Functional studies of p.N258K and p.G279D variants in the KCNQ2 gene

Abstract

Mutations in the KCNQ2 gene encoding for voltage-gated K channel subunits underlying the neuronal M-current, have been associated with infantile-onset epileptic disorders. The clinical spectrum ranges from self-limited neonatal seizures to epileptic encephalopathy and delayed development. Although gain- and loss-of-function mutations of KCNQ2 leads to indistinguishable phenotypes, different therapeutic approaches are required. To better understand genotype-phenotype correlation, more reports of patients and their mutations with elucidated molecular mechanism are needed. Here, we report nine unrelated patients with KCNQ2-related epilepsy carrying de novo heterozygous KCNQ2 p.N258K or p.G279D identified via Trio exome sequencing that has never been previously reported. To investigates the molecular mechanisms by electrophysiological techniques combined with immunofluorescence analysis and western blotting. The Cellular localization and protein expression analysis demonstrates that p.N258K and p.G279D impairs surface membrane expression of Kv 7.2. Whole-cell patch-clamp analyses revealed that both variants significantly impaired Kv7.2 M-current amplitude and density, conductance depolarizing shift in voltage dependence of activation, membrane resistance, and membrane time constant (Tau), indicating a loss-of-function in both the homotetrameric and heterotetrameric with Kv7.3 channels. In addition, both variants also exerted dominant-negative effects in heterotetrameric with Kv7.3 channels. Thus, novel variants are loss-of-function on Kv7.2 channels, causing infantile-onset epilepsy.