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

Abstract

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in "gating-defective" or "expression-defective" mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.

Keywords: ClC-1 chloride channel; drug repurposing; myotonia congenita; niflumic acid; precision medicine.

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 V_0.5 (half-maximal activation potential) of WT, A531V, and V947E channels before and after the application of NFA. Each dot represents the V_0.5 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).

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 V_0.5 (half-maximal activation potential) of WT, A531V, and V947E channels before and after the incubation of NFA. Each dot represents the V_0.5 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).

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).