Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability

Resurgent and Gating Pore Currents Induced by De Novo SCN2A Epilepsy Mutations

Over 150 mutations in the SCN2A gene, which encodes the neuronal Nav1.2 protein, have been implicated in human epilepsy cases. Of these, R1882Q and R853Q are two of the most commonly reported mutations. This study utilized voltage-clamp electrophysiology to characterize the biophysical effects of the R1882Q and R853Q mutations on the hNav1.2 channel, including their effects on resurgent current and gating pore current, which are not typically investigated in the study of Nav1.2 channel mutations. HEK cells transiently transfected with DNA encoding either wild-type (WT) or mutant hNav1.2 revealed that the R1882Q mutation induced a gain-of-function phenotype, including slowed fast inactivation, depolarization of the voltage dependence of inactivation, and increased persistent current. In this model system, the R853Q mutation primarily produced loss-of-function effects, including reduced transient current amplitude and density, hyperpolarization of the voltage dependence of inactivation, and decreased persistent current. The presence of a Navβ4 peptide (KKLITFILKKTREK-OH) in the pipette solution induced resurgent currents, which were increased by the R1882Q mutation and decreased by the R853Q mutation. Further study of the R853Q mutation in Xenopus oocytes indicated a reduced surface expression and revealed a robust gating pore current at negative membrane potentials, a function absent in the WT channel. This not only shows that different epileptogenic point mutations in hNav1.2 have distinct biophysical effects on the channel, but also illustrates that individual mutations can have complex consequences that are difficult to identify using conventional analyses. Distinct mutations may, therefore, require tailored pharmacotherapies in order to eliminate seizures

Navβ4 regulates fast resurgent sodium currents and excitability in sensory neurons

BACKGROUND: Increased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navβ4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navβ4 regulates INaR in DRG sensory neurons. RESULTS: Our immunocytochemistry studies show that Navβ4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navβ4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navβ4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navβ2 nor overexpression of a Navβ4-mutant, predicted to be an inactive form of Navβ4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navβ4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navβ4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navβ4 decreased INaR generation. CONCLUSION: INaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navβ4 as an important regulator of INaR and excitability in sensory neurons. As such, Navβ4 is a potential target for the manipulation of pain sensations

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

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 Nav1.6, but not Nav1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy., 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 Nav1.6, but not Nav1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy. , 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

Effects of the Navβ4 peptide on use-dependent reduction with 10 Hz stimulation.

<p>A, Representative traces of use-dependent reduction traces generated by hNav1.1 (left) and hNav1.6 (right) when pulsed at +30mV. Use-dependent reduction was examined with an initial and final 20ms step pulse to -10mV from -80mV to assess the current available before and after 19 consecutive step depolarization from -80mV for 80ms to +30mV or +60mV for 20ms. B, Percent inhibition was calculated between the initial and final currents for cells in the absence (dark grey bars) and presence (light grey bars) of Navβ4 peptide (<i>n</i> = 8–9). <i>Inset</i>, Abbreviated protocol used to measure use-dependent reduction. *p < 0.05 compared to corresponding hNav1.1 group.</p

Rate and fraction of recovery from fast inactivation by hNav1.1 and hNav1.6.

<p>A, Representative traces of recovery from fast inactivation measured by first inducing fast inactivation from a holding potential of -100mV with a 20ms step pulse to 0mV and then applying a 20ms test pulse to 0mV subsequent to various recovery times at -70mV. <i>Inset</i>, Protocol used to measure recovery from fast inactivation. B, hNav1.6 (blue circles; <i>n</i> = 17) has a smaller time constant for recovery at -70mV compared to hNav1.1 (black squares; <i>n</i> = 19). C, Maximal fraction recovered from fast inactivation was greater for hNav1.1 at voltage ranging from -90mV to -70mV compared to hNav1.6 (Unpaired t-test, *p < 0.05).</p

Resurgent current generation and kinetics of inactivation of hNav1.1 and hNav1.6.

<p>A, Representative family of resurgent current traces from hNav1.1 (left) and hNav1.6 (right) with Navβ4 peptide in the pipette solution. Currents were elicited by depolarization to +60mV for 20ms followed by repolarization to potentials ranging from +25mV to -80mV for 50ms. B, Protocol used to measure resurgent currents. C, Current-voltage curve of peak resurgent current normalized to peak transient current measured at +10mV by hNav1.1 (purple squares; <i>n</i> = 15) and hNav1.6 (green circles; <i>n</i> = 15). D, Representative normalized current traces elicited by a step depolarization from -100mV to -10mV by hNav1.1 (black) and hNav1.6 (blue) without Navβ4 peptide in the pipette solution. E, Averaged decay time constants measured at potentials ranging from -20mV to +20mV from hNav1.1 (black square; <i>n</i> = 14) and hNav1.6 (blue circles; <i>n</i> = 14) (Unpaired t-test, *p < 0.05).</p

Summary of activation and inactivation gating parameters.

<p>* p < 0.05 Compared to hNav1.1</p><p>Summary of activation and inactivation gating parameters.</p

Current-voltage relationship, voltage-dependence of steady-state activation and deactivation kinetics of hNav1.1 and hNav1.6.

<p>A, Representative current traces recorded from hNav1.1 (left) and hNav1.6 (right) expressed in HEK293T cells. The currents were elicited by applying 50ms step-depolarization to potentials ranging from -80mV to +80mV from a holding potential of -100mV. <i>Inset</i>, Protocol used to obtain current-voltage traces. B, Normalized peak current-voltage relationship for hNav1.1 (black squares; n = 14) and hNav1.6 (blue circles; n = 14). C, Voltage-dependence of steady-state activation shows no difference in conductance between hNav1.1 and hNav1.6. D, Representative traces showing hNav1.1 (black) and hNav1.6 (blue) deactivation tail currents at -70mV. <i>Inset</i>, Protocol used to elicit deactivation tail currents. E, Time constants of channel deactivation were similar for hNav1.1 and hNav1.6 at voltages ranging from -100mV to -60mV. Time constants were obtained by a brief 0.5ms depolarization to +10mV followed by a series of repolarizations to potentials from -100mV to -60mV eliciting tail currents that were fit to a single exponential function.</p

Sodium influx in response to fast and slow AP waveforms with and without Navβ4 peptide.

<p>A, Fast and slow voltage command waveforms modeled using NEURON. B, Area under the curve for currents elicited by the fast (left) and slow waveform (right) from cells expressing hNav1.1 (purple bars) and hNav1.6 (green bars) measured between 20 and 35ms (Unpaired t-test, *p < 0.05). Currents generated in response to fast and slow AP waveforms were normalized and then averaged. C, hNav1.1 generated sodium current in response to a fast voltage command waveform in the absence (black traces; <i>n</i> = 17) and presence (purple traces; <i>n</i> = 14) of Navβ4 peptide. D, Response of hNav1.1 to a slow voltage command waveform. E, hNav1.6 generated sodium current in response to a fast voltage command waveform in the absence (blue trace; <i>n</i> = 14) and presence (green trace; <i>n</i> = 15) of Navβ4 peptide. F, Response of hNav1.6 to a slow voltage command waveform. Grey boxes represent regions of statistically significant differences (Unpaired t-test, p < 0.05).</p