Biophysical and structural mechanisms of epilepsy-associated mutations in the S4-S5 Linker of KCNQ2 channels

Abstract

Mutations in KCNQ2 are linked to various neurological disorders, including neonatal-onset epilepsy. The severity of these conditions often correlates with the mutation's location and the biochemical properties of the altered amino acid side chains. Two mutations affecting aspartate at position 212 (D212) in the S4-S5 linker of KCNQ2 have been identified. Interestingly, while the charge-conserved D212E mutation leads to severe neonatal-onset developmental and epileptic encephalopathy (DEE), the more dramatic substitution to glycine (D212G) results in self-limited familial neonatal epilepsy (SLFNE), a much milder pathology. To elucidate the underlying mechanisms, we performed electrophysiological studies and in silico simulations to investigate these mutations' biophysical and structural effects. Our findings reveal that the D212E mutation stabilizes the channel in the voltage sensor down-state and destabilizes the up-state, leading to a rightward shift in the voltage-dependent activation curve, slower activation kinetics, and accelerated deactivation kinetics. This disruption in KCNQ2 voltage sensitivity persists even in the more physiologically relevant KCNQ2/3 heterotetrameric channels. In contrast, the D212G mutation primarily destabilizes the up-state, but its impact on voltage sensitivity is significantly reduced in KCNQ2/3 heterotetrameric channels. These findings provide key insights into the biophysical and structural basis of KCNQ2 D212 mutations and their contribution to epilepsy-related symptoms, offering a clearer understanding of how these mutations drive the varied clinical outcomes observed in patients.

Keywords: KCNQ; channelopathy; developmental and epileptic encephalopathy; gating; self-limited familial neonatal epilepsy.

Figure 1.

Functional characterization of dee-causing KCNQ2-D212E and SLFNE-causing KCNQ2-D212G mutations. (a-c) Representative current traces of human KCNQ2 channels in HEK293 cells transfected with KCNQ2 WT (a), KCNQ2-D212E (b), or KCNQ2-D212G (c). Cells were held at −114.2 mV and subjected to 3-second voltage steps ranging from −114.2 mV to 65.8 mV in 10 mV increments, followed by a return to −14.2 mV. (d) Steady-state current densities measured at the end of the 3-second voltage steps plotted against test voltage. KCNQ2-D212E and KCNQ2-D212G were activated at more depolarized potentials than WT. (e) Normalized conductance densities measured between 55.8 and 65.8 mV showed comparable current densities among WT, KCNQ2-D212E, and KCNQ2-D212G (n = 10, 10, and 10 for WT, D212E, and D212G, respectively; one-way ANOVA). (f-h) Activation curves of WT, KCNQ2-D212E, and KCNQ2-D212G reveal rightward shifts for both mutants, indicating elevated V_1/2 values (g) (eq.1) (n = 10, 12, and 6 for WT, D212E, and D212G, respectively; p < 0.001 for WT vs D212E and p < 0.001 for WT vs D212G; one-way ANOVA). Both KCNQ2-D212E and KCNQ2-D212G exhibited increased k values, suggesting impaired voltage sensing (H) (n = 10, 12, and 6 for WT, D212E, and D212G, respectively; p < 0.05 for WT vs D212E, p < 0.05 for D212E vs D212G, and p < 0.001 for WT vs D212G; one-way ANOVA).

Figure 2.

KCNQ2-D212E and KCNQ2-D212G accelerate KCNQ2 channel closure. (a) Representative normalized current traces from HEK293 cells transfected with KCNQ2 WT (black), KCNQ2-D212E (red), and KCNQ2-D212G (blue). After depolarization to 25.8 mV, the voltage was stepped back to −34.2 mV. (b) The activation time constant (τ) plotted against test voltage pulses shows that neither KCNQ2-D212E nor KCNQ2-D212G affected KCNQ2 channel activation (n = 10, 11, and 5 for WT, D212E, and D212G, respectively; Two-way ANOVA). (c) Both KCNQ2-D212E and KCNQ2-D212G significantly accelerated channel deactivation across all test voltages (eq.2)(n = 10, 10, and 6 for WT, D212E, and D212G, respectively; Two; p < 0.001 for WT vs D212E, p < 0.001 for WT vs D212G; one-way ANOVA).

Figure 3.

Dee-causing KCNQ2-D212E mutation moderately affects KCNQ2/3 heterotetrameric channels. (a) Activation curves of heterotetrameric KCNQ2/3 channels containing WT-KCNQ2, KCNQ2-D212E, or KCNQ2-D212G. KCNQ2-D212E-containing KCNQ2/3 heterotetrameric channels exhibited moderately impaired activation, as indicated by a rightward shift in the activation curve. In contrast, KCNQ2-D212G-containing KCNQ2/3 heterotetrameric channels were comparable to WT, with a slightly flattened activation curve at more depolarized voltages (n = 14, 14, and 7 for WT, D212E, and D212G, respectively). (b) Elevated V_1/2 values were observed in KCNQ2-D212E-containing KCNQ2/3 heterotetrameric channels compared to WT (n = 14, 14, and 7 for WT, D212E, and D212G, respectively; p < 0.05, one-way ANOVA). (c) KCNQ2-D212G-containing KCNQ2/3 heterotetrameric channels exhibited increased k values, indicating impaired voltage sensing (n = 14, 14, and 7 for WT, D212E, and D212G, respectively; p < 0.05 for WT vs. D212E and D212E vs. D212G, one-way ANOVA).

Figure 4.

Predicted structures of WT, KCNQ2-D212E, and KCNQ2-D212G channels. (A) Side view of the predicted WT KCNQ2 channel structure. The voltage-sensing domain and the S4-S5 linker region, part of the pore domain, are highlighted in the boxed area. (B – D) Close-up views of the S4-S5 linker region: (B) for WT KCNQ2, in the down-state, the region containing D212 forms a loosely packed, unstructured loop. In the up-state, the S4-S5 linker stretches, and the carboxyl group of D212 forms up to five potential hydrogen bonds with the peptide backbone of G215 and G216. (C) For KCNQ2-D212E, in the down-state, the carboxyl group of glutamate forms two hydrogen bonds with G215 and G216, stabilizing the closed conformation. In the up-state, the bulkier glutamate side chain bends inward, allowing only one hydrogen bond with G215, resulting in a less stable conformation. (D) For KCNQ2-D212G, in the down-state, the region forms a loosely packed loop without hydrogen bonds, resembling the WT KCNQ2 in the down-state. In the up-state, the smaller glycine residue forms only one potential hydrogen bond with G215, leading to a less stable conformation compared to WT.

Figure 5.

Schematic representation of D212 in voltage-dependent gating of KCNQ2 channels. In the voltage-sensor down-state, the S4-S5 linker forms an unstructured loop, and D212 does not establish stable bonds with potential interacting partners. Upon depolarization, the voltage sensor moves upward, restructuring the S4-S5 linker. In the up-state, the S4-S5 linker stretches, allowing D212 to form stable bonds with multiple partners in the linker region. For the dee-causing D212E mutation, the glutamate residue stabilizes the down-state by creating a stable structure with nearby partners. However, in the up-state, the bulkier glutamate side chain prevents stable bond formation, destabilizing the up-state conformation. For the slfne-causing D212G mutation, the glycine residue is too small to interact effectively with potential binding partners, resulting in a less stable down-state. In the up-state, the short glycine residue similarly fails to form stable bonds with the S4-S5 linker, reducing the stability of the voltage-sensor up-state.