Pathomechanisms of a CLCN1 Mutation Found in a Russian Family Suffering From Becker's Myotonia

Abstract

Objective: Myotonia congenita (MC) is a rare muscle disease characterized by sarcolemma over-excitability inducing skeletal muscle stiffness. It can be inherited either as an autosomal dominant (Thomsen's disease) or an autosomal recessive (Becker's disease) trait. Both types are caused by loss-of-function mutations in the CLCN1 gene, encoding for ClC-1 chloride channel. We found a ClC-1 mutation, p.G411C, identified in Russian patients who suffered from a severe form of Becker's disease. The purpose of this study was to provide a solid correlation between G411C dysfunction and clinical symptoms in the affected patient. Methods: We provide clinical and genetic information of the proband kindred. Functional studies include patch-clamp electrophysiology, biotinylation assay, western blot analysis, and confocal imaging of G411C and wild-type ClC-1 channels expressed in HEK293T cells. Results: The G411C mutation dramatically abolished chloride currents in transfected HEK cells. Biochemical experiments revealed that the majority of G411C mutant channels did not reach the plasma membrane but remained trapped in the cytoplasm. Treatment with the proteasome inhibitor MG132 reduced the degradation rate of G411C mutant channels, leading to their expression at the plasma membrane. However, despite an increase in cell surface expression, no significant chloride current was recorded in the G411C-transfected cell treated with MG132, suggesting that this mutation produces non-functional ClC-1 chloride channels. Conclusion: These results suggest that the molecular pathophysiology of G411C is linked to a reduced plasma membrane expression and biophysical dysfunction of mutant channels, likely due to a misfolding defect. Chloride current abolition confirms that the mutation is responsible for the clinical phenotype.

Keywords: ClC-1; chloride channel; intracellular trafficking; myotonia congenita; patch-clamp.

Figure 1

Probands' EMG, pedigree, and ClC-1 mutation. (A) Repetitive nerve stimulation (RNS) on abductor digiti minimi muscle. (B) Pedigrees of the Russian family affected by Becker disease. Squared symbols indicate men and round symbols indicate women. Empty symbols denote healthy individuals and dark filled symbols denote affected individuals. The arrow indicates the proband in the family. (C) Amino acid alignment of the ClC-1 chloride channel among different species and human CLC members. (D) Three-dimensional representation of hClC-1 channel modeled upon the X-ray structure of a eukaryotic Cl⁻/H⁺ exchanger CmClC showing the localization of G411C mutation.

Figure 2

Chloride currents of WT and G411C hClC-1 channels expressed in HEK293T cells. (A) Representative chloride currents recorded from HEK293T cells transfected with 5 μg WT or G411C hClC-1 cDNAs in high-chloride intracellular solution. The G411C currents were also measured after 16 h incubation with 20 μM MG132. The holding potential (HP) was set at 0 mV. Voltage steps of 400 ms were applied from −150 to +150 mV in 10 mV intervals, each followed by a voltage step at −105 mV to record tail currents. Voltage steps were applied every 3 s to allow complete recovery of current amplitude at the HP between two pulses. (B) Instantaneous currents were measured at the beginning (~1 ms) of voltage step and normalized with respect to cell capacitance (pA/pF) to calculate current density. (C) Steady-state current densities were measured at the end of voltage step (~390 ms). Each data point is the mean ± S.E.M. from 10 to 12 cells.

Figure 3

Confocal microscopy images of HEK293T cells expressing wild-type YFP-ClC-1 channel and YFP-ClC-1 G411C mutant. (A) Representative confocal single optical plane image showing localization of wild-type YFP-ClC-1 (green signal), plasma membrane marker WGA-555 (red signal), and colocalization (yellow signal) in a dividing cell doublet. (B) Representative dividing cell doublet showing localization of YFP-G411C (green signal), plasmalemma WGA-555 (red signal), and colocalization (yellow signal). (C,D) Scatter-plots of pixel intensity in images (A,B), showing the relationship between WT or G411C and WGA. Linear regressions (black lines) demonstrate significant colocalization of WT hClC-1 with WGA [Pearson's correlation coefficient (P) of 0.7] and anticolocalization of G411C with WGA (P = 0.4). (E) Histograms reporting the Pearson's coefficient (P) of pixel intensity scatter-plot linear regressions (as in C,D), and the Mander's overlap coefficients (M1 and M2) of the two markers (WGA-555 and YFP-ClC-1). M1 indicates summed intensities of red pixels overlapping with green to the total red intensity (proportion of WGA overlapping with ClC-1). M2 indicates summed intensities of green pixels overlapping with red to the total green intensity (proportion of ClC-1 overlapping with WGA). Each bar is the mean ± SD from 64 (WT) and 34 (G411C) cell clusters. *p < 0.001 between WT and G411C hClC-1 with unpaired Student's t-test.

Figure 4

ClC-1 protein expression in HEK293T cells transfected with WT or G411C ClC-1 channels and effects of MG132 incubation. (A) Representative western blot of total ClC-1 and β-actin proteins from transfected HEK293T incubated for 16 h in absence or presence of 20 μM MG132. (B) Quantification of ClC-1 total protein expression level. The ClC-1 signal of each column was standardized to 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 significant difference (p < 0.04) between control condition and MG132 treatment using two-way ANOVA. (C) Representative western blot of surface and cytoplasmic ClC-1 and β-actin proteins obtained from biotinylation assay of transfected HEK293T cells incubated for 16 h in absence or presence of 20 μM MG132. (D) Quantification of surface and cytoplasmic ClC-1 protein distribution. Each bar is the mean ± S.E.M. from 3 to 4 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Sidak's multiple comparison test (* at least p < 0.05 vs. WT and GC + MG132).