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. 2014 Sep 17;9(9):e108021.
doi: 10.1371/journal.pone.0108021. eCollection 2014.

Perampanel inhibition of AMPA receptor currents in cultured hippocampal neurons

Chao-Yin Chen  1 , Lucas Matt  1 , Johannes Wilhelm Hell  1 , Michael A Rogawski  2
Affiliations

Perampanel inhibition of AMPA receptor currents in cultured hippocampal neurons

Chao-Yin Chen et al. PLoS One. .

Abstract

Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM), concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M-1 s-1 and 0.58 s-1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM) was similar at all kainate concentrations (3-100 µM), demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics.

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Conflict of interest statement

Competing Interests: This research was supported, in part, by a grant from Eisai. MAR has served as a consultant to Eisai. None of the authors is an employee of Eisai. There are no restrictions on the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Perampanel inhibition of AMPA-evoked currents in cultured hippocampal neurons.
(A) Sample currents evoked by 100 µM AMPA in 4 neurons in the absence (left panels) and presence (right panels) of perampanel at the concentrations indicated demonstrating a concentration-dependent reduction in current. (B) Perampanel (1 µM) did not alter the mean values of the ratio of the peak to the late amplitude of currents evoked by 10, 30 and 100 µM AMPA in 7, 8 and 7 neurons, respectively, and (C) did not affect the mean rise time constant values or mean decay time constant values of the currents. Peak (D) and late (E) current values evoked by various AMPA concentrations expressed as percent of control prior to perampanel. Curves represent logistic fits to the data for each AMPA concentration. Peak current is the maximum current level; late current is the current during the last 100 ms of the perfusion. Data points in D and E represent mean ± S.E.M. of values from 3 to 9 neurons. Control values for peak/late ratio: 10 µM AMPA: 1.5±0.1; 30 µM AMPA: 1.7±0.1; 100 µM AMPA: 2.7±0.3. Control values for decay time constant (ms): 10 µM AMPA: 141±13; 30 µM AMPA: 93±7; 100 µM AMPA: 50±2. Control values for rise time (ms): 10 µM AMPA: 51±2; 30 µM AMPA: 45±3; 100 µM AMPA: 32±1.
Figure 2
Figure 2. Perampanel inhibition of kainate-evoked currents in cultured hippocampal neurons.
(A) Sample currents evoked by 100 µM kainate in 4 neurons in the absence (left panels) and presence (right panels) of perampanel at the concentrations indicated. The percent inhibition values (derived from the current amplitude in the absence of perampanel and the corresponding current amplitude in the presence of perampanel) are 20%, 41%, 55%, and 80% at concentrations of 0.1, 0.3, 1, and 3 µM, respectively. (B) Current amplitude values evoked by various kainate concentrations expressed as percent of control prior to perampanel. Curves represent logistic fits to the data for each kainate concentration. Data points represent mean ± S.E.M. of values from 3 to 6 neurons.
Figure 3
Figure 3. Slow onset and recovery from perampanel block of kainate- and AMPA-evoked currents.
(A) Currents evoked by 100 µM kainate in the absence (A1) and with pre-application (A2), co-application (A2) and post-application (A3) of perampanel (3 µM) in the same neuron. (B) Currents evoked by 100 µM AMPA in the absence (B1) and with pre-application (B2), co-application (B2) and post-application (A3) of perampanel (3 µM) in a different neuron from (A). Open arrows indicate peak current levels in the case of AMPA-evoked currents and the current at 100 ms after onset of agonist application in the case of kainate-evoked currents; closed arrows indicate late current levels (end of agonist application). Scale for current is 300 pA in (A) and 500 pA in (B). (C) Mean ± S.E.M. values of peak (C1) and late (C2) current levels as a percent of control (as in A1) with perampanel pre-application (as in A2), co-application (as in A3) and post-application (as in A4) in 3–4 neurons (D) Mean ± S.E.M. values of peak (D1) and late (D2) current levels as a percent of control (as in B1) with perampanel pre-application (as in B2), co-application (as in B3) and post-application (as in B4) in 4 neurons. *p<0.05 vs. A2 or B2.
Figure 4
Figure 4. Kinetics of peramapanel block of AMPA-evoked currents.
(A) Time constants for block and unblock of 100 µM AMPA-evoked currents determined from the best single exponential fits to current traces at the onset (τapp) and termination (τoff) of application of 1, 3 and 10 µM perampanel. (B) Reciprocal mean τapp values ( = k app) plotted against perampanel concentration. The best-fit straight line to the data is shown. The slope and the intercept values are 1.5±0.1×105 M−1 s−1 and 0.58±0.6 s−1, respectively.
Figure 5
Figure 5. Perampanel does not inhibit NMDA-evoked currents in cultured hippocampal neurons.
(A) Sample currents evoked by 10 and 100 µM NMDA in the absence (left panels) and presence (right panels) of perampanel (30 µM). (B) Mean ± S.E.M. peak amplitude of currents evoked by 10 and 100 µM NMDA during control conditions (bath perfusion) and in the presence of perampanel. (C) Mean ± S.E.M. late amplitude of currents evoked by 10 and 100 µM NMDA during control conditions (bath perfusion) and in the presence of perampanel. Each bar represents data from 6 neurons.
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