Gain of function due to increased opening probability by two KCNQ5 pore variants causing developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are neurodevelopmental diseases characterized by refractory epilepsy, distinct electroencephalographic and neuroradiological features, and various degrees of developmental delay. Mutations in KCNQ2, KCNQ3, and, more rarely, KCNQ5 genes encoding voltage-gated potassium channel subunits variably contributing to excitability control of specific neuronal populations at distinct developmental stages have been associated to DEEs. In the present work, the clinical features of two DEE patients carrying de novo KCNQ5 variants affecting the same residue in the pore region of the Kv7.5 subunit (G347S/A) are described. The in vitro functional properties of channels incorporating these variants were investigated with electrophysiological and biochemical techniques to highlight pathophysiological disease mechanisms. Currents carried by Kv7.5 G347 S/A channels displayed: 1) large (>10 times) increases in maximal current density, 2) the occurrence of a voltage-independent component, 3) slower deactivation kinetics, and 4) hyperpolarization shift in activation. All these functional features are consistent with a gain-of-function (GoF) pathogenetic mechanism. Similar functional changes were also observed when the same variants were introduced at the corresponding position in Kv7.2 subunits. Nonstationary noise analysis revealed that GoF effects observed for both Kv7.2 and Kv7.5 variants were mainly attributable to an increase in single-channel open probability, without changes in membrane abundance or single-channel conductance. The mutation-induced increase in channel opening probability was insensitive to manipulation of membrane levels of the critical Kv7 channel regulator PIP2. These results reveal a pathophysiological mechanism for KCNQ5-related DEEs, which might be exploited to implement personalized treatments.

Distinct epilepsy phenotypes and response to drugs in KCNA1 gain- and loss-of function variants.

A wide phenotypic spectrum of neurological diseases is associated with KCNA1 (Kv1.1) variants. To investigate the molecular basis of such a heterogeneous clinical presentation and identify the possible correlation with in vitro phenotypes, we compared the functional consequences of three heterozygous de novo variants (p.P403S, p.P405L, and p.P405S) in Kv1.1 pore region found in four patients with severe developmental and epileptic encephalopathy (DEE), with those of a de novo variant in the voltage sensor (p.A261T) identified in two patients with mild, carbamazepine-responsive, focal epilepsy. Patch-clamp electrophysiology was used to investigate the functional properties of mutant Kv1.1 subunits, both expressed as homomers and heteromers with wild-type Kv1.1 subunits. KCNA1 pore mutations markedly decreased (p. P405S) or fully suppressed (p. P403S, p. P405L) Kv1.1-mediated currents, exerting loss-of-function (LoF) effects. By contrast, channels carrying the p.A261T variant exhibited a hyperpolarizing shift of the activation process, consistent with a gain-of-function (GoF) effect. The present results unveil a novel correlation between in vitro phenotype (GoF vs LoF) and clinical course (mild vs severe) in KCNA1-related phenotypes. The excellent clinical response to carbamazepine observed in the patients carrying the A261T variant suggests an exquisite sensitivity of KCNA1 GoF to sodium channel inhibition that should be further explored.