Effect of carbamazepine on tetrodotoxin-resistant Na+ channels in trigeminal ganglion neurons innervating to the dura

Abstract

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na⁺ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na⁺ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na⁺ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na⁺ currents, it strongly inhibited steady-state currents of transient as well as persistent Na⁺ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na⁺ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na⁺ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na⁺ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.

Keywords: Carbamazepine; Dural afferent neurons; Migraine; Patch clamp; Sodium channel; Tetrodotoxin-resistant.

Fig. 1. Effects of carbamazepine on TTX-R Na⁺ currents.

(A) Typical phase contrast (Ph, left), fluorescent (DiI, middle), and superimposed (right) images of medium-sized DiI-positive neurons. (B) Typical traces of TTX-R Na⁺ currents in the absence and presence of 100 µM carbamazepine (CBZ). The TTX-R Na⁺ currents was elicited by electrical stimulation from a V_H of −80 mV to −10 mV (100 ms duration) every 5 s (0.2 Hz). Note that carbamazepine more potently decreased the steady-state than peak amplitudes of transient TTX-R Na⁺ currents. (C) Typical traces of TTX-R persistent Na⁺ currents in the absence and presence of various concentrations of carbamazepine. The TTX-R persistent Na⁺ currents were elicited by slow voltage ramp stimulation from a V_H of −80 mV to +10 mV (15 mV/s) every 15 s. (D) Concentration-inhibition relationships of carbamazepine for the peak (I_peak) and steady-state (I_steady) amplitudes of transient TTX-R Na⁺ currents and slow voltage ramp-induced persistent currents (I_ramp). The continuous curve was fitted using a least-squares method. Each point represents the mean and SEM from 10–12 experiments.

Fig. 2. Effects of carbamazepine on the current-voltage relationship of TTX-R Na⁺ channels.

(A) A schematic illustration of voltage step pulses. The TTX-R Na⁺ currents were induced from a V_H of −80 mV by 50 ms depolarization pulses from −80 to +20 mV in 10 mV increments. (B) Typical traces of the TTX-R Na⁺ currents elicited by voltage step pulses in the absence (left) and presence (right) of 100 µM carbamazepine (CBZ). (C) (a) Current-voltage relationships of TTX-R Na⁺ channels in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Each point represents the mean and SEM from 12 experiments. (b) Conductance-voltage relationships of TTX-R Na⁺ channels in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Continuous lines represent the best fit of the Boltzmann function. Each point represents the mean and SEM from 12 experiments. (D) Carbamazepine-induced changes in the half-maximal voltage for activation (V_{50, activation}; a) and the slope factor k (b) of TTX-R Na⁺ channels. Each column represents the mean and SEM from 12 experiments. n.s, not significant.

Fig. 3. Effects of carbamazepine on the voltage dependence of steady-state fast inactivation of TTX-R Na⁺ channels.

(A) A schematic illustration of voltage step pulses. The TTX-R Na⁺ currents were induced by 50 ms depolarization pulses to −10 mV following 500 ms prepulses from −120 to −20 mV in 10 mV increments. (B) Typical traces of the TTX-R Na⁺ currents elicited by voltage step pulses in the absence (left) and presence (right) of 100 µM carbamazepine (CBZ). (C) Current-voltage relationships of TTX-R Na⁺ channels in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Continuous lines represent the best fit of the Boltzmann function. Each point represents the mean and SEM from 10 experiments. (D) Carbamazepine-induced changes in the half-maximal voltage for inactivation (V₅₀, inactivation; a) and the slope factor k (b) of TTX-R Na⁺ channels. Each column represents the mean and SEM from 10 experiments. n.s, not significant.

Fig. 4. Effects of carbamazepine on the voltage-dependence of slow inactivation of TTX-R Na⁺ channels.

(A) A schematic illustration of voltage step pulses. The TTX-R Na⁺ currents were induced by 50 ms depolarization pulses to −10 mV after 5000 ms prepulses from −100 to −10 mV in 10 mV increments. (B) Typical traces of the TTX-R I_Na elicited by voltage step pulses in the absence (left) and presence (right) of 100 µM carbamazepine (CBZ). (C) Current-voltage relationships of TTX-R Na⁺ channels in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Continuous lines represent the best fit of the Boltzmann function. Each point represents the mean and SEM from 12 experiments. (D) Carbamazepine-induced changes in the half-maximal voltage for slow inactivation (V_{50, slow inactivation}; a) and the slope factor k (b) of TTX-R Na⁺ channels. Each column represents the mean and SEM from 12 experiments. ^**p<0.01.

Fig. 5. Effects of carbamazepine on the use-dependent inactivation of TTX-R Na⁺ channels.

(A) Typical traces of TTX-R Na⁺ currents elicited by 20 successive voltage step pulses (−80 mV to −10 mV, 30 ms duration, 5 Hz) in the absence (left) and presence (right) of 100 µM carbamazepine (CBZ). (B) Time course of the peak amplitude of TTX-R Na⁺ currents during a train of 20 pulses (5 Hz) in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Each point represents the mean and SEM from 11 experiments. (C) Carbamazepine-induced changes in the P₂₀/P₁ ratio of TTX-R Na⁺ currents (a, 1 Hz; b, 5 Hz). Each column represents the mean and SEM from 11 experiments. ^**p<0.01.

Fig. 6. Effects of carbamazepine on the onset of inactivation of TTX-R Na⁺ channels.

(A) A schematic illustration of voltage step pulses. The TTX-R Na⁺ currents were induced by a two-pulse protocol. Conditioning pulses (P₁; −10 mV depolarization, 2–8000 ms duration) were followed by the second test pulses (P₂; −10 mV depolarization, 50 ms duration). The second TTX-R Na⁺ currents were recovered with an interpulse interval of 20 ms at a potential of −80 mV. (B) Typical traces of TTX-R I_Na elicited by the second test pulses of the two-pulse protocol in the absence (left) and presence (right) of 100 µM carbamazepine (CBZ). (C) Kinetics for the recovery from inactivation of TTX-R Na⁺ channels in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Each point represents the mean and SEM from 8 experiments. (D) Carbamazepine-induced changes in the P₂/P₁ ratio of TTX-R Na⁺ currents. Each point represents the mean and SEM from 8 experiments. (E) Carbamazepine-induced changes in the kinetic parameters [a, fast time constants (τ_fast); b, slow time constants (τ_slow); c, weighted time constants (τ_weighted)] for the recovery from inactivation of the TTX-R Na⁺ currents. Each column represents the mean and SEM from 8 experiments. ^**p<0.01.

Fig. 7. Effects of carbamazepine on the recovery from inactivation of TTX-R Na⁺ channels.

(A) A schematic illustration of voltage step pulses. The TTX-R Na⁺ currents were induced by a two-pulse protocol. Conditioning pulses (P₁; −10 mV depolarization, 500 ms duration) were followed by the second test pulses (P₂; −10 mV depolarization, 50 ms duration). The second TTX-R Na⁺ currents were recovered with various interpulse intervals of 1 to 5000 ms at a potential of −80 mV. (B) Typical traces of TTX-R I_Na elicited by the second test pulses of the two-pulse protocol in the absence (left) and presence (right) of 100 µM carbamazepine (CBZ). (C) Kinetics for the recovery from inactivation of TTX-R Na⁺ channels in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Each point represents the mean and SEM from 10 experiments. (D) Carbamazepine-induced changes in the P₂/P₁ ratio of TTX-R I_Na. Each point represents the mean and SEM from 10 experiments. (E) Carbamazepine-induced changes in the kinetic parameters [a, fast time constants (τ_fast); b, slow time constants (τ_slow); c, weighted time constants (τ_weighted)] for the recovery from inactivation of the TTX-R Na⁺ currents. Each column represents the mean and SEM from 10 experiments. ^**p<0.01; n.s, not significant.

Fig. 8. Effects of carbamazepine on the excitability of dural afferent neurons.

(A) Effect of carbamazepine on voltage responses to depolarizing current injection in the absence (upper) and presence (lower) of 100 µM carbamazepine (CBZ). Four representative raw voltage traces were elicited by 1-fold threshold (1T; 200 pA) to 4-fold threshold (4T; 800 pA) depolarizing current injection. Note that the number of action potentials increases with stronger depolarizing current injection. (B) Changes in the number of action potentials elicited by depolarizing current injection (1T to 4T) in the absence (open circles) and presence (closed circles) of 100 µM carbamazepine. Each point represents the mean and SEM from 8 experiments. (C) Carbamazepine-induced changes in the number of action potentials elicited by depolarizing current injection (1T to 4T). Each column represents the mean and SEM from 8 experiments. ^**p<0.01, n.s; not significant. (D) Carbamazepine-induced changes in the amplitude of rheobase currents. Each column represents the mean and SEM from 8 experiments. n.s; not significant. (E) Typical voltage responses to hyperpolarizing current injection in the absence (upper) and presence (lower) of 100 µM carbamazepine. DiI-positive neurons were current-clamped and hyperpolarizing step pulses (−200 pA increments, up to −800 pA, 1 s duration) were applied to patched neurons. Note that the sag potentials (arrows), which are typical properties mediated by HCN channels, were not affected by adding carbamazepine.