Gain of function of sporadic/familial hemiplegic migraine-causing SCN1A mutations: Use of an optimized cDNA

Abstract

Introduction Familial hemiplegic migraine 3 is an autosomal dominant headache disorder associated with aura and transient hemiparesis, caused by mutations of the neuronal voltage-gated sodium channel Nav1.1. While a gain-of function phenotype is generally assumed to underlie familial hemiplegic migraine, this has not been fully explored. Indeed, a major obstacle in studying in vitro neuronal sodium channels is the difficulty in propagating and mutagenizing expression plasmids containing their cDNAs. The aim of this work was to study the functional effect of two previously uncharacterized hemiplegic migraine causing mutations, Leu1670Trp (L1670W) and Phe1774Ser (F1774S). Methods A novel SCN1A containing-plasmid was designed in silico and synthesized, and migraine mutations were inserted in this background. Whole-cell patch clamp was performed to investigate the functional properties of mutant Nav1.1 transiently expressed in Human Embryonic Kidney 293 cells. Results and conclusions We generated an optimized Nav1.1 expression plasmid that was extremely simple to handle and used the novel plasmid to study the functional effects of two migraine mutations. We observed that L1670W, but not F1774S, reduced current density and that both mutations led to a dramatic increase in persistent sodium currents, a depolarizing shift of the steady state-inactivation voltage-dependence, and a faster recovery from inactivation. The results are consistent with a major gain-of function effect underlying familial hemiplegic migraine 3. Our optimization strategy will help to characterize in an efficient manner the effect in vitro of mutations of neuronal voltage-gated sodium channels.

GABAergic neurons and NaV1.1 channel hyperactivity: a novel neocortex-specific mechanism of Cortical Spreading Depression

“…With wholecell patch-clamp recordings of Na + currents in cell lines, we first confirmed that also in our conditions Hm1a at low concentration (10 nM) selectively targets NaV1.1 over the two other NaV isoforms expressed in the adult cortex, NaV1.2 and NaV1.6 ( Supplementary Fig.S1). Importantly, Hm1a induced a 12-fold increase of persistent current, effect that is comparable to that previously observed with FHM3 mutations Bertelli et al, 2019;Cestele et al, 2008;Cestele et al, 2013b;Dhifallah et al, 2018), making it a good pharmacological tool for modeling the effect of these mutations.…”

“…CSD was induced in the neocortex both by optogenetic activation of GABAergic neurons and by activation of NaV1.1 with the specific toxin Hm1a (Osteen et al, 2016). This is consistent with the key role of NaV1.1 in GABAergic neurons' excitability and with the effect of FHM3 mutations, which cause gain of function of NaV1.1 and can induce an increase of the persistent Na + current similar to that observed with Hm1a Bertelli et al, 2019;Cestele et al, 2013a;Cestele et al, 2008;Cestele et al, 2013b;Fan et al, 2016;Mantegazza and Broccoli, 2019;Mantegazza and Cestele, 2017). Notably, it has been recently reported that Hm1a rescued the hypoexcitability of hippocampal GABAergic neurons and the severity of the epileptic phenotype in epileptic Scn1a +/knock-out mice, but it did not modify firing properties of wild type hippocampal GABAergic neurons in brain slices (Richards et al, 2018).…”

“…The direct overactivation of the GABAergic neuron, with conditions similar to those that we used in (Desroches et al, 2019), generated simulations that were similar to those obtained in (Desroches et al, 2019) (not shown). Then, we tested the effect of an increase of the persistent Na + current of the GABAergic neuron, which mimics the common effect of most FHM3 mutations Bertelli et al, 2019;Cestele et al, 2013a;Cestele et al, 2008;Cestele et al, 2013b;Dhifallah et al, 2018;Fan et al, 2016;Mantegazza and Broccoli, 2019;Mantegazza and Cestele, 2017), as well as that of Hm1a. We modeled the control physiologic condition implementing a persistent conductance equal to 1% of the maximal sodium conductance, and we increased it up to 6% to simulate a pathological condition or the presence of Hm1a.…”

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients

“…Another folding/trafficking defective FHM mutation, Na v 1.1-L1670W, showed that functional effect can be switched from a complete loss of function to the gain of function by expression in neurons 47 . Epileptogenic mutations cause a variable degree of loss of function, whereas FHM mutations can, in some cases of functional studies, appear as loss of function because of rescuable folding/trafficking defects; other studies, however, confirmed that FHM mutations cause a gain of function of Na v 1.1 48,49 . Several studies have also suggested that environmental factors and genetic modifiers may influence the clinical phenotype of SCN1A-related epilepsy [41][42][43][44][45]50,51 .…”

SCN1A/NaV1.1 channelopathies: Mechanisms in expression systems, animal models, and human iPSC models

“…Notably, when this mutation was studied in the cardiac Na V 1.5 channel, it did not cause folding defects and induced mild modifications of functional properties that are consistent with moderate gain‐of‐function . Other reports have now confirmed that FHM mutations cause gain‐of‐function of Na V 1.1 . One of these reports identified another folding/trafficking defective FHM mutation for which the rescue induced by expression in neurons switched the functional effect from complete loss‐of‐function to large gain‐of‐function because of the modifications of gating properties .…”