The Novel Activity of Carbamazepine as an Activation Modulator Extends from NaV1.7 Mutations to the NaV1.8-S242T Mutant Channel from a Patient with Painful Diabetic Neuropathy

Abstract

Neuropathic pain in patients carrying sodium channel gain-of-function mutations is generally refractory to pharmacotherapy. However, we have shown that pretreatment of cells with clinically achievable concentration of carbamazepine (CBZ; 30 μM) depolarizes the voltage dependence of activation in some Na_V1.7 mutations such as S241T, a novel CBZ mode of action of this drug. CBZ reduces the excitability of dorsal root ganglion (DRG) neurons expressing Na_V1.7-S241T mutant channels, and individuals carrying the S241T mutation respond to treatment with CBZ. Whether the novel activation-modulating activity of CBZ is specific to Na_V1.7, and whether this pharmacogenomic approach can be extended to other sodium channel subtypes, are not known. We report here the novel Na_V1.8-S242T mutation, which corresponds to the Na_V1.7-S241T mutation, in a patient with neuropathic pain and diabetic peripheral neuropathy. Voltage-clamp recordings demonstrated hyperpolarized and accelerated activation of Na_V1.8-S242T. Current-clamp recordings showed that Na_V1.8-S242T channels render DRG neurons hyperexcitable. Structural modeling shows that despite a substantial difference in the primary amino acid sequence of Na_V1.7 and Na_V1.8, the S242 (Na_V1.8) and S241 (Na_V1.7) residues have similar position and orientation in the domain I S4-S5 linker of the channel. Pretreatment with a clinically achievable concentration of CBZ corrected the voltage dependence of activation of Na_V1.8-S242T channels and reduced DRG neuron excitability as predicted from our pharmacogenomic model. These findings extend the novel activation modulation mode of action of CBZ to a second sodium channel subtype, Na_V1.8.

Fig. 1.

(A) Sequence alignment and position of mutation in the DI S4-S5 linker in the Na_V1.8 channel. The sequence of the DI S4-S5 linker is invariant in the nine sodium channels from humans. The substitution of S242T is highlighted in red type, and the substitution of Na_V1.7-S241T is highlighted in blue type. (B) Structural modeling of human Na_V1.8 and human Na_V1.7 channels. The two channel structures were superimposed, and the membrane-spanning segments showed good alignment. DI is colored blue in Na_V1.8, and red in Na_V1.7. The overall structure is tilted and rotated to give a clearer view of the DI S4-S5 linker where the S242T and Na_V1.7-S241T substitution occurs. A box is drawn to indicate the region of the structure that is zoomed out to better show the Na_V1.8-T242 residue (blue carbons) and Na_V1.7-T241 residue (red carbons). The serine-to-threonine substitution in the DI S4-S5 linker in Na_V1.7 and Na_V1.8 maintains the same position and orientation of the side chain.

Fig. 2.

Voltage-clamp analysis of Na_V1.8/S242T mutant channels. Representative current traces recorded from Na_V1.8-null DRG neurons expressing WT (A) or S242T (B) channels. (C) Point-to-point curve of the mean G value of WT and S242T channels, which shows that the V_1/2,act of S242T (n = 30) channels is hyperpolarized by 6.1 mV compared with WT (n = 26) channels (two-tailed t test, P = 0.0006). (D) Point-to-point curve for mean time-to-peak shows that mutant S242T channels (n = 29) opens faster than WT (n = 25) at voltages from −40 to −10 mV (two-tailed t test with Bonferroni correction, *P < 0.05). (E) S242T (n = 24) channels deactivate significantly slower than WT (n = 21) channels at voltages from −30 to −20 mV (two-tailed t test with Bonferroni correction, *P < 0.05). (F) Compared with WT (n = 24), the S242T mutation (n = 25) shifts V_1/2 for fast inactivation by −3.8 mV in a hyperpolarized direction. (G) Compared with WT (n = 14), the S242T mutation (n = 13) enhances steady-state slow-inactivation with the V_1/2 shifting by −8.9 mV. (H) Representative ramp currents for WT and S242T mutant. Compared with WT (n = 25) channels, S242T (n = 16) shifts the voltage at which the peak of ramp current occurs. (I) Comparison of persistent currents between cells expressing WT (n = 26) channels and cells expressing S242T (n = 30) mutant channels for activation depolarization step pulses from −70 to 20 mV. Two-tailed t test with Bonferroni correction, *P < 0.05.

Fig. 3.

Current-clamp analysis of effects of S242T mutant channels on DRG neuron excitability. (A and B) Representative action potential (AP) traces recorded from DRG neuron expressing WT (A) or S242T mutant (B) channels. Action potentials were elicited by 200-millisecond step depolarizing current injections from resting membrane potential (V_m). (C) Comparison of current threshold for DRG neurons expressing WT (n = 31) and S242T (n = 29) mutant channels. Expression of S242T channels reduces the current threshold significantly. Two-tailed t test, *P < 0.05. Responses of a representative DRG neuron expressing WT channels (D–F) or S242T mutant channels (G–I) to 500-millisecond depolarization current steps that are 150 pA (left), 300 pA (middle), and 450 pA (right). (J) Comparison of responses (number of impulses evoked by a 500-millisecond stimulus) for the populations of DRG neurons expressing WT (n = 29) or S242T (n = 24) channels across a range of step current injections from 50 to 500 pA. Two-way ANOVA, ***P < 0.001.

Fig. 4.

Clinically relevant concentration of CBZ rescued the hyperpolarizing shift of activation of S242T mutant channels. Representative traces of current families recorded from DRG neurons expressing S242T mutant channel treated with DMSO (A) or with 30 μM CBZ (B). (C) Treatment with CBZ (n = 17) significantly shifted the voltage-dependence of activation of the S242T mutant channel compared with the treatment with DMSO (n = 12); two-tailed t test. (D) Treatment with 30 μM CBZ (n = 11) did not affect the voltage dependence of steady-state fast inactivation compared with the treatment with DMSO (n = 7).

Fig. 5.

A clinically relevant concentration of CBZ had no effect on WT channels. Representative traces of current families recorded from DRG neurons expressing WT channels treated with DMSO (A) or with 30 μM CBZ (B). (C) Treatment with CBZ (n = 14) did not shift the voltage- dependence of activation of WT channels compared with treatment with DMSO (n = 10). (D) Treatment with 30 μM CBZ (n = 14) did not affect the voltage-dependence of steady-state fast inactivation compared with the treatment with DMSO (n = 10).

Fig. 6.

Current-clamp analysis of the effects of CBZ treatment on DRG neurons expressing S242T mutant channels. Representative action potential (AP) traces recorded from DRG neuron expressing S242T mutant channels after treatment with DMSO (A) or 30 μM CBZ (B). (C) Comparison of current threshold for DRG neurons expressing S242T mutant channels after treatment with CBZ (n = 29) or DMSO (n = 28) control. CBZ treatment increased current threshold significantly. Two-tailed t test, *P < 0.05. Responses of a representative DRG neurons expressing S242T mutant channels after the treatment with DMSO (D–F) or CBZ (G–I) to 500-millisecond depolarization current steps of 150 pA (left), 300 pA (middle), and 450 pA (right). (J) Comparison of responses (number of impulses evoked by a 500-millisecond stimulus) in the population of DRG neurons expressing S242T mutant channels with treatment with CBZ (n = 26) or DMSO (n = 23) control across a range of step current injections from 50 to 500 pA. Two-way ANOVA, ***P < 0.001. V_m, membrane potential.

Fig. 7.

Current-clamp analysis of the effects of CBZ treatment on DRG neurons expressing WT channels. Representative action potential (AP) traces recorded from DRG neurons expressing WT channels after treatment with DMSO (A) or 30 μM CBZ (B). (C) Comparison of current threshold for DRG neurons expressing WT channels after treatment of CBZ (n = 27) or DMSO (n = 25) control. CBZ treatment did not have a significant effect on current threshold. Responses of the representative DRG neurons expressing WT channels after treatment with DMSO (D–F) or CBZ (G–I) to 500-millisecond depolarization current steps of 150 pA (left), 300 pA (middle), and 450 pA (right). (J) Comparison of responses (number of impulses evoked by a 500-millisecond stimulus) in the population of DRG neurons expressing WT channels with treatment with CBZ (n = 25) or DMSO (n = 20) control across a range of step current injections from 50 to 500 pA. Two-way ANOVA, *P < 0.05. V_m, membrane potential.