Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

Abstract

Background and purpose: Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

Experimental approach: Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K(IR)3 and K(V)7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na(+), Ca(2+), inward rectifier K(+), K(V)7 K(+), and TRPV1 channels, as well as GABA(A), glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

Key results: Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na(+) or Ca(2+) channels, inward rectifier K(+) channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K(V)7 K(+) channels to more negative potentials and the gating of GABA(A) receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K(V)7 channels and GABA(A) receptors was comparable, whereas in hippocampal neurons the effects on K(V)7 channels were more pronounced.

Conclusions and implications: These results indicate that flupirtine exerts its analgesic action by acting on both GABA(A) receptors and K(V)7 channels.

Figure 1

Effects of flupirtine on recombinant and native G-protein coupled inwardly rectifying K channels. (A, B) K_IR3.1/3.2 concatemers and P2Y₁₂ receptors were co-expressed in tsA cells. Currents were evoked by ramp depolarizations from −140 to 40 mV during periods of 200 ms. (A) Representative traces of recordings in the absence (control) or presence of ADP 100 µM, either with solvent or with 30 µM flupirtine. (B) Amplitudes of inward currents were determined at −140 mV; normalized amplitudes of currents obtained either in solvent or in 30 µM flupirtine, either alone (control) or together with 100 µM ADP, are shown (n = 7). (C, D) Currents were evoked in hippocampal neurons by 200 ms hyperpolarizations from a holding potential of −70 mV to potentials ranging from −80 to −140 mV with 10 mV increments as published previously (Jakob and Krieglstein, 1997). This pulse protocol was applied in the presence of either solvent or 100 µM baclofen, 10 µM flupirtine and 1 mM BaCl₂, respectively. (C) Representative original traces. (D) Summary of results obtained in nine different neurons; for each neuron, all amplitude values were normalized to the amplitude determined in the presence of solvent at a potential of −80 mV. *** indicates statistically significant differences versus currents in the presence of solvent at P < 0.001 as determined by a two way ANOVA.

Figure 2

Effects of flupirtine on heterologously expressed K_V7 channels. Human K_V7.2 and 7.3 were co-expressed in tsA cells. Currents were evoked by ramp hyperpolarizations from −25 to −100 mV during periods of 1 s. (A) Representative current traces in the presence of solvent and 3 or 30 µM flupirtine, respectively. (B) Current amplitudes were determined at −30 mV in the presence of solvent or increasing concentrations of flupirtine; the average of normalized current amplitudes is shown for seven cells.

Figure 3

Effects of flupirtine on various ligand-gated ion channels. The indicated concentrations of (A) glutamate (Glu) or (B) NMDA were applied to hippocampal neurons. (C) Capsaicin was applied to DRG neurons. (D) Rat α3 and β4 subunits of nicotinic acetylcholine receptors were expressed in tsA cells, and the indicated concentration of ACh was applied. All cells were clamped at a voltage of −70 mV using the perforated patch-clamp technique. The representative current traces shown were obtained in the presence of either solvent (control) or 30 µM flupirtine. A summary of the effects of flupirtine is given in Table 2.

Figure 4

Effects of flupirtine on GABA_A and glycine receptors in hippocampal neurons. Neurons were clamped at −70 mV and increasing concentrations of GABA or glycine were applied for 3 to 5 s in the presence of either solvent or 30 µM flupirtine. (A and C) Original traces evoked by 3 µM GABA and 300 µM glycine, respectively. (B and D) Concentration–response curves for currents induced by GABA (n = 5 to 11) and glycine (n = 5), respectively. Amplitudes evoked by different agonist concentrations in solvent or flupirtine were normalized to those evoked in solvent in the very same neuron by 30 µM GABA and 100 µM glycine, respectively. Values for statistical differences (F-test) between EC₅₀ values in the presence of either solvent or flupirtine are P < 0.001 for GABA and P > 0.8 for glycine.

Figure 5

Comparison of the effects of flupirtine on K_V7 channels in hippocampal, DRG and dorsal horn neurons. Cultured hippocampal (HC), DRG or dorsal horn (DH) neurons were clamped at −30 mV and hyperpolarized to −55 mV for 1 s periods every 10 s in order to de-activate K_V7 channels. Drugs were present for at least 8 s before their effects on the currents were determined. (A) Representative traces measured in a DRG neuron in the presence of solvent, 30 µM flupirtine or 30 µM linopirdine. The shift in the outward current at −30 mV is indicated by the arrows. (B) Normalized amplitudes of outward currents at −30 mV were measured in the presence of solvent or 30 µM linopirdine in hippocampal (n = 5), DRG (n = 6) and dorsal horn (n = 4) neurons. (C) Normalized amplitudes of currents measured at −30 mV in the absence and presence of increasing concentrations of flupirtine were determined in hippocampal (n = 9), DRG (n = 10) and dorsal horn (n = 8) neurons, respectively. EC₅₀ values were 6.1, 4.4 and 5.4 µM in hippocampal, DRG and dorsal horn neurons, respectively. The maximal effects observed in hippocampal neurons were significantly different from those in DRG or dorsal horn neurons at P < 0.01.

Figure 6

Comparison of the effects of flupirtine on GABA_A receptors in DRG, dorsal horn and SCG neurons. Neurons were clamped at −70 mV and increasing GABA concentrations were applied for 3 to 5 s in the presence of either solvent or 30 µM flupirtine. (A–C) Original traces evoked by 3 µM GABA in DRG, dorsal horn and SCG neurons, respectively. (D–F) Concentration–response curves obtained in DRG (n = 5 to 15), dorsal horn (n = 6 to 16) and SCG (n = 5 to 13) neurons. Amplitudes evoked by different GABA concentrations in solvent or flupirtine were normalized to those evoked by 30 µM GABA in solvent in the very same neuron. Values for statistical differences (F test) between EC₅₀ values in the presence of either solvent or flupirtine are P < 0.001 for DRG, P < 0.05 for dorsal horn and P < 0.001 for SCG neurons, respectively.

Figure 7

Comparison of the effects of therapeutic flupirtine concentrations in hippocampal, DRG and dorsal horn neurons. (A) The concentration-dependence of the effects of flupirtine on GABA_A receptors was investigated in hippocampal (HC; n = 6), DRG (n = 6 to 8) and dorsal horn (n = 5 to 8) neurons clamped at −70 mV using GABA concentrations corresponding to the calculated EC₅ values to induce currents; these EC₅ values were 0.4, 3 and 1 µM for hippocampal (HC), DRG and dorsal horn (DH) neurons, respectively. * indicates a significant difference versus the corresponding result in DRG and dorsal horn neurons at P < 0.05. (B) Outward currents at −30 mV or currents induced by GABA at the above EC₅ concentrations were determined in hippocampal (n = 6), DRG (n = 6 to 8) and dorsal horn (n = 4 to 5) neurons in the presence of either solvent or 3 µM flupirtine. The graph shows normalized current amplitudes in the presence of flupirtine; *** indicates a significant difference between the effects on the two types of currents as determined by a Kruskal–Wallis ANOVA followed by Dunn's multiple comparison test.