A Novel Kv7.3 Variant in the Voltage-Sensing S4 Segment in a Family With Benign Neonatal Epilepsy: Functional Characterization and in vitro Rescue by β-Hydroxybutyrate.

Louise Bier,Lidia Carotenuto,Tristan T Sands,Erin L Heinzen,Francesco Miceli,Maria Virginia Soldovieri,Edward C Cooper,Arthur M Mandel,David B Goldstein,Maria Roberta Cilio,Natalie Lippa,Maurizio Taglialatela,Vincenzo Barrese

doi:10.3389/fphys.2020.01040

Abstract

Pathogenic variants in KCNQ2 and KCNQ3, paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K+ channel subunits, are responsible for early−onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early−onset developmental and epileptic encephalopathy (DEE). KCNQ2 variants account for the majority of pedigrees with BFNE and KCNQ3 variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novel KCNQ3 variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S4 transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to β-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S4 segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits.

Highlights

Voltage-gated potassium (K+) channels (Kv channels) regulate the resting membrane potential and set the threshold and duration of the action potential in excitable cells
Some variants have been identified in patients with benign familial neonatal epilepsy (BFNE), an autosomal-dominant epilepsy with seizures affecting otherwise healthy infants in the first days of life and spontaneously disappearing over the several months, with mostly normal neurocognitive development (Jentsch, 2000; Singh et al, 2003; Soldovieri et al, 2006)
We report a BFNE family carrying the first variant in KCNQ3 located in the S4 helix of the voltagesensing domain (VSD) (M240R)

Summary

Introduction

Voltage-gated potassium (K+) channels (Kv channels) regulate the resting membrane potential and set the threshold and duration of the action potential in excitable cells. Kv7.2 and Kv7.3 voltage-gated K+ subunits, encoded by the KCNQ2 and KCNQ3 genes, are expressed in the central and peripheral nervous system (Brown and Adams, 1980; Wang et al, 1998) These subunits form homoand heterotetrameric channels underlying the M-current (IKM), a non-inactivating K+ current with slow activation and deactivation kinetics that activates at the threshold potential of about −60/−50 mV, regulating the resting membrane potential and suppressing repetitive neuronal firing (Brown and Adams, 1980). De novo variants in KCNQ3 have been rarely described in children with DEE (Allen et al, 2013; Grozeva et al, 2015; Miceli et al, 2015; Ambrosino et al, 2018; Lauritano et al, 2019), intellectual disability (ID) apparently without epilepsy (Rauch et al, 2012; Deciphering Developmental Disorders, 2017), cortical visual impairment (Bosch et al, 2016), and in patients with ID and autism (Sands et al, 2019)

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