A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia

Abstract

Objective: Human and animal studies have shown that Na(v)1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Na(v)1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ).

Methods: We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Na(v)1.7 complementary DNA. Wild-type Na(v)1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents.

Results: Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Na(v)1.7 but does not affect these parameters in wild-type Na(v)1.7.

Interpretation: Our results demonstrate a normalizing effect of CBZ on mutant Na(v)1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Na(v)1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders.

Fig 1

Inheritance pattern of the V400M mutation in Na_v1.7 associated with carbamazepine (CBZ)-responsive inherited erythromelalgia (IEM). A three-generation Canadian family with the affected patients all having similar symptoms; the proband is indicated by an arrow. Circles denote female subjects; squares denote male subjects; black symbols indicate subjects affected with inherited erythromelalgia; plus signs denote subjects heterozygous for the V400M mutation in exon 9; and minus sign denotes subjects without the mutation. CBZ+ = pain relief from CBZ treatment; CBZ ND = the effect of CBZ on the patient’s symptoms is unknown.

Fig 2

V400M mutation shifts activation. (A, B) Representative traces from human embryonic kidney (HEK) 293 cells stably expressing wild-type (WT; A) and V400M (B) mutant Na_v1.7 channels resulting from voltage steps for generation of activation curves as described in Subjects and Methods. (C) Normalized current-voltage relations generated from activation curves from cells expressing V400M (n = 18; black squares) or WT (n = 15; blue circles) Na_v1.7 channels. (D) Voltage-dependent hyperpolarizing shift in activation is demonstrated by Boltzmann fits of activation curves from cells expressing V400M (n = 18) or WT (n = 15) Na_v1.7 channels (V400M: V_1/2 = −50.4 ± 1.64mV; WT: V_1/2 = −43.9 ± 1.47mV; p < 0.01).

Fig 3

Further characterization of the V400M mutation. (A) Voltage dependence of steady-state inactivation shows a depolarizing shift in cells expressing V400M mutants (n = 19; black squares) as compared with wild-type (WT) channels (n = 25; blue circles) (V400M: V_1/2 = −82.8 ± 1.44mV; WT: V_1/2 = −90.1 ± 1.44mV; p < 0.001). (B) Composite graph showing Boltzmann fits of activation and steady-state inactivation curves of V400M mutant versus WT channels shows an increase in overlap window current (enlarged in C: blue represents WT and black represent V400M). (D) Kinetics of fast inactivation as measured by fits of the time constant (τ) are unchanged in cells expressing V400M mutants (n = 18; black squares) versus WT (n = 15; blue circles) channels (not significant). (E) Progressive slowing of the deactivation time constant (τ) is observed in cells expressing V400M mutant channels (n = 20) compared with WT channels (n = 24) (**p < 0.0005; *p < 0.005; ⁺⁺p < 0.02; ⁺p < 0.05). (inset) Representative currents and prolonged deactivation at −50mV. Blue solid line represents 1.7R WT; black dashed line represents 1.7R V400M. (F) Voltage dependence of slow inactivation is unchanged in cells expressing V400M mutant channels (n = 18; black squares) versus WT channels (n = 16; blue circles) (V400M: V_1/2 = −76.2 ± 3.9mV; WT: V_1/2 = 73.9 ± 3.9 mV; p value is not significant).

Fig 4

Response to slow-voltage-ramp currents was assessed in human embryonic kidney (HEK) cells expressing V400M mutant channels (black line) or wild-type (WT; blue line) channels. Shown are representative traces of inward currents (expressed as percentage of peak current obtained with the activation protocol) in response to slow-voltage depolarization from −120 to 20mV over a 600-millisecond time course (0.23mV/msec).

Fig 5

Block of wild-type (WT) and V400M mutant Na currents by carbamazepine (CBZ). (A) The holding potential was set to −120mV to maintain the channels in the resting (closed) state; then the current available for activation was elicited by a 20-millisecond pulse to 0mV. The fractional block is given by dividing the current measured in the presence of the indicated concentration of CBZ by the current elicited in the same cell just before compound addition. The IC₅₀ fitted to the WT data (blue circles) is 1,350 ± 230µM (adjusted R² = 0.98). The IC₅₀ fitted to the V400M data (black squares) is 970 ± 30µM (adjusted R² = 0.99). (B) To evaluate block of inactivated Na channels, we applied a 10-second conditioning pulse to −50mV followed by a 100-millisecond pulse to −120mV to recover from fast inactivation; then the test pulse to 0mV was applied. The IC₅₀ fitted to the WT data (blue circles) is 1,100 ± 80µM (adjusted R² = 0.98). The IC₅₀ fitted to the V400M data (black squares) is 1,030 ± 160µM (adjusted R² = 0.89).

Fig 6

Carbamazepine (CBZ) normalizes the gating properties of the V400M mutation. (A) Depolarizing shifts in the voltage dependence of activation are observed in human embryonic kidney (HEK) 293 cells stably expressing the V400M mutation in response to increasing concentrations of CBZ (V_{1/2 DMSO} = −47.9 ± 2.93mV; V_{1/2 10µM CBZ} = −36.9 ± 1.2mV; V_{1/2 100µM CBZ} = −37.3 ± 2.72mV). Black circles designate V400M + DMSO (n = 6); green upward triangles designate V400M + 10µM CBZ (n = 19); blue downward triangles designate V400M + 100µM CBZ (n = 8). (B) CBZ treatment of HEK 293 cells expressing wild-type channels does not affect the voltage dependence of activation (V_{1/2 DMSO} = −36.6 ± 2.63mV; V_{1/2 10µM CBZ} = −37.9 ± 1.7mV; V_{1/2 100µM CBZ} = −40.5 ± 3.28mV). Black squares designate WT + DMSO (n = 9); orange circles designate WT + 10µM CBZ (n = 10); blue upward triangles designate V400M + 100µM CBZ (n = 7). (C) Hyperpolarizing shifts in the voltage dependence of steadystate fast inactivation are observed with CBZ treatment of V400M-expressing cells (V_{1/2 DMSO} = −87.2 ± 1.97mV; V_{1/2 10µM CBZ} = −91.7 ± 1.5mV; V_{1/2 100µM CBZ} = −92.9 ± 2.65mV). Black circles designate V400M + DMSO (n = 8); green upward triangles designate V400M + 10µM CBZ (n = 19); blue downward triangles designate V400M + 100µM CBZ (n = 8). (D) Voltage dependence of steady-state fast inactivation is unchanged by CBZ treatment of cells expressing wild-type channels (V_{1/2 DMSO} = −93.1 ± 3.32mV; V_{1/2 10µM CBZ} = −94.1 ± 0.76mV; V_{1/2 100µM CBZ} = −94.7 ± 1.63mV). Black squares designate WT + DMSO (n = 5); orange circles designate WT + 10µM CBZ (n = 9); blue upward triangles designate V400M + 100µM CBZ (n = 8). (E) The concentration–response curve for the CBZ-induced shifts of the V_1/2 of activation is plotted for a range of CBZ pretreatment concentrations. Data points are the averages of the activation V_1/2 values obtained by fitting a Boltzmann function to the GV (conductance-voltage) curves (n = 5–19). A logistic fit of the data suggests that the EC₅₀ for CBZ to normalize the V_1/2 of V400M toward the WT value is 3.4 ± 0.5µM (adjusted R² = 0.98) with a power coefficient of 3.7. DMSO = dimethylsulfoxide.