Mutational Electrophysiological Characterization Of The Antiepileptic Target Nav1.6 Channel And Small Molecule Inhibitor Discovery

Epilepsy is a chronic brain disorder characterized by malfunction of the brain caused by abnormal synchronous discharges of neurons.The sodium channel Nav 1.6is highly concentrated at glutamatergic excitatory neurons of the central nervous system and is essential for the efficient initiation and propagation of action potential firing.Human genetic studies have shown that gain of function mutations of Nav1.6 is related to several types of epileptics such as benign familial neonatal infantile epilepsy and developmental epileptic encephalopathy.The conditional mouse with gain-of-function mutations resulted in early-onset seizures and death.Scn8 a knockdown reduced the threshold and development of spontaneous seizures in mice compared to wild type.Therefore,the Nav1.6 channel is considered as a potential antiepileptic drug target,and the discovery of channel-selective inhibitors could help in the development of antiepileptic drugs.Although over 200 Nav1.6 mutations have been linked to epileptic encephalopathy,only few mutations have ever been functionally characterized.In addition,there are few small molecule inhibitors targeting Nav1.6,which hinders the development of antiepileptic drugs targeting this channel.Most of the mutated residues in patients are located in the highly conserved portions of the protein: the domains containing transmembrane segments,inactivation gate,and proximal 2/3 of the C-terminus of Nav1.6.Most of them are gain-of-function mutations that usually include premature opening or impaired inactivation and increase the persistent current.We constructed 25 Nav1.6 mutants located in highly conserved portions of the protein by site-directed mutagenesis techniques.Combined with membrane clamp techniques,we performed systematic electrophysiological characterization of the mutations that have not been functionally reported.Consistent with previous reports,we found that most of the epilepsy-associated mutants both improved premature opening,impaired inactivation,or increased persistent sodium currents.However,few mutants(e.g.,N1466 T,N1466K,and F1754S)only significantly increased the persistent current of the channel without significant effects on the other channel characteristics.This result suggests that persistent current may be an important channel feature for epilepsy-related mutants.The N1466 T and N1466 K mutations occurred at the same residue and were located at the inactivation gate.The clinical study showed that patients with the N1466T/K mutations have different onset times and clinical features.In addition to significantly increasing the persistent current and ramp current of the channel,the two mutations shift voltage-dependent slow inactivation toward the hyperpolarized direction.The current density and voltage-dependent activation and inactivation curves failed to show statistically significant differences among WT and two mutants.The N1466 K mutation significantly increased the time constant of fast inactivation and slow inactivation and increased the recovery from fast inactivation.However,the N1466 T only increased the recovery from fast inactivation but had no significant effect on the time constant of inactivation.These data suggest that N1466 T and N1466 K are gain of function mutations.Nonselective sodium channel inhibitors are commonly used as antiepileptic drugs in clinical practice,and topiramate was shown to be clinically effective in patients carrying N1466 T or N1466 K mutations.We investigated the effect of topiramate on N1466 T and N1466 K and found that topiramate selectively inhibited persistent current and ramp current without effect on transient current.This indicated that inhibition of persistent current and ramp current may be an important mechanism for the treatment of these two mutations.Based on this,we further evaluated the effects of other antiepileptic drugs oxcarbazepine,lacosamide,carbamazepine,phenytoin,lamotrigine and valproic acid.The results showed that carbamazepine and phenytoin significantly enhanced the inhibition of persistent current and ramp current in N1466 T and N1466 K mutants compared with topiramate.Our findings suggested that carbamazepine and phenytoin may act as alternative therapies for epilepsy patients,particularly for those carrying N1466 T and N1466 K mutations.Based on the important role of Nav1.6 channels in the generation and treatment of epilepsy,we have collaborated with medicinal chemists to discover small molecule inhibitors.Firstly,we detected compounds with structural similarity to the clinical drug and identified several small molecules with inhibitory efficacy on the Nav1.6 channel.Systematic conformational optimization was carried out using these active molecules as prototypes,and several inhibitors with strong inhibitory effects on Nav1.6 channels,represented by NK728,were further identified.Those inhibitors can reduce the excitability of primary neurons in vitro.The antiepileptic activity of the above molecules was evaluated by the maximal electroshock model,and several compounds were identified that could effectively inhibit seizures in mice.This part of the work lays the foundation for the development of antiepileptic candidate compounds targeting the Nav1.6 channel.