Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol

Abstract

Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 μM Navβ4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.

Keywords: Dravet syndrome; GEFS+; VGSC; cannabidiol; resurgent current.

Resurgent sodium currents arise from channel reopening during repolarisation, and are predicted to increase neuronal excitability. Patel et al. show that epilepsy-associated mutations in the voltage-gated sodium channel Na_v1.6, but not Na_v1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy.

Figure 1

Biophysical characterization of hNa_v1.1 wild-type, R1648H and N1788K mutant channels. (A) Linear schematic of the structure of the VGSC α-subunit depicting the locations of the hNa_v1.1 R1648H (green circle) and hNa_v1.1 N1788K (orange circle) mutations. (B) Representative family of current traces generated by hNa_v1.1 wild-type (WT), R1648H and N1788K expressing HEK293T cells. Currents were elicited with step depolarizations ranging from −80 mV to +80 mV for 50 ms from a holding potential of −100 mV. Peak current traces are in bold. (C) Current density–voltage curve for hNa_v1.1 wild-type (blue squares), R1648H (green circles) and N1788K (orange triangles). Current density values were calculated by normalizing the peak sodium current at each voltage to the cell capacitance and subsequently averaged across cells. (D) Voltage dependence of steady-state activation and inactivation curves fit with a Boltzmann function. Steady-state inactivation was measured using a protocol in which cells were held at a series of voltages ranging from −120 mV to +30 mV for 500 ms followed by a 20-ms step pulse to +10 mV to measure channel availability (inset). (E) Persistent current amplitude plotted as a function of voltage. Persistent current was measured at 180 ms into current traces elicited by 200-ms step depolarizations ranging from −80 mV to +30 mV from a holding potential of −100 mV (inset). (F) Resurgent currents were elicited with a step depolarization from −100 mV to +60 mV for 20 ms to open channels allowing them to undergo open-channel block and subsequently repolarizing to a series of potentials ranging from +25 mV to −80 mV for 50 ms to allow the blocker to unbind. (G) Representative family of resurgent current traces generated by hNa_v1.1 wild-type (top), R1648H (middle) and N1788K (bottom). Peak resurgent current traces are bolded. (H) Per cent resurgent current plotted as a function of the repolarization voltage for hNa_v1.1 wild-type (blue squares; n = 29), R1648H (green circles; n = 17) and N1788K (orange triangles; n = 11). Per cent resurgent current was calculated by normalizing peak resurgent current at each voltage to peak transient current measured with a single step pulse from −120 mV to +10 mV. *P < 0.05; two-way ANOVA.

Figure 2

Biophysical characterization of hNa_v1.6 wild-type, L1331V and N1768D mutant channels. (A) Linear schematic of the structure of the VGSC α-subunit depicting the locations of the hNa_v1.6 L1331V (purple circle) and N1768D (pink circle) mutations. (B) Representative family of current traces generated by HEK293T cells expressing hNa_v1.6 wild-type (WT), L1331V and N1768D channels. Peak current traces are in bold. (C) Plot of current density versus voltage. (D) Steady-state inactivation and activation curves fit with a Boltzmann function. Inset: Magnification of the voltage-dependence of inactivation curve between −20 mV to +30 mV depicting incomplete inactivation of mutant channels. (E) Peak persistent current amplitude plotted as a function of voltage. (F) Representative family of resurgent current traces generated by hNa_v1.6 wild-type (top), L1331V (middle) and N1768D (bottom). Peak resurgent current traces are in bold. (G) Per cent resurgent current plotted as a function of the repolarization voltage for hNa_v1.6 wild-type (black squares; n = 20), L1331V (purple triangles; n = 11) and N1768D (pink circles; n = 14). *P < 0.05; two-way ANOVA.

Figure 3

Resurgent and persistent current generation by reciprocal epilepsy-associated mutation in non-native channel isoform. (A) Position of epilepsy-associated mutations in the linear schematic of the VGSC α- subunit and in the sequence alignment of hNa_v1.1 and hNa_v1.6. (B and C) Per cent resurgent current and peak persistent current amplitude generated by hNa_v1.1 wild-type (WT) (blue squares), hNa_v1.1 N1788K (orange triangles) and the reciprocal N1788D mutation in hNa_v1.1 (open pink circles). (D and E) Per cent resurgent current and peak persistent current amplitude generated by hNa_v1.6 wild-type (black squares), hNa_v1.6 N1768D (pink circles) and the reciprocal N1768K mutation in hNa_v1.6 (open orange triangles).

Figure 4

Effects of anandamide and cannabidiol on peak current density and resurgent current generated by wild-type hNa_v1.1 and hNa_v1.6 channels. (A and B) Effects of 5 μM anandamide (AEA) (n = 14) and 1μM cannabidiol (CBD) (n = 26–28) on peak current density and peak resurgent current generated by wild-type hNa_v1.1. (C and D) Effects of 5 μM AEA (n = 12–13) and 1 μM CBD (n = 26–30) on peak current density and peak resurgent current generated by hNa_v1.6. *P < 0.05, **P < 0.01; unpaired t-test.

Figure 5

Effects of 1 µM cannabidiol on hNa_v1.6 L1331V generated currents. (A) Representative family of current traces generated by hNa_v1.6 L1331V in presence of vehicle (left) and 1 μM cannabidiol (CBD) (right). (B) Current density curve showing no statistical difference in the peak current density between vehicle (purple triangles; n = 9) and CBD (black triangles; n = 10). (C) Representative family of resurgent current traces generated by hNa_v1.6 L1331V in presence of vehicle (left) and 1 μM CBD (right). Peak resurgent current traces are bolded. (D) Per cent resurgent current plotted against voltage. (E) Steady-state activation and inactivation curves fit with a Boltzmann function. (F) Normalized available current plotted against recovery duration and fit with an exponential function. Recovery from fast inactivation was measured by applying an initial depolarizing step to 0 mV to assess the peak current and then repolarizing to −80 mV for increasing durations followed by a final test pulse 0 mV to measure channel availability (inset). (G) Persistent current amplitude plotted versus voltage. *P < 0.05; two-way ANOVA.

Figure 6

Effects of 1 µM cannabidiol on hNa_v1.6 N1768D generated currents. (A) Representative family of current traces generated by hNa_v1.6 N1768D in the presence of vehicle (left) and cannabidiol (CBD) (right). (B) Current density curves for hNa_v1.6 N1768D in the presence of vehicle (pink circles; n = 20) and the presence of CBD (black circles; n = 19). (C) Representative family of resurgent current traces generated by hNa_v1.6 N1768D in the presence of vehicle (left) and CBD (right). Peak resurgent current traces are in bold. (D) Summary data showing the per cent resurgent current versus the repolarization voltage. (E) Steady-state activation and inactivation curves fit with a Boltzmann function. (F) Normalized available current plotted versus recovery duration and fit with an exponential function. (G) Persistent current amplitude plotted against voltage. *P < 0.05, two-way ANOVA.

Figure 7

Effects of 1 µM cannabidiol on endogenous sodium currents recorded from striatal neurons. (A) Peak transient current for vehicle (red) and 1 μM cannabidiol (CBD) (black). Peak transient current was measured with a step pulse from −80 mV to +10 mV from the steady-state inactivation protocol. (B) Representative family of resurgent current traces from striatal neurons in the presence of vehicle (left) and 1 μM cannabidiol (right). Peak resurgent current traces are in bold and magnified below. To elicit resurgent current in striatal neurons an initial prepulse to +30 mV for 20 ms was applied and followed by repolarizing steps ranging from +25 mV to −90 mV for 200 ms (inset). (C) Percent resurgent current plotted versus the repolarization voltage. (D) Steady-state inactivation curve fit with a Boltzmann function. Steady-state inactivation was measured using a prepulse ranging from −80 mV to +30 mV for 500 ms followed by a test pulse to +10 mV to measure channel availability (inset). (E) Normalized available current plotted versus recovery duration and fit with an exponential function. (F) Persistent current amplitude plotted as a function of voltage. *P < 0.05; two-way ANOVA.

Figure 8

Effects of 1 µM cannabidiol on excitability of striatal neurons. (A) Representative traces of activity evoked with a 200 ms stimulus of 100 pA and 200 pA for vehicle (top) and 1 μM cannabidiol (CBD) (bottom) from a holding potential of −60 mV (grey and black traces) and −80 mV (red and black traces). (B) Resting membrane potential of striatal neurons in the presence of vehicle (open, red) and 1 μM cannabidiol (CBD) (open, black) from all recorded cells (n = 47 and 45, respectively). (C) Input resistance calculated from the change in voltage with a 200 ms, −200 pA stimulus according to V = IR for vehicle (open, red) and 1 μM cannabidiol (open, black). (D) Number of action potentials elicited by a 200 ms stimulus of increasing intensity from 20 pA to 200 pA in 20 pA steps from a holding potential of −60 mV for vehicle (grey circles; n = 14) and cannabidiol (black squares; n = 12). (E) Action potential peak was measured at the current threshold with a holding potential of −60 mV for vehicle (grey) and 1 μM cannabidiol (black). (F) Current threshold measured using a 1 ms stimulus increasing incrementally from 0 pA to 1 nA in 20 pA steps with a holding potential of −60 mV. (G) Number of action potentials elicited by a 200 ms stimulus of increasing intensity from 20 pA to 200 pA in 20 pA steps from a holding potential of −80 mV for vehicle (red circles; n = 33) and cannabidiol (black squares; n = 33). (H) Action potential peak was measured at the current threshold with a holding potential of −80 mV for vehicle (red) and 1 μM cannabidiol (black). (I) Current threshold measured using a 1 ms stimulus increasing incrementally from 0 pA to 2 nA in 40 pA steps with a holding potential of −80 mV. *P < 0.05, **P < 0.01, ^##P < 0.0001; unpaired t-test and two-way ANOVA.