm. <b>B.</b> Quantification by luminometry of FLAG-tagged Na_v1.5/WT channels in the presence (gray bars) or the absence (white bars) of R1432G subunits in HEK293T cells. Surface and total amount of WT-FLAG channels was measured from non-permeabilized (solid bars) and permeabilized (hatched bars) cells respectively. Data are presented as relative light unit (RLU) normalized to the total WT-FLAG/(-) condition. Data are expressed as mean ± SEM of six independent experiments. *, P<0.05, indicates significant difference with surface WT-FLAG/( ) condition (<i>t</i>-test). All experiments were realized in the presence of the β₁-subunit.</p

Functional impact of R1432G mutant co-expression upon WT sodium channels in the presence of β₁-subunit.

<p><b>A.</b> Representative whole-cell sodium current recordings from HEK293T cells expressing WT and/or R1432G Na_v1.5 channels and the β₁ auxiliary subunit. Current traces are presented as membrane current density. The inset shows the voltage-clamp protocol. <b>B.</b> Current-voltage relationships of Na_v1.5 currents measured from 7–11 cells. <b>C.</b> Average peak current densities of WT and/or R1432G channels at -40 mV. **, P<0.01 <i>versus</i> WT/R1432G (Student <i>t</i>-test). <b>D.</b> Voltage-dependence of activation (open symbols, n = 11) and inactivation (filled symbols, n = 7–9) of Na_v1.5 channels. Inactivation and activation data were measured with standard pulse protocols shown in inset and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048690#pone-0048690-g001" target="_blank">Figure 1A</a> respectively. Activation and inactivation curves were generated and fitted with a Boltzmann equation to obtain biophysical parameters summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048690#pone-0048690-t001" target="_blank">Table 1</a>.</p

Gating parameters of WT and R1432G Na_v1.5 channels expressed in HEK293T cells.

<p>Abbreviations are: <i>n</i>, number of cells per group; <i>k</i>, slope factor of voltage dependence of (in)activation (mV) and V_1/2, voltage of half-maximal (in)activation (mV). Statistically significant results were determined using one-way analysis of variance (ANOVA) with Bonferroni's post hoc tests. ^*, P<0.05; ^**, P<0.01 (<i>vs</i> WT/(-) + β₁); ^†, P<0.05; ^††, P<0.01 (<i>vs</i> WT/R1432G + β₁).</p

Abolition of R1432G dominant-negative effect in the absence of the β₁-subunit.

<p><b>A.</b> IV curves of Na_v1.5 currents recorded from HEK293T cells transfected with WT and/or R1432G channels without β₁-subunit (n = 9–10). <b>B.</b> Average peak current densities WT and/or R1432G channels at -40 mV. n.s., p>0.05 <i>versus</i> WT/R1432G condition (Student <i>t</i>-test). <b>C.</b> Voltage-dependence of activation (open symbols, n = 9–10) and inactivation (filled symbols, n = 7) of WT and/or R1432G Na_v1.5 expressed in HEK293T without β₁. Inactivation and activation data were obtained using standard pulse protocols shown in inset and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048690#pone-0048690-g001" target="_blank">Figure 1A</a> respectively. (In)activation parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048690#pone-0048690-t001" target="_blank">Table 1</a>. <b>D.</b> Quantification by luminometric ELISA assay of FLAG-tagged Na_v1.5/WT channels alone (white bars, n = 6) or with R1432G (grey bars, n = 6) in the absence of β₁-subunit. The bar chart shows surface and total amount of WT-FLAG channels from nonpermeabilized cells (solid bars) and permeabilized cells (hatched bars) respectively in the absence of β₁-subunit. Data are presented as relative light unit (RLU) normalized to the total WT-FLAG/(-) condition and are expressed as mean ± SEM. n.s. indicates no significant difference with surface WT-FLAG/(-) condition (<i>t</i>-test).</p

Na_v1.5 α-α subunits interaction is dependent on the auxiliary β₁-subunit.

<p>HEK293T cells were transfected with FLAG-tagged R1432G and/or GFP-tagged WT channels in the absence (bottom panel) or the presence (top panel) of β₁-subunit. Immunoprecipitations were performed with anti-FLAG M2 antibodies. Equal amounts of total lysates (15 µg) and immunoprecipitated proteins were immunoblotted with anti-pan-Na_v and anti-GFP antibodies to respectively detect both tagged forms or GFP-WT channels. Negative and positive controls are shown in the right panel. (WB: Western Blot; IP: immunoprecipitation).</p