The Novel Direct Modulatory Effects of Perampanel, an Antagonist of AMPA Receptors, on Voltage-Gated Sodium and M-type Potassium Currents

Abstract

Perampanel (PER) is a selective blocker of AMPA receptors showing efficacy in treating various epileptic disorders including brain tumor-related epilepsy and also potential in treating motor neuron disease. However, besides its inhibition of AMPA-induced currents, whether PER has any other direct ionic effects in different types of neurons remains largely unknown. We investigated the effects of PER and related compounds on ionic currents in different types of cells, including hippocampal mHippoE-14 neurons, motor neuron-like NSC-34 cells and U87 glioma cells. We found that PER differentially and effectively suppressed the amplitude of voltage-gated Na⁺ currents (I_Na) in mHippoE-14 cells. The IC₅₀ values required to inhibit peak and late I_Na were 4.12 and 0.78 μM, respectively. PER attenuated tefluthrin-induced increases in both amplitude and deactivating time constant of I_Na. Importantly, PER also inhibited the amplitude of M-type K⁺ currents (I_K(M)) with an IC₅₀ value of 0.92 μM. The suppression of I_K(M) was attenuated by the addition of flupirtine or ZnCl₂ but not by L-quisqualic acid or sorafenib. Meanwhile, in cell-attached configuration, PER (3 μM) decreased the activity of M-type K⁺ channels with no change in single-channel conductance but shifting the activation curve along the voltage axis in a rightward direction. Supportively, PER suppressed I_K(M) in NSC-34 cells and I_Na in U87 glioma cells. The inhibitory effects of PER on both I_Na and I_K(M), independent of its antagonistic effect on AMPA receptors, may be responsible for its wide-spectrum of effects observed in neurological clinical practice.

Keywords: M-type K+ current; Na+ current; Perampanel; glioma cell; hippocampal cell; motor neuron.

Figure 1

Inhibitory effects of perampanel (PER) on the peak and late components of voltage-gated Na+ current (INa) in mouse hippocampal mHippoE-14 cells. In these experiments, we immersed the cells in Ca²⁺-free Tyrode’s solution containing 10 mM tetraethylammonium chloride (TEA) and the recording pipette was filled with a Cs+-containing solution. Once the whole-cell mode had been firmly established, the voltage protocol from −80 mV to different voltages with a duration of 80 msec was applied at a rate of 0.5 Hz. (A) Superimposed INa traces obtained in the controls (a) and presence (b) of 3 μM PER. The uppermost part shows the voltage protocol used. The lower part depicts an expanded record of late INa (INa,L) as indicated in the dashed box. (B) Concentration-response relationships for PER-induced suppression of INa measured at the beginning (□) and end (■) of the depolarizing pulses (mean ± SEM; n = 13 for each point). Smooth lines represent the least-squares fit to a modified Hill function as described in the Materials and Methods.

Figure 2

Effects of tefluthrin (Tef) and Tef plus PER on INa in mHippoE-14 cells. These experiments used the same protocol as described above. Tef (10 μM) was added to the bath and in the continued presence of Tef, PER was subsequently applied to the investigated cells at different concentrations. (A) Original INa traces obtained in the control (a) or in the presence of 10 μM Tef alone (b), 10 μM Tef plus 1 μM PER (c) and 10 μM Tef plus 3 μM PER (d). The upper part indicates the voltage-step protocol used; that is, the examined cells were depolarized from −80 to 0 mV for 30 msec, then repolarized to −30 mV. (B) and (C), respectively, depict the summary bar graphs indicating the peak amplitude and deactivation time constant (τdeact) of depolarization-induced INa obtained in the controls and during the exposure to Tef and Tef plus PER (mean ± SEM; n = 12 for each point). (1): controls (in the absence of Tef or PER); (2): 10 μM Tef alone; (3): 10 μM Tef plus 1 μM PER; (4): 10 μM Tef plus 3 μM PER. * Significantly different from the controls (i.e., bars (1)) (p < 0.05) and †significantly different from the 10 μM Tef alone group (i.e., bars (2)) (p < 0.05).

Figure 3

Concentration-dependent effect of PER on M-type K⁺ current (IK(M)) in mHippoE-14 cells. In these experiments, the cells were immersed in high-K⁺, Ca²⁺-free Tyrode’s solution and each pipette was filled with K⁺-containing solution. (A) Original IK(M) traces elicited by a long-lasting membrane depolarization from −50 to −10 mV (indicated in the upper part of (A)). a: controls; b: 0.1 μM PER; c: 0.3 μM PER; d: 1 μM PER. (B) Concentration-response relation for PER-induced inhibition of IK(M). Each point represents the mean ± SEM (n = 12–14). The smooth line indicates the best fit to the Hill equation. The IC50 value, the maximally inhibited percentage of the current and Hill coefficient were 0.92 μM, 100% and 1.2, respectively. (C) Summary bar graph showing the effects of PER, PER plus flupirtine, PER plus ZnCl2, PER plus L-quisqualic acid (QA) and PER plus sorafenib on IK(M) amplitude (mean ± SEM; n = 11 for each bar). Current amplitude was measured at the end of each depolarizing step. (1): control; (2): 1 μM PER; (3): 1 μM PER plus 10 μM flupirtine; (4): 1 μM PER plus 10 μM ZnCl2; (5) 1 μM PER plus 10 μM L-quisqualic acid (QA); (6) 1 μM PER plus 10 μM sorafenib. * Significantly different from the control (i.e., bar (1)) (p < 0.05) and Ϯsignificantly different from the 1 μM PER alone group (i.e., bar (2)) (p < 0.05). (D) Summary bar graph showing the effect of PER (0.1, 0.3 and 1 µM) on the τ_deact value of IK(M) (mean ± SEM; n = 9 for each bar). * Significantly different from the control (p < 0.05). (1): control; (2) 0.1 µM PER; (3) 0.3 µM PER; (4) 1 µM PER.

Figure 3

Concentration-dependent effect of PER on M-type K⁺ current (IK(M)) in mHippoE-14 cells. In these experiments, the cells were immersed in high-K⁺, Ca²⁺-free Tyrode’s solution and each pipette was filled with K⁺-containing solution. (A) Original IK(M) traces elicited by a long-lasting membrane depolarization from −50 to −10 mV (indicated in the upper part of (A)). a: controls; b: 0.1 μM PER; c: 0.3 μM PER; d: 1 μM PER. (B) Concentration-response relation for PER-induced inhibition of IK(M). Each point represents the mean ± SEM (n = 12–14). The smooth line indicates the best fit to the Hill equation. The IC50 value, the maximally inhibited percentage of the current and Hill coefficient were 0.92 μM, 100% and 1.2, respectively. (C) Summary bar graph showing the effects of PER, PER plus flupirtine, PER plus ZnCl2, PER plus L-quisqualic acid (QA) and PER plus sorafenib on IK(M) amplitude (mean ± SEM; n = 11 for each bar). Current amplitude was measured at the end of each depolarizing step. (1): control; (2): 1 μM PER; (3): 1 μM PER plus 10 μM flupirtine; (4): 1 μM PER plus 10 μM ZnCl2; (5) 1 μM PER plus 10 μM L-quisqualic acid (QA); (6) 1 μM PER plus 10 μM sorafenib. * Significantly different from the control (i.e., bar (1)) (p < 0.05) and Ϯsignificantly different from the 1 μM PER alone group (i.e., bar (2)) (p < 0.05). (D) Summary bar graph showing the effect of PER (0.1, 0.3 and 1 µM) on the τ_deact value of IK(M) (mean ± SEM; n = 9 for each bar). * Significantly different from the control (p < 0.05). (1): control; (2) 0.1 µM PER; (3) 0.3 µM PER; (4) 1 µM PER.

Figure 4

Effect of PER on the activity of M-type K⁺ (KM) channels recorded from mHippoE-14 cells. In these cell-attached single-channel recordings, the cells were bathed in high-K⁺, Ca²⁺-free solution, the recording pipette was filled with low-K⁺ (5.4 mM) solution and the potential was held at +30 mV relative to the bath. (A) Superimposed KM-channel traces in the absence (upper) and presence (lower) of 1 μM PER. Of note, the opening events caused the upper deflections of the trace in this on-cell patch. (B) Summary bar graph showing the effects of PER and PER plus flupirtine on the probabilities of KM channels that would be open (mean ± SEM; n = 11 for each bar). (1): controls; (2): 1 μM PER; (3): 3 μM PER; (4) 3 μM PER plus 10 μM flupirtine. * Significantly different from the controls (p < 0.05) and Ϯsignificantly different from the PER (3 μM) alone group (p < 0.05).

Figure 4

Effect of PER on the activity of M-type K⁺ (KM) channels recorded from mHippoE-14 cells. In these cell-attached single-channel recordings, the cells were bathed in high-K⁺, Ca²⁺-free solution, the recording pipette was filled with low-K⁺ (5.4 mM) solution and the potential was held at +30 mV relative to the bath. (A) Superimposed KM-channel traces in the absence (upper) and presence (lower) of 1 μM PER. Of note, the opening events caused the upper deflections of the trace in this on-cell patch. (B) Summary bar graph showing the effects of PER and PER plus flupirtine on the probabilities of KM channels that would be open (mean ± SEM; n = 11 for each bar). (1): controls; (2): 1 μM PER; (3): 3 μM PER; (4) 3 μM PER plus 10 μM flupirtine. * Significantly different from the controls (p < 0.05) and Ϯsignificantly different from the PER (3 μM) alone group (p < 0.05).

Figure 5

Effect of PER on current-voltage (I-V) relationships and voltage-dependent activation of KM channels in mHippoE-14 cells. In these single-channel recordings, the cells were bathed in high K⁺, Ca²⁺-free solution and the recording pipette was filled with low-K⁺ solution. (A) The I-V curves of KM channels in the absence (■) and presence (□) of 3 μM PER (mean ± SEM; n = 11 for each point). Each dashed line is pointed toward the level of the resting potential (i.e., −70 mV which is indicated by the arrowhead). Of note, the single-channel conductance (i.e. I-V relationship) of the channel in the controls was virtually overlaid with that during exposure to PER (3 μM). (B) The activation curve (i.e., relative channel open probability versus Δvoltage) of KM channels with or without the addition of 3 μM PER (mean ± SEM; n = 11 for each point). The smooth curve was least-squares fit to a Boltzmann function as described in the Materials and Methods. ■: controls; □: in the presence of 3 μM PER.

Figure 6

Effect of PER on delayed-rectifier K⁺ current (IK(DR)) in mHippoE-14 cells. The whole-cell current recordings were conducted in cells bathed in Ca²⁺-free Tyrode’s solution containing 1 μM tetrodotoxin (TTX). (A) Superimposed current traces in the absence (upper) and presence (lower) of 10 μM PER. The upper part indicates the voltage protocol applied. (B) Averaged I-V relationships of IK(DR) obtained in the controls (■) and during exposure to 10 μM PER (□) (mean ± SEM; n = 11 for each point). * Significantly different from the controls at the same level of membrane potential (p < 0.05). (C) Concentration-response curve for PER-induced inhibition of IK(DR) in mHippoE-14 cells. The examined cells were depolarized from −50 to +50 mV with a duration of 1 s and current amplitudes was measured at the end of depolarizing pulses during exposure to different PER concentrations was compared with the control value (mean ± SEM; n = 7–9 for each point). The continuous line represents the best fit to a Hill function. The values for IC50, maximally inhibited percentage of IK(DR) and the Hill coefficient were 25.3 µM, 100% and 1.2, respectively.

Figure 7

Effect of PER on IK(M) in motor neuron-like NSC-34 cells. In this set of experiments, the cells were bathed in high-K⁺, Ca²⁺-free solution and the recording pipette was filled with K⁺-containing solution. (A) Superimposed IK(M) traces obtained in the controls (a) and during cell exposure to 0.3 µM PER (b) and 1 μM PER (c). The upper part indicates the step protocol applied. (B) Summary bar graph showing the effects of PER, PER plus 9-phenanthrol and PER plus ZnCl₂ on IK(M) amplitude (mean ± SEM; n = 12 for each bar). IK(M) amplitude was measured at the end of each depolarizing step. (1): controls; (2): 0.3 μM PER; (3): 1 μM PER; (4): 1 μM PER plus 3 μM 9-phenanthrol; (5): 1 μM PER plus 10 μM ZnCl2. * Significantly different from the controls (p < 0.05) and †significantly different from the PER (1 μM) alone group (p < 0.05).

Figure 8

Inhibitory effect of PER on INa in U87 glioma cells. The experimental protocol used was similar to that described above for mHippoE-14 cells. (A) Superimposed INa traces obtained in the controls (a) and during cell exposure to 3 μM PER (b). Inset indicates the voltage-clamp protocol used. (B) Summary bar graph showing the inhibitory effects of 1 or 3 μM PER on peak amplitude of INa in response to the rapid depolarizing step (mean ± SEM; n = 11 for each bar). * Significantly different from the controls (p < 0.05).