Commentary
Aberrant Epilepsy-Associated Mutant Nav1.6 Sodium Channel Activity Can Be Targeted With Cannabidiol.
Patel RR, Barbosa C, Brustovetsky T, Brustovetsky N, Cummins TR. Brain 2016;139(pt 8):2164–2168. [PubMed]
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.
Mutations in voltage-gated sodium channel genes are responsible for several epilepsy syndromes with a wide spectrum of clinical severity. For example, genetic epilepsy with febrile seizures plus (GEFS+) is relatively mild and has been associated with SCN1A missense mutations that encode NaV1.1 channels with subtle biophysical defects (1–4). At the other end of the spectrum, Dravet syndrome is a severe epileptic encephalopathy with multiple seizure types and impairment of psycho-motor and cognitive development, most often caused by de novo SCN1A mutations resulting in loss of NaV1.1 function (4, 5). SCN8A epileptic encephalopathy is an emerging syndrome with multiple seizure types and cognitive impairment caused by de novo gain-of-function mutations in the SCN8A gene that encodes the NaV1.6 voltage-gated sodium channel (6, 7). The gain-of-function effects include increased resurgent sodium current that is predicted to increase neuronal excitability.
Cannabidiol has received abundant media attention as a potential therapy for intractable epilepsy, based mainly on anecdotal evidence. Although it has shown some promise in an open-label trial of severe childhood-onset intractable epilepsy (8), the results of a randomized double-blind placebo-controlled trial are yet to be published. Another point of uncertainty surrounding cannabidiol is the mechanism underlying the potential anticonvulsant effect. Interestingly, other cannabinoids have been shown to inhibit voltage-gated sodium channels and, in some cases, preferentially inhibit resurgent current over peak transient current (9,10). In the current study, Patel and colleagues perform a detailed neuro-physiological characterization of epilepsy-associated NaV1.1 and NaV1.6 mutations to determine if they result in aberrant resurgent current using heterologous expression of wild-type and mutant sodium channels. Additionally, they examined whether cannabidiol preferentially inhibits resurgent sodium current of both wild-type and mutant sodium channels. Finally, they probed the effect of cannabidiol on intrinsic firing properties of cultured striatal neurons, a neuronal population with significant resurgent current.
A major question of this study is whether epilepsy syndromes resulting from mutations in NaV1.1 or NaV1.6 arise via distinct mechanisms—specifically, the effects of these mutations on resurgent current. To measure channel biophysical properties, the authors expressed either wild-type NaV1.1 or NaV1.6, or NaV1.1-R1648H, NaV1.1-N1788K, NaV1.6-L1331V, or NaV1.6-N1768D in HEK293T cells. The authors found that mutations in NaV1.1 showed predominantly loss-of-function effects consistent with previous observations for epilepsies that result from SCN1Amutations (3–5). The Na V1.1-R1648H mutation, associated with GEFS+, showed a slight hyperpolarized shift in the voltage-dependence of inactivation, while other biophysical properties —peak current density; voltage-dependence of activation; voltage-dependence, recovery from and onset of slow inactivation—remained indistinguishable from wild-type channels. The Dravet syndrome mutation, NaV1.1-N1788K, showed more overt loss-of-function, including reduced peak current density, a depolarized shift in voltage-dependence of activation, and slowed recovery from slow inactivation. Neither mutation showed altered resurgent current, suggesting that resurgent current may not significantly contribute to SCN1A-related epilepsies.
In contrast, mutations in SCN8A showed a mix of gain- and loss-of-function phenotypes. NaV1.6-L1331V showed incomplete channel inactivation, as well as a depolarized voltage-dependence and slower onset of slow inactivation, all of which are consistent with enhanced channel function. NaV1.6-N1768D, showed a depolarized shift in voltage-dependence of fast inactivation, consistent with a gain-of-function phenotype, but enhanced entry into and slowed recovery from slow inactivation, consistent with reduced function. Importantly, however, both NaV1.6 mutations showed elevated resurgent current, while NaV1.6-N1768D also showed increased persistent sodium current compared to WT channels, both of which would contribute to enhanced neuronal excitability. These results suggest that SCN1A– and SCN8A-related epilepsies occur via distinct mechanisms. It is interesting to note that NaV1.1-N1788 and NaV1.6-N1768 lie in analogous positions, and analysis of the reciprocal mutations NaV1.1-N1788D showed increased resurgent current, while NaV1.6-N1768K showed resurgent current indistinguishable from wild-type channels. This suggests that the nature of the mutation—not the channel isoform—underlies the development of resurgent sodium current.
Patel and colleagues next tested whether endogenous cannabinoids or cannabidiol could preferentially inhibit resurgent current mediated by either WT or mutant sodium channels. Anandamide, an endocannabinoid that has been shown to inhibit resurgent current mediated by NaV1.7, surprisingly had no effect on resurgent current mediated by NaV1.6, although peak sodium current was significantly inhibited. However, cannabidiol (1μM) significantly inhibited resurgent current, while having no effect on peak transient current, suggesting that selective targeting of resurgent current may be therapeutic. To further corroborate this hypothesis, Patel et al. tested the effects of cannabidiol on the NaV1.6 mutations L1331V and N1768D. For both mutations, cannabidiol selectively inhibited resurgent current, having only modest effects on peak transient current mediated by NaV1.6-L1331V. Interestingly, cannabidiol also impaired some biophysical properties of mutant channels that were unaffected when wild-type channels were exposed to cannabidiol. Cannabidiol elicited a depolarized shift in the voltage-dependence of channel activation for NaV1.6-L1331V and slowed recovery from channel inactivation for both NaV1.6-L1331V, and NaV1.6-N1768D. Additionally, cannabidiol inhibited persistent sodium current mediated by NaV1.6-N1768D. This suggests that cannabidiol exerts multiple effect on sodium channel biophysical properties, all of which would reduce neuronal excitability.
The reported experiments show that cannabidiol is able to selectively inhibit resurgent sodium current mediated by wild-type and mutant sodium channels using a heterologous expression system. However, it must be noted that resurgent current in native cells is mediated by the NaVβ4 subunit, while it is induced in heterologous systems by inclusion of a small peptide fragment of the β4 subunit. Therefore, in a final set of experiments, the authors examined the effects of cannabidiol on cultured striatal neurons, a neuronal population with significant native resurgent current. Similar to observations in heterologous cell experiments, Patel et al. found that cannabidiol selectively inhibited resurgent and persistent sodium current in striatal neurons, induced a slight depolarized shift in the voltage-dependence of activation, and slowed recovery from fast inactivation. Additionally, current clamp experiments revealed that cannabidiol impaired excitability, reducing the number of action potentials in response to a depolarizing stimulus when measured from a membrane potential of either −60 or −80 mV, as well as reducing action potential height and increasing action potential threshold only when measured from a membrane potential of −60 mV. This suggests that the slight shift in the voltage-dependence of inactivation may play a significant role in how cannabidiol regulates neuronal excitability.
Overall, this study is a step forward in understanding the potential contribution of resurgent sodium current to the pathophysiology of some epilepsy syndromes. Additionally, it may help explain how cannabidiol provides benefit to some epilepsy patients, and it suggests resurgent current as a therapeutic target for development of alternative therapies with less baggage than cannabidiol. Further experiments are needed to examine the effects of cannabidiol on specific sodium channel mutations in native neuronal populations important to epileptogenesis. Finally, whole animal studies will be critical, as cannabidiol likely exerts effects on a number of other neuronal proteins.
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