Pharmacological approaches in drug-resistant pediatric epilepsies caused by pathogenic variants in potassium channel genes

Abstract

Variants in genes encoding for voltage-gated K⁺ (Kv) channels are frequent cause of drug-resistant pediatric epilepsies. Obtaining a molecular diagnosis gives the opportunity to assess the efficacy of pharmacological strategies based on in vitro features of mutant channels. In this retrospective observational study, we selected patients with drug-resistant pediatric epilepsies caused by variants in potassium channel encoding genes, followed at the Fondazione IRCCS Istituto Neurologico Carlo Besta of Milan, Italy. After the experimental characterization of variants' functional properties in transiently transfected Chinese Hamster Ovary (CHO) cells, we identified drugs to be used as pharmacological approaches. We recruited six patients carrying different missense variants in four Kv channels (Kv7.2, Kv7.3, Kv3.1, and K_Na1.1). In vitro experiments demonstrated that variants in Kv7 channels induced loss-of-function (LoF) effects, while those affecting Kv3.1 or K_Na1.1 led to gain-of-function (GoF). Moreover, we found that the Kv7 channels activator gabapentin was able to revert the LoF effects caused by Kv7.2/Kv7.3 variants, and the potassium channel-blocker fluoxetine counteracted the GoF effects in Kv3.1 or K_Na1.1 variants. According to experimental data, patients carrying Kv7 variants were treated with gabapentin. While this treatment resulted successful in two patients (#1, Kv7.2 G310S variant; #3, Kv7.3 V359L + Kv7.3 D542N), it resulted detrimental in the remaining case (#2, Kv7.2 D535E), requiring drug withdrawal. The application in vivo of fluoxetine to counteract GoF effects induced by Kv3.1 or K_Na1.1 variants determined a significant reduction of both seizure frequency and behavior disturbances in patient #4 (Kv3.1 V425M), and in both subjects carrying K_Na1.1 variants (#5, S937G and #6, R262Q). However, for the latter case, this drug was halted due to severe behavioral side effects. For most of the patients herein reported, pharmacological strategies, selected according to the in vitro functional properties of Kv-channels pathogenic variants, resulted in a significant improvement of both epileptic and cognitive features.

Keywords: epilepsy; fluoxetine (FLX); functional study; gabapentin (GBP); potassium channel.

Figure 1

Functional properties of channels carrying the newly-identified Kv7.2 and KCNT1 variants. Representative family traces (A) and quantification of maximal current densities (B) recorded in CHO cells transiently expressing the indicated channels in response to the voltage protocol shown below the leftmost traces. Current scales: 500 pA; time scales: 200 ms. In panel (B): *p < 0.05 versus Kv7.2, **p < 0.05 versus Kv7.2 + Kv7.3 (1:1). Representative current traces (C), quantification of current densities (D), conductance-voltage curves (E), and quantification of I_INST/I_SS ratios (F) recorded in CHO cells transiently expressing the indicated channels in response to the voltage protocol shown below the leftmost traces. Current scales: 500 pA; time scales: 100 ms. In panel (F): *p < 0.05 versus K_Na1.1. In panels (B,F), gray symbols indicate single data points used for quantification.

Figure 2

Pharmacological properties of channels incorporating Kv7.2, Kv7.3, or KCNT1 variants. Representative current traces (A), quantification of AUC (B), and (C) pre-post AUC values measured in single cells expressing the indicated channels at the transfection ratios specified in parenthesis upon exposure to the voltage protocol shown above the leftmost traces in control solution (ctl), upon exposure to 10 μM gabapentin (GBP), or upon drug washout (wo). Current scale: 200 pA; time scale: 200 ms. Representative currents traces (D), quantification of AUC (E), and (F) pre-post AUC values measured in single cells transiently expressing the indicated channels, upon application of the indicated voltage protocol, in control solution (ctl), upon exposure to 10 μM fluoxetine (FLX), or upon drug washout (wo), as indicated. Current scale: 500 pA; time scale: 200 ms. In panels (B,E): *p < 0.05 versus each respective control.