Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita

Figures

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  FIGURE 1. Acute application of NFA on WT, A531V, and V947E ClC-1 chloride channels. (A). Representative traces of WT, A531V, and V947E chloride currents at −90 and +60 mV before (in black) and after (in red) the application of 50 µM NFA. (B). Percentage of block of instantaneous and steady-state chloride current for WT, A531V, and V947E mutants measured at −90 mV and +60 mV induced by NFA 50 µM. (C). Values of V0.5 (half-maximal activation potential) of WT, A531V, and V947E channels before and after the application of NFA. Each dot represents the V0.5 value obtained from a single recording. Data are shown as box-and-whisker plots. Median (solid line); mean (dash line); and 10th, 25th, 75th, and 90th percentiles are indicated. The number of examined cells is reported in brackets. Asterisk denotes a significant difference compared with the relative control condition (*p < 0.05).

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  FIGURE 2. Chaperone effect of NFA on WT, A531V, and V947E ClC-1 chloride channels. (A). Representative traces of WT, A531V, and V947E chloride currents before and after the incubation of 50 µM NFA for 24 h. (B). Values of instantaneous and steady-state chloride currents of WT, A531V, and V947E mutants measured at −90 and +60 mV before and after the incubation with NFA 50 µM. (C). Values of V0.5 (half-maximal activation potential) of WT, A531V, and V947E channels before and after the incubation of NFA. Each dot represents the V0.5 value obtained from a single recording. Data are shown as box-and-whisker plots. Median (solid line); mean (dash line); and 10th, 25th, 75th, and 90th percentiles are indicated. The number of examined cells is reported in brackets. (*at least p < 0.05 vs WT CTRL, # at least p < 0.05 vs its relative CTRL).

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  FIGURE 3. ClC-1 protein expression in HEK293T cells transfected with WT and V947E ClC-1 channels and effects of 50 µM NFA incubation for 24 h. (A). Representative western blot of total ClC-1 and β-actin proteins from transfected HEK293T with WT and V947E in the absence or presence of 50 μM NFA. (B). Quantification of ClC-1 total protein expression level. The ClC-1 signal of each column was standardized to the β-actin signal and normalized to WT on the same blot. Each bar is the mean ± S.E.M. from three independent experiments. Asterisks denote a significant difference (p < 0.05) between V947E in the control condition and after NFA treatment. (C). Representative western blot of surface and cytoplasmic ClC-1 and β-actin proteins obtained from the biotinylation assay of transfected HEK293T cells incubated for 24 h in the absence or presence of 50 μM NFA. (D). Quantification of surface and cytoplasmic ClC-1 protein distribution. Each bar is the mean ± SD from three independent experiments (*at least p < 0.05 vs WT in the control condition, # at least p < 0.05 vs V947E in the control condition).