De novo BK channel variant causes epilepsy by affecting voltage gating but not Ca2+ sensitivity

Abstract

Epilepsy is one of the most common neurological diseases and it causes profound morbidity and mortality. We identified the first de novo variant in KCNMA1 (c.2984 A > G (p.(N995S)))-encoding the BK channel-that causes epilepsy, but not paroxysmal dyskinesia, in two independent families. The c.2984 A > G (p.(N995S)) variant markedly increased the macroscopic potassium current by increasing both the channel open probability and channel open dwell time. The c.2984 A > G (p.(N995S)) variant did not affect the calcium sensitivity of the channel. We also identified three other variants of unknown significance (c.1554 G > T (p.(K518N)), c.1967A > C (p.(E656A)), and c.3476 A > G (p.(N1159S))) in three separate patients with divergent epileptic phenotypes. However, these variants did not affect the BK potassium current, and are therefore unlikely to be disease-causing. These results demonstrate that BK channel variants can cause epilepsy without paroxysmal dyskinesia. The underlying molecular mechanism can be increased activation of the BK channel by increased sensitivity to the voltage-dependent activation without affecting the sensitivity to the calcium-dependent activation. Our data suggest that the BK channel may represent a drug target for the treatment of epilepsy. Our data highlight the importance of functional electrophysiological studies of BK channel variants in distinguishing whether a genomic variant of unknown significance is a disease-causing variant or a benign variant.

Fig. 1

Identification and electrophysiological characterization of a BK channel variant p.(N995S) causing epilepsy. a Pedigrees of the two independent families. Squares, males; circles, females; filled symbol, affected individual; open symbols, unaffected individuals. b The p.(N995) residue, shown in bold and italic, of KCNMA1 is evolutionally conserved among different species. c Structure of the BK channel with the p.(N995S) variant indicated. The p.(D434G) variant identified previously in a large family with generalized epilepsy and paroxysmal dyskinesia, and three other variants identified in this study are also shown. d Representative macroscopic currents of WT and p.(N995S) mutant BK channels from inside-out patches in the presence of 10 μM Ca²⁺ by the protocol as indicated. e Activation time constants of WT and p.(N995S) mutant BK channels at 10 μM Ca²⁺ are plotted against membrane potentials. f G–V curves of WT and p.(N995S) mutant BK channels at 10 μM Ca²⁺. All G–V curves are fitted by Boltzmann function (solid lines) with V_1/2 (14.1 ± 6.6 mV for WT and −44.6 ± 8.2 mV for p.(N995S)). The data are presented as mean ± SD (n = 6, P < 0.05). g The representative traces of WT and p.(N995S) mutant BK channels from inside-out patches evoked by a test pulse of 100 mV in the presence of 10 μM Ca²⁺ before (solid) and after (dash) application of 200 nM paxilline. h, i Normalized currents of WT and p.(N995S) mutant BK channels are plotted vs. time. The horizontal bar indicates the treatment duration of 200 nM paxilline

Fig. 2

The β4 subunit slightly augments the effect of the p.(N995S) variant on channel activation. a Representative macroscopic currents of WT and p.(N995S) mutant BK channels with hβ4 from inside-out patches in the presence of 10 μM Ca²⁺ by the protocol as indicated. b G–V curves of WT + hβ4 and p.(N995S) + hβ4 at 10 μM Ca²⁺. All G–V curves are fitted by Boltzmann function (solid lines) with V_1/2 (24.1 ± 8.7 mV for WT + hβ4 and −47.1 ± 7.9 mV for p.(N995S) + hβ4). The data are presented as mean ± SD (n = 6, P < 0.05)

Fig. 3

Variant p.(N995S) has little effect on the Ca²⁺-dependent activation mechanism of the BK channel. a Representative macroscopic currents of WT (upper panel) and p.(N995S) mutant (lower panel) BK channels from inside-out patches at nominal 0 μM Ca²⁺ (left), 1 μM Ca²⁺ (middle), and 10 μM Ca²⁺ (right) by the protocol as indicated. b G–V curves of WT and p.(N995S) mutant BK channels at nominal 0 μM Ca²⁺, 1 μM Ca²⁺, and 10 μM Ca²⁺. All G–V curves are fitted by Boltzmann function (solid lines) with V_1/2 at nominal 0 μM Ca²⁺ (158.2 ± 6.3 mV for WT and 115.8 ± 6.9 mV for p.(N995S), P < 0.05), at 1 μM Ca²⁺ (102.8 ± 2.6 mV for WT and 15.8 ± 3.5 mV for p.(N995S), P < 0.05), and at 10 μM Ca²⁺ (14.1 ± 6.6 mV for WT and −44.6 ± 8.2 mV for p.(N995S), P < 0.05). c V_1/2 of G–V curves vs. [Ca²⁺]_i for WT and p.(N995S) mutant BK channels. d G–V curves of p.2D2A/p.5D5N and p.(N995S)/p.2D2A/p.5D5N mutant BK channels at nominal 0 μM Ca²⁺, 1 μM Ca²⁺, and 10 μM Ca²⁺. All G–V curves are fitted by Boltzmann function (solid lines) with V_1/2 and slope factor at nominal 0 μM Ca²⁺ (169.8 ± 7.1 mV, 24.6 ± 2.9 for p.2D2A/p.5D5N and 92.8 ± 4.9 mV, 22.0 ± 2.6 for p.(N995S)/p.2D2A/p.5D5N, P_(V1/2) < 0.05), at 1 μM Ca²⁺ (168.6 ± 6.6 mV, 26.2 ± 3.0 for p.2D2A/p.5D5N and 92.7 ± 5.1 mV, 23.2 ± 2.1 for p.(N995S)/p.2D2A/p.5D5N, P_(V1/2) < 0.05) and at 10 μM Ca²⁺ (164.0 ± 6.3 mV, 25.5 ± 3.2 for p.2D2A/p.5D5N and 89.7 ± 5.0 mV, 23.6 ± 2.5 for p.(N995S)/p.2D2A/p.5D5N, P_(V1/2) < 0.05). e V_1/2 of G–V curves vs. [Ca²⁺]_i for p.2D2A/p.5D5N and p.(N995S)/p.2D2A/p.5D5N mutant BK channels. The data are presented as mean ± SD (n = 6, P < 0.05)

Fig. 4

Single-channel electrophysiological analysis of the p.(N995S) variant of BK channel causing epilepsy. a Representative single-channel currents of WT and p.(N995S) mutant BK channels at 1 μM Ca²⁺ and 10 μM Ca²⁺ recorded at 100 mV. C and O indicate the closed and open state of the channel, respectively. The sample rate is 50 kHz, and the low-pass filter is 5 kHz. b Open probability P_o–V curves of WT and p.(N995S) mutant BK channels at 1 μM Ca²⁺ and 10 μM Ca²⁺. c Representative open dwell time histograms of WT and p.(N995S) mutant BK channels at 1 μM Ca²⁺ and 10 μM Ca²⁺ measured at 100 mV. Histograms are plotted in log-bin timescales and fitted by exponential function (solid lines). d Time constants of open dwell time for WT and p.(N995S) mutant BK channels at 1 μM Ca²⁺ (1.9 ± 0.5 ms for WT and 3.9 ± 1.1 ms for p.(N995S), P < 0.05) and at 10 μM Ca²⁺ (2.8 ± 0.5 ms for WT and 4.9 ± 1.1 ms for p.(N995S), P < 0.05). The data are presented as mean ± SD (n = 5, P < 0.05)