Constitutive opening of the Kv7.2 pore activation gate causes KCNQ2-developmental encephalopathy

Abstract

Pathogenic variants in KCNQ2 encoding Kv7.2 voltage-gated potassium channel subunits cause developmental encephalopathies (KCNQ2-encephalopathies), both with and without epilepsy. We herein describe the clinical, in vitro, and in silico features of two encephalopathy-causing variants (A317T, L318V) in Kv7.2 affecting two consecutive residues in the S₆ activation gate that undergoes large structural rearrangements during pore opening; the disease-causing A356T variant in KCNQ3, paralogous to the A317T variant in KCNQ2, was also investigated. Currents through KCNQ2 mutant channels displayed increased density, hyperpolarizing shifts in activation gating, faster activation and slower deactivation kinetics, and resistance to changes in the cellular concentrations of phosphatidylinositol 4,5-bisphosphate (PIP₂), a critical regulator of Kv7 channel function; all these features are consistent with a strong gain-of-function effect. An increase in the probability of single-channel opening, with no change in membrane abundance or single-channel conductance, was responsible for the observed gain-of-function effects. All-atom molecular dynamics simulations revealed that the mutations widened the inner pore gate and stabilized a constitutively open channel configuration in the closed state, with minimal effects on the open conformation. Thus, mutation-induced stabilization of the inner pore gate open configuration is a molecular pathogenetic mechanism for KCNQ2-related encephalopathies.

Keywords: Genotype–phenotype correlations; Molecular dynamics; channel gating; developmental and epileptic encephalopathies; potassium channels.

Fig. 1.

Biophysical properties of K⁺ currents from Kv7.2 channels harboring variants in the pore AG. (A) Kv7.2 structure; only the PD from two opposite subunits is shown for clarity. The enlargement highlights the A317 (purple) and the L318 (blue) residues. The SF, CC, and AG regions are indicated. (B) Sequence alignment of the indicated Kv subunits in the S₆ region; the investigated variants are highlighted. The region in yellow indicates the pore AG. (C) Macroscopic whole-cell currents from Kv7.2 (green), Kv7.2 A317T (purple), and Kv7.2 L318V (blue) homomeric channels in response to the indicated voltage protocol. In red are the traces recorded at −40 mV, corresponding to the threshold voltage in Kv7.2 currents (see Inset). Current scale: 200 pA; time scale: 200 ms. (D) Quantification of the current densities recorded from cells transfected with the indicated cDNA constructs. * = P < 0.05. (E) Superimposed normalized current traces at 0 mV from the Kv7.2, Kv7.2 A317T, and Kv7.2 L318V homomeric channels. Note the instantaneous current component in the Kv7.2 A317T (purple) and Kv7.2 L318V channels at the beginning of the pulse. (F) Conductance/voltage curves for the indicated channels; continuous lines represent Boltzmann fits of the experimental data to equation 1 in the Methods section. (G) Quantification of current densities recorded from cells transfected with the indicated cDNA constructs. * = P < 0.05. (H) Conductance/voltage curves for the indicated channels; continuous lines represent Boltzmann fits of the experimental data to equation 1 in the Methods section.

Fig. 2.

Nonstationary noise analysis of K⁺ currents from Kv7.2 channels carrying variants in the pore AG. (A) Representative average response to 100 pulses at +20 mV (Top traces), variance (Middle traces), and variance versus current mean plot (Bottom traces) for Kv7.2 and Kv7.2 L318V, as indicated. The continuous lines in the variance/mean plots are the parabolic fits of the experimental data to equation 2 in the Methods section. (B–D) Quantification of the number of channels divided by the capacitance (b), the single-channel current (c), and the opening probability at 20 mV (d) for the indicated channels. * = P < 0.05. (E) Representative western blot image of total, cytosolic, or plasma membrane protein fractions from CHO cells transfected with pcDNA3.1 (empty vector, C), Kv7.2 (WT), Kv7.2 A317T (A/T), or Kv7.2 L318V (L/V) subunits. On the right, the positions of the estimated molecular masses of the Kv7.2 (95 kDa) and GAPDH (37 kDa) bands are shown. (F) Densitometric quantification of the 95 kDa band intensity in the indicated experimental groups. The data are expressed as the means ± SEMs.

Fig. 3.

Effect of PIP₂ levels manipulations on Kv7.2, Kv7.2 A317T, and Kv7.2 L318V currents. (A) Macroscopic currents recorded in the presence of 100 µM Dic8-PIP₂ in an intracellular pipette solution at 0 min (immediately after patch rupture, black traces) and after 4 min of whole-cell intracellular dialysis (colored traces). (B) Normalized whole-cell currents for the three channels are indicated as a function of time; 100 µM Dic8-PIP₂ was added to the pipette solution. (C) V_½ values comparison between cells recorded in the absence (−) or in the presence (+) of 100 µM Dic8-PIP₂ in the intracellular solution after 4 min of whole-cell intracellular dialysis. * = P < 0.05. (D) Currents recorded in response to the indicated voltage protocol in cells expressing DrVSP and Kv7.2, Kv7.2 A317T, or Kv7.2 L318V. Time scale, 2 s. (E) Time-dependent current changes in cells coexpressing the indicated channels and DrVSP. The data are 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 as a function of time.

Fig. 4.

MD simulations of the A317T Kv7.2 variant. (A) Superposition of WT (green) and A317T (purple) representative Kv7.2 closed structures after equilibration and before MD production simulations. (B) Channel radius profiles along the pore axis of the two proteins, averaged over all the simulated replicas. Shaded regions indicate standard deviations (SD). (C) Distribution of water molecules along the channel axis. Averages and SD were calculated for all replicates. (D–F) Same as (A–C) but for the open structures.

Fig. 5.

Close-up cytosolic views of AG conformations from WT and A317T Kv7.2 channel simulations. (A) Closed WT. (B) Closed A317T variant. (C) Open WT. (D) Open A317T variant. Cross distances are indicated as gray dashed lines. Where present, black dashed lines indicate HBs.

Fig. 6.

MD simulations of the L318V Kv7.2 variant. (A) Superposition of WT (green) and L318V (blue) representative Kv7.2 closed structures after equilibration and before MD production simulations. (B) Channel radius profiles along the pore axis of the two proteins averaged over all the simulated replicas. Shaded regions indicate SD. (C) Distribution of water molecules along the channel axis. Averages and SD were calculated for all replicates. (D–F) Same as (A–C) but for the open structures.