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

Abstract

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.

Keywords: developmental and epileptic encephalopathies; genotype–phenotype correlations; potassium channels.

Fig. 1.

Topological location of the G347 (G1) residue and functional characterization of Kv7 .5, Kv7.5 G1S, and Kv7.5 G1A channels. (A) Schematic representation of a single Kv7.5 subunit, showing the position of the G1 residue herein investigated (gray highlighting) and of the currently known Kv7.5 pathogenic variants whose functional characteristics have been studied (bold characters) or are yet unknown (regular characters). (B) Sequence alignment of the bottom part of the S₆ segments of the indicated Kv subunits (https://www.ebi.ac.uk/Tools/psa/). The shaded region highlights the -GSG- motif, with the G1 residue indicated by the box. (C) Macroscopic currents from Kv7.5, Kv7.5 G1S, and Kv7.5 G1A homomeric channels, in response to the indicated voltage protocol. Current scale, 200 pA; time scale, 200 ms. (D) Superimposed normalized current traces from Kv7.5 (black), Kv7.5 G1S (dark blue), and Kv7.5 G1A (light blue) homomeric channels. A and B represent time points at which instantaneous and steady-state currents were measured, respectively; (B − A)/B × 100 represents the fraction of voltage-dependent, time-dependent current (I_VDEP); and the fraction of voltage-independent, instantaneous current (I_INST) was calculated as (A/B) × 100 (or 1 − I_VDEP). (E) Conductance/voltage curves for the indicated channels. Continuous lines are Boltzmann fits of the experimental data. (F) Ramp currents from the indicated channels using the voltage protocol shown at the bottom in 5 and 15 mM extracellular K⁺ ions, as indicated. Current scales, 200 pA for Kv7.5 and 500 pA for Kv7.5 G1S; time scale, 20 ms. (G) Effect of 100 mM TEA_e (Upper) or 1 to 10 mM Ba²⁺_e (Lower) on Kv7.5 G1S currents recorded with the voltage protocol shown at the bottom. A₁ and A₂ indicate the I_INST current values measured in control and drug-containing solutions, respectively; B₁ and B₂ indicate the steady-state (I_INST + I_VDEP) current values measured in control and drug-containing solutions, respectively. Current scales, 200 pA for Upper (100 mM TEA_e) and 500 pA for Lower (1 to 10 mM Ba²⁺_e); time scale, 100 ms. (H) Quantification of current densities recorded from cells transfected with the indicated cDNA constructs. Asterisks indicate values significantly different (*P < 0.05) from their respective control (Kv7.3 + Kv7.5). (I) Conductance/voltage curves for the currents recorded from cells transfected with the indicated cDNA constructs. Continuous lines are Boltzmann fits of the experimental data.

Fig. 2.

Functional characterization of Kv7.2 and Kv7.5 channels carrying naturally and nonnaturally occurring variants at position G1. (A) Macroscopic currents from Kv7.2 and Kv7.2 G1S homomeric channels, in response to the indicated voltage protocol. Current scale, 200 pA; time scale, 200 ms. (B) Current densities from the indicated homomeric channels. Asterisks indicate values significantly different (*P < 0.05) from respective controls (Kv7.2 or Kv7.5 channels). (C) Conductance/voltage curves for the indicated channels. Continuous lines are Boltzmann fits of the experimental data.

Fig. 3.

Nonstationary noise analysis of Kv7.5, Kv7.5 G1S, Kv7.2, and Kv7.2 G1S channels. (A) Representative average response to 100 pulses at +20 mV (Top), of the corresponding variance (Middle), and variance versus current mean plot (Bottom) from Kv7.5, Kv7.5 G1S, Kv7.2, and Kv7.2 G1S channels, as indicated. The continuous lines in Bottom are parabolic fits of the experimental data (Materials and Methods). Current and variance scales are 100 pA and 50 pA² for Kv7.5, 750 pA and 100 pA² for Kv7.5 G1S, 100 pA and 50 pA² for Kv7.2, and 750 pA and 100 pA² for Kv7.2 G1S. Time scale, 100 ms. P_o and I estimates for the four cells shown were 0.18 and 0.20 for Kv7.5, 0.57 and 0.19 for Kv7.5 G1S, 0.25 and 0.38 for Kv7.2, and 0.79 and 0.39 for Kv7.2 G1S. (B–D) Quantification of the number of channels divided by capacitance (B), of the single-channel current (C), and of the opening probability at +20 mV (D). Asterisks indicate values significantly different (*P < 0.05) from respective controls (Kv7.5 channels, Left; Kv7.2 channels, Right).

Fig. 4.

Effect of DrVSP or PIP5K on Kv7.2 and Kv7.2 G1S homomeric channels. (A) Currents recorded in response to the indicated voltage protocol in cells expressing DrVSP and Kv7.2 or Kv7.2 G1S channels. Time scale, 1 s. (B) Time-dependent current decrease in cells coexpressing the indicated channels and DrVSP, expressed as the ratio between the current values recorded at 0 mV immediately after (t₂) and before (t₁) the Dr-VSP–activating +100 mV depolarizing step. (C) Macroscopic currents recorded in response to the indicated voltage protocol in cells expressing Kv7.2 or Kv7.2 G1S channels in the absence or presence of PIP5K. Current scale, 500 pA; time scale, 200 ms. (D) Current densities at 0 mV from cells expressing Kv7.2 or Kv7.2 G1S channels alone or in combination with PIP5K, as indicated. The asterisk indicates a value significantly different (*P < 0.05) from Kv7.2 currents.