A novel role of the antidepressant paroxetine in inhibiting neuronal Kv7/M channels to enhance neuronal excitability

Figures

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  Fig. 1. Concentration-dependent inhibition of Kv7.2/Kv7.3 currents by paroxetine. A Left panel, representative Kv7.2/Kv7.3 current traces elicited by depolarizing potential at 0 mV before and after application of paroxetine (3 μM) or positive control XE991 (3 μM). The dotted line represents zero current level; Right panel, summary for normalized current from left panel (n = 5), paired t-test, ***P < 0.001. B Left panel, concentration-dependent inhibition of Kv7.2/Kv7.3 currents by paroxetine. Representative current traces from each concentration of paroxetine are shown in the inset; Right panel, data from the left panel were fitted with Logistic function and IC50 of 3.6 ± 0.2 μΜ was obtained (n = 5). C Left panels, representative current traces from Kv7.2/Kv7.3 channels in response to depolarizing voltage steps from −110–30 mV with an increment of 10 mV followed by a pulse at 0 mV in the absence (left panel) and presence (middle panel) of paroxetine; Right panel, activation curves of Kv7.2/Kv7.3 currents in the absence or presence of paroxetine measured at the beginning of the 0 mV and fitted with Boltzmann function (n = 8). D Superimposed activation traces generated at 0 mV (left panel)…

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  Fig. 2. Reduction of Kv7.2/Kv7.3 single channel open probability by paroxetine. A Representative single-channel (cell-attached) current traces recorded at 0 mV with or without 3 μM paroxetine. Letters O and C indicate channel open state (O) and closed state (C), respectively. B All-point amplitude histograms of single-channel currents in panel A. C Summary for effects of paroxetine on Kv7.2/Kv7.3 single-channel open probability and conductance. Paired t-test, ***P < 0.001; ns, no signification; n = 5.

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  Fig. 3. Concentration-dependent inhibition of Kv7.2 currents by paroxetine. A Left panel, Representative current traces for concentration-dependent inhibition of Kv7.2 by paroxetine. The dotted line represents zero current level; Right panel, data from the left panel were fitted with Logistic function and IC50 of 12.3 ± 6.5 μM was obtained (n = 9). B Representative current traces of Kv7.2 channels in response to depolarizing voltage steps followed by a pulse at 0 mV in the absence (left panel) and presence (middle panel) of paroxetine; Right panel, activation curves of Kv7.2 in the absence or presence of paroxetine measured at the beginning of the 0 mV and fitted with Boltzmann function (n = 6). C Superimposed activation traces generated at 0 mV (left panel) and deactivation traces at −120 mV (middle panel) before and after paroxetine (10 μΜ). The current traces from paroxetine were scaled up to the same current amplitude of the control (blue dotted line); Right panel, summary of the effect of paroxetine on activation and deactivation time constants of Kv7.2 fitted with a single exponential function (n = 6). All data were expressed as the mean±SEM. Paired t-test, *P < 0.05.

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  Fig. 4. Reduction of Kv7.2 single channel open probability by paroxetine. A Representative single-channel (cell-attached) current traces recorded at 0 mV with or without 10 μM paroxetine. B All-point amplitude histograms of single-channel currents in panel A. C Representative single-channel current traces before and after application of XE991 (3 μΜ). D Left and middle panels, all-point amplitude histograms of single-channel currents in panel C. Right panel, summary for effects of paroxetine and XE991 on Kv7.2 single-channel open probability and conductance. Paired t-test, ***P < 0.001, n = 4–5.

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  Fig. 5. Concentration-dependent inhibition of other Kv7 subtypes by paroxetine. A–C, Representative current traces of Kv7.1 (A), Kv7.4 (B), Kv7.5 (C) expressed in HEK293T cells before and after application of different concentrations of paroxetine. The dotted lines represent zero current level. D Concentration-dependent inhibition effect of paroxetine on Kv7 channels and fitted with Logistic function (n = 5–10).

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  Fig. 6. The effects of paroxetine on the voltage-dependent activation of other Kv7 channels. A–C, Left and middle panels show a family of voltage-dependent currents of each Kv7 channel in the absence (left panels) and presence (middle panels) of paroxetine. The dotted lines represent zero current level; Right panels, activation curves of Kv7 channels fitted by Boltzmann function without or with paroxetine (n = 6–7).

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  Fig. 7. Inhibition of native M-current and increase of firings in hippocampal neurons by paroxetine. A Left panel, representative M-current traces without or with 3 μM XE991, 10 μM paroxetine and 10 μM RTG recorded in hippocampal neurons. The dotted line represents the zero current level; Right panel, the inhibition effect of paroxetine (3 μM, n = 5; 10 μM, n = 16) and XE991 (3 μM, n = 10) on M-current measured at −20 mV. One-way ANOVA followed by Tukey test, ***P < 0.001. B Left panel, depolarization shift of resting membrane potentials (RMP) of hippocampal neurons by paroxetine (3 μM, n = 6; 10 μM, n = 21). Paired t-test, **P < 0.01, ***P < 0.001. Right panel, statistics for changes of RMP with paroxetine (3 μM and 10 μM), XE991 (1 μM and 3 μM) and RTG (10 μM). C Representative action potential traces before and after application of paroxetine (3 μM and 10 μM) and XE991 (3 μM). D Summary for increased firings by paroxetine (3 μM, n = 5; 10 μΜ, n = 7; left) and 3 μM XE991 (n = 6; right) in hippocampal neurons. Paired t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 8. The docking results of paroxetine to Kv7.2. A Side (left) and top (right) views of the predicted pocket of paroxetine binding to Kv7.2. B Overview of paroxetine binding to pocket and its zoom-in view of the binding pocket formed by residues F100 (S1), D212 (S4) and Y237 (S5, Adjacent α-subunit), the energy of amino acid residues in binding pocket is shown in the inset. C Left panels, representative current traces for concentration-dependent inhibition of D212A Kv7.2 and D212A Kv7.2/Kv7.3 by paroxetine. The dotted lines represent zero current level; Right panel, right shift of concentration-dependent inhibition of D212A Kv7.2 and D212A Kv7.2/Kv7.3 mutant channels by paroxetine in different concentrations (n = 5–8). D Representative current traces of WT Kv7.2, W236A, F240A, L299A, F304A and F305A Kv7.2 channel expressed in HEK293T cells before and after application of 10 or 30 µM paroxetine and summary of normalized inhibition effect is shown in the bottom right (n = 3–6). The dotted lines represent the zero current level. One-way ANOVA followed by Tukey test, **P < 0.01, ns, no signification.