M-Channel Activation Contributes to the Anticonvulsant Action of the Ketone Body β-Hydroxybutyrate

Abstract

Ketogenic diets are effective therapies for refractory epilepsy, yet the underlying mechanisms are incompletely understood. The anticonvulsant efficacy of ketogenic diets correlates positively to the serum concentration of β-hydroxybutyrate (BHB), the primary ketone body generated by ketosis. Voltage-gated potassium channels generated by KCNQ2-5 subunits, especially KCNQ2/3 heteromers, generate the M-current, a therapeutic target for synthetic anticonvulsants. Here, we report that BHB directly activates KCNQ2/3 channels (EC₅₀ = 0.7 µM), via a highly conserved S5 tryptophan (W265) on KCNQ3. BHB was also acutely effective as an anticonvulsant in the pentylene tetrazole (PTZ) seizure assay in mice. Strikingly, coadministration of γ-amino-β-hydroxybutyric acid, a high-affinity KCNQ2/3 partial agonist that also acts via KCNQ3-W265, similarly reduced the efficacy of BHB in KCNQ2/3 channel activation in vitro and in the PTZ seizure assay in vivo. Our results uncover a novel, unexpected molecular basis for anticonvulsant effects of the major ketone body induced by ketosis. SIGNIFICANCE STATEMENT: Ketogenic diets are used to treat refractory epilepsy but the therapeutic mechanism is not fully understood. Here, we show that clinically relevant concentrations of β-hydroxybutyrate, the primary ketone body generated during ketogenesis, activates KCNQ2/3 potassium channels by binding to a specific site on KCNQ3, an effect known to reduce neuronal excitability. We provide evidence using a mouse chemoconvulsant model that KCNQ2/3 activation contributes to the antiepileptic action of β-hydroxybutyrate.

Graphical abstract

Fig. 1.

BHB activates KCNQ2/3 potassium channels. (A) Schematic illustration of the generation of BHB during ketosis. FFA, free fatty acids; LPL, lipoprotein lipase; red areas, blood vessel; TCA, tricarboxylic acid cycle; TG, triglycerides. (B) Electrostatic surface potentials (red, electron dense; blue, electron poor; green, neutral) and structures calculated and plotted using Jmol. (C) TEVC recording of water-injected Xenopus laevis oocytes. Mean traces show no effect of BHB (100 µM) on endogenous currents (n = 5). Dashed line here and throughout, zero current level. Upper inset, voltage protocol. (D) Left panel, mean I/V relationship from water-injected traces as in (C) (n = 5). Right panel, mean current fold change in response to BHB (100 µM) in water-injected oocytes (n = 5). Error bars indicate S.E.M. (E–J) TEVC recordings of KCNQ2/3 expressed in Xenopus oocytes in the absence (control) or presence of BHB (n = 5). Dashed line here and throughout, zero current level. Voltage protocol as in (C). Error bars indicate S.E.M. (E) Mean traces. (F) Mean tail current vs. prepulse voltage [recorded here and throughout at arrow in (E)]. (G) Normalized tail currents [conductance/maximal conductance (G/G_max); recorded here and throughout at arrow in (E)] vs. prepulse voltage. (H) Voltage dependence of BHB augmentation of KCNQ2/3 activity. (I) Dose-dependent fold increase in current at −60 mV in response to BHB. (J) Dose-dependent changes in resting membrane potential (E_M) in response to BHB.

Fig. 2.

BHB does not alter homomeric KCNQ2 activity. TEVC recording of KCNQ2 expressed in Xenopus oocytes in the absence (control) or presence of BHB (100 µM or 1 mM) (n = 5). Error bars indicate S.E.M. (A) Mean traces (voltage protocol, upper inset). (B) Mean peak I/V relationships. (C) Mean tail current vs. prepulse voltage. (D) Mean normalized tail current [conductance/maximal conductance (G/G_max)] vs. prepulse voltage.

Fig. 3.

BHB activates homomeric KCNQ3* potassium channels. TEVC recording of KCNQ3* expressed in Xenopus oocytes in the absence (control) or presence of BHB (10 nM–10 mM) (n = 6). Error bars indicate S.E.M. (A) Mean traces (voltage protocol, upper inset). (B) Mean tail current vs. prepulse voltage. (C) Mean normalized tail current [conductance/maximal conductance (G/G_max)] vs. prepulse voltage. (D) Dose response of BHB vs. KCNQ3* current fold increase at −60 mV. (E) Voltage dependence of BHB effects on KCNQ3* activity.

Fig. 4.

BHB activation of KCNQ2/3 requires an S5 tryptophan. (A) Upper panel, chimeric KCNQ1/KCNQ3 structural model (red pentagon, KCNQ3-W265); lower panel, schematic illustration showing membrane topology; domain coloring: blue, VSD; pink, S4-5 linker; purple, S5; light gray, S6. VSD, voltage-sensing domain; pentagon, KCNQ3-W265. (B) Close-up side view of KCNQ structure as in (A) showing results of SwissDock docking of retigabine in the structural model. (C) Close-up side view of KCNQ structure as in (A) showing results of SwissDock docking of BHB in the structural model. (D) Close-up view of KCNQ structure from extracellular face, showing results of SwissDock docking of BHB in the structural model. (E–G) TEVC recordings of Xenopus laevis oocytes showing effects of BHB (100 µM–4 mM) on heteromeric KCNQ2-W236L/KCNQ3-W265L channels; n = 6; voltage protocol as in Fig. 3. Error bars indicate S.E.M. (E) Mean tail current vs. prepulse voltage. (F) Mean normalized tail current [conductance/maximal conductance (G/G_max)] vs. prepulse voltage. (G) Dose response of BHB vs. KCNQ2-W236L/KCNQ3-W265L current fold increase at −60 mV.

Fig. 5.

GABOB diminishes BHB activation of KCNQ2/3. (A) GABOB electrostatic surface potential (red, electron dense; blue, electron poor; green, neutral) and structure calculated and plotted using Jmol. (B) Left panel, side view of KCNQ1/3 chimera model structure, showing results of SwissDock docking of GABOB; right panel, view of KCNQ1/3 chimera model structure from the extracellular face, showing results of SwissDock docking of GABOB. (C–G) TEVC recordings of Xenopus laevis oocytes showing effects of BHB (10 µM) in the presence of GABOB (100 µM) on heteromeric KCNQ2/3 channels; n = 5. Error bars indicate S.E.M. (C) Mean traces (voltage protocol, upper inset). (D) Peak current. (E) Mean tail current vs. prepulse voltage. (F) Fold change in tail current vs. prepulse for 10 µM BHB with 100 µM GABOB vs. without 100 µM GABOB. (G) Mean normalized tail current [conductance/maximal conductance (G/G_max)] vs. prepulse voltage.

Fig. 6.

GABOB diminishes anticonvulsant effects of BHB. Comparison of anticonvulsant effects of vehicle vs. 40 or 200 mg/kg BHB, in the absence (n = 8) or presence (n = 7–11) of 200 mg/kg GABOB, intraperitoneally injected 30 minutes before intraperitoneal injection of 80 mg/kg PTZ. Seizures were quantified during the first 20 minutes post-PTZ injection: (A) latency to first tail flick or seizure; ***P = 0.001 versus vehicle control. (B) clonic seizure episodes; (C) tonic seizure episodes; *P = 0.02 versus vehicle control. (D) seizure-related mortality; *P = 0.02 versus vehicle control. All other comparisons P>0.05 versus vehicle control.