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The voltage-sensitive sodium channel Na V 1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate Na V 1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of Na V 1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na + currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing Na V 1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous Na V 1.1 in primary cultures were identified by expressing a fluorescent protein under the Na V 1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (Na V 1.1), lead to DS. Na V 1.1 is primarily found in the axon initial segment of fast-spiking GABAergic inhibitory interneurons in the brain, and the principle mechanism proposed to underlie seizure genesis in DS is loss of inhibitory input due to dysfunctional firing of GABAergic interneurons. We hypothesised that DS symptoms could be ameliorated by a drug that activates the reduced population of functional Na V 1.1 channels in DS interneurons. We recently identified two homologous disulfide-rich spider-venom peptides (Hm1a and Hm1b) that selectively potentiate Na V 1.1, and showed that selective activation of Na V 1.1 by Hm1a restores the function of inhibitory interneurons in a mouse model of DS. Here we produced recombinant Hm1b (rHm1b) using an E. coli periplasmic expression system, and examined its selectivity against a panel of human Na V subtypes using whole-cell patch-clamp recordings. rHm1b is a potent and highly selective agonist of Na V 1.1 and Na V 1.3 (EC 50 ~12 nM for both). rHm1b is a gating modifier that shifts the voltage dependence of channel activation and inactivation to hyperpolarised and depolarised potentials respectively, presumably by interacting with the channel's voltage-sensor domains. Like Hm1a, the structure of rHm1b determined by using NMR revealed a classical inhibitor cystine knot (ICK) motif. However, we show that rHm1b is an order of magnitude more stable than Hm1a in human cerebrospinal fluid. Overall, our data suggest that rHm1b is an exciting lead for a precision therapeutic targeted against DS., (Copyright © 2020 Elsevier Inc. All rights reserved.)