A Novel KCNA2 Variant in a Patient with Non-Progressive Congenital Ataxia and Epilepsy: Functional Characterization and Sensitivity to 4-Aminopyridine

Figures

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  Figure 1. Brain MRI of the patient performed at 23 years. T1-weighted midsagittal section (left) and T2-weighted coronal (right).

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  Figure 2. (A) Position of identified mutations in the Kv1.2 channel structure and localization of the E236K variant. (B) Amino acid alignment of Kv1 channels. LoF, loss-of-function; GoF, gain-of-function; ND, not detected.

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  Figure 3. (A–C) Representative current traces evoked by 200 ms depolarizing steps from a holding potential of −80 to +60 mV from Kv1.2 WT (A), E236K (B), and Kv1.2 WT+E236K (C) channels expressed in HEK293 cells. The voltage protocol is indicated in the upper panel in (A). (D) Current–voltage relationship for Kv1.2 WT (7 μg), E236K (7 μg), and Kv1.2 WT+E236K (3.5 + 3.5 μg) channels (n = 14–36).

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  Figure 4. (A) The current–voltage relationships for Kv1.2 WT, E236K, and Kv1.2 WT+E236K channels were obtained by plotting the normalized peak tail currents measured at −20 mV as a function of the prepulse potentials and fitting data points with a Boltzmann function (n = 10–18 cells). (B) Deactivation and activation kinetics measured for Kv1.2 WT, E236K, and Kv1.2 WT+E236K channels. The time constants, resulting from the fit of the activating and deactivating current traces with a single exponential function, were plotted as a function of voltage (n = 10–19 cells). (C) The voltage-dependence of steady-state inactivation for Kv1.2 WT, E236K, and Kv1.2 WT+E236K channels was obtained by plotting the normalized peak currents measured at +40 mV as a function of the prepulse potentials and fitting data points with a Boltzmann function (n = 7–13 cells). (D) Bar graphs showing the time constants of the C-type inactivation for the indicated channels calculated by fitting current decay with a single exponential function (n = 9–11 cells).

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  Figure 5. (A,B) Representative current traces evoked by 200 ms depolarizing steps from a holding potential of −80 to +60 mV from Kv1.2 WT (A) and E236K (B), before and after the application of 4-aminopyridine (4-AP) 300 μM. (C) % of inhibition of potassium current measured at +60 mV as a function of the concentration of 4-AP for Kv1.2 WT and E236K channels. The Hill equation was fitted to the dose-response curves and used to calculate the IC50 for each channel type (n = 4 cells/dose).

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  Figure 6. Three-dimensional (3D) protein structure of the Kv1.2 channel shown as a ribbon model and localization of the E236K missense variant. Close-up views of the Kv1.2 WT (A) and E236K subunit (B). The residues E236, E236K, K306 and E154 are shown in stick representation. Whereas E236 (red) in the WT subunit remains interacting with the 5th gating charge in S4 during the simulation, K306 (blue), the E236K (cyan) does no longer interact with the charges in S4 but rather with E154 (magenta) in the T1-S1 linker close to S1. (C) Distances between the Kv1.2 WT or E236K and K306 (S4; upper panel) or E154 (T1-S1; lower panel). The distance to the S4 gating charge is much larger for the mutants, and they flip within the first 15 ns to the E154.