Mechanism of increased BK channel activation from a channel mutation that causes epilepsy

Abstract

Concerted depolarization and Ca(2+) rise during neuronal action potentials activate large-conductance Ca(2+)- and voltage-dependent K(+) (BK) channels, whose robust K(+) currents increase the rate of action potential repolarization. Gain-of-function BK channels in mouse knockout of the inhibitory beta 4 subunit and in a human mutation (alpha(D434G)) have been linked to epilepsy. Here, we investigate mechanisms underlying the gain-of-function effects of the equivalent mouse mutation (alpha(D369G)), its modulation by the beta 4 subunit, and potential consequences of the mutation on BK currents during action potentials. Kinetic analysis in the context of the Horrigan-Aldrich allosteric gating model revealed that changes in intrinsic and Ca(2+)-dependent gating largely account for the gain-of-function effects. D369G causes a greater than twofold increase in the closed-to-open equilibrium constant (6.6e(-7)-->1.65e(-6)) and an approximate twofold decrease in Ca(2+)-dissociation constants (closed channel: 11.3-->5.2 microM; open channel: 0.92-->0.54 microM). The beta 4 subunit inhibits mutant channels through a slowing of activation kinetics. In physiological recording solutions, we established the Ca(2+) dependence of current recruitment during action potential-shaped stimuli. D369G and beta 4 have opposing effects on BK current recruitment, where D369G reduces and beta 4 increases K(1/2) (K(1/2) microM: alpha(WT) 13.7, alpha(D369G) 6.3, alpha(WT)/beta 4 24.8, and alpha(D369G)/beta 4 15.0). Collectively, our results suggest that the D369G enhancement of intrinsic gating and Ca(2+) binding underlies greater contributions of BK current in the sharpening of action potentials for both alpha and alpha/beta 4 channels.

Figure 1.

D369G shifts mslo steady-state G-V relation to hyperpolarizing membrane potentials. (A) A family of currents from wild-type (top) or D369G mutant (bottom) BK channels composed of only the pore forming α subunits. Recorded in 2.1 μM Ca²⁺, currents were evoked in response to 200-ms depolarizations at the indicated membrane potentials. (B) Alignment of amino acid sequence flanking the lysine (D) to glycine (G) epilepsy mutation. (C) Mean G-V relations at different Ca²⁺ for α_WT and α_D369G. Each point represents mean data from 5 to 26 experiments. Solid curves represent fits to the Boltzmann function. (D) Mean V_1/2 and (E) mean effective gating charge (Q) values plotted as a function of Ca²⁺. Error bars represent SEM. (F) D434G shifts G-V to more negative membrane potentials at 41 µM Ca²⁺ compared to D369G (hslo_α_WT: n = 9; hslo_α_D434G: n = 10; mslo_α_WT: n = 19; mslo_α_D369G: n = 18). (G) D434G shifts G-V to more negative membrane potentials at nominal Ca²⁺ compared to D369G (hslo_α_WT: n = 5; hslo_α_D434G: n = 5; mslo_α_WT: n = 12; mslo_α_D369G: n = 14). Symbols represent mean G/G_max data, curves represent fits to the Boltzmann function, and error bars represent SEM.

Figure 2.

D369G decreases the energetic barrier for channel to open. (A; left) According to the dual-allosteric mechanism (Horrigan et al., 1999; Horrigan and Aldrich, 2002), BK channel transitions between closed (C) and open (O) conformation is allosterically regulated by the state of four independent and identical voltage sensors. Sub-Scheme a represents BK channel’s gating scheme at 0 Ca²⁺. The channel resides in either the open or closed conformation, with 0–4 voltage sensors in the activated state. The equilibrium between C-O transitions is allosterically regulated by the states of the voltage sensors. Sub-Scheme b represents BK channel’s gating scheme at 0 Ca²⁺ and very negative voltages. With all voltage sensors in the resting state, the channel resides in one of two conformations, C₀ and O₀. The equilibrium between the C₀-O₀ transition is described by L, the intrinsic equilibrium for channel opening in the absence of Ca²⁺ and voltage sensor activation. (Right) This illustrates how two components of L (L₀ and z_L) can be estimated by logPo-V data at 0 Ca²⁺ and negative voltages. The curve represents simulated logPo versus voltage curve in nominally 0 Ca²⁺. The gating parameters used for simulation are as follows: L₀ = 2.5 × 10⁻⁶, z_L = 0.25 e₀, z_J = 0.54e₀, Vh_C = 173 mV, and Vh_O = 25 mV. Dashed line represents fit for logPo-V at limiting slope using Eq. 4. L₀ and z_L can be derived from the fit (Horrigan and Aldrich, 2002). (B) Single-channel BK currents recorded in nominally 0 [Ca²⁺] at the indicated voltages. α_WT and α_D369G data were obtained from patches containing estimated 64 and 317 channels, respectively. All traces were filtered at 5 kHz. (C) D369G increases L₀. Mean logPo plotted as a function of voltage in nominally 0 Ca²⁺ (α_WT: n = 5–13; α_D369G: n = 6–11). Error bars represent SEM. L₀ was estimated by linear fits (dashed lines) of logPo-V relations between −100 to −50 mV using Eq. 1. z_L value was set as 0.25 e₀.

Figure 3.

D369G increases channel’s Ca²⁺-binding affinities. (A; left) According to the dual-allosteric mechanism (Horrigan and Aldrich, 2002), BK channel transitions between closed (C) and open (O) conformation are allosterically regulated by the state of four independent and identical Ca²⁺-binding sites. Sub-Scheme c represents BK channel’s gating scheme at very negative voltages, where voltage sensors remain in the resting states. The channel resides in either closed or open conformations, with 0–4 Ca²⁺-binding sites occupied. The equilibrium between the C-O transitions is allosterically regulated by the states of the Ca²⁺-binding sites. In the absence of voltage sensor activation, voltage dependence of the C-O transition is entirely dependent on z_L. (Right) This illustrates how logL₀′ can be estimated by logPo-V data at high Ca²⁺ and very negative voltages. Curves are simulated logPo-V curves in nominally 0 Ca²⁺ and 100 μM Ca²⁺ according to Sub-Scheme c. The gating parameters used for simulation are as follows: L₀ = 2.5 × 10⁻⁶, z_L = 0.25 e₀, z_J = 0.54 e₀, Vh_C = 173 mV, Vh_O = 25 mV, K_C = 13.9 µM, and K_O = 1.4 µM. Dashed lines represent fits for logPo-V at limiting slopes using Eq. 8. L₀′ and z_L can be derived from the fits (Horrigan and Aldrich, 2002). (B) Symbols represent averaged logPo-V relations at various Ca²⁺. Error bars represent SEM. Dashed lines are fits for mean logPo-V at limiting slope using Eq. 2 and z_L of 0.25 e₀. (C) Open and closed symbols are logL₀′ versus Ca²⁺ for α_WT and α_D369G BK channels, respectively. Curves represent fits of logL₀′-Ca²⁺ using Eq. 3.

Figure 4.

Changes in Ca²⁺ affinities and intrinsic gating are sufficient to account for changes between α_WT (A) and α_D369G (B). Fits (curve and gating parameters listed in Table II) are compared with average P_O-V and logPo-V data (symbols).

Figure 5.

Ca²-dependent effects of β4 on mutant BK channel G-V. (A) A family of currents from α_D369G BK channels composed of either the pore forming alone (top) or with the β4 auxiliary subunits (bottom). Recorded in 2.1 µM Ca²⁺, currents were evoked in response to 200-ms depolarizations. (B, top) Effect of β4 on mslo and hslo mutant G-V relations at 2.1 µM Ca²⁺ (hslo_α_D434G: n = 7; hslo_α_D434G β4: n = 9; mslo_α_D369G: n = 22; mslo_α_D369G β4: n = 32). (B, bottom) Effect of β4 on mslo and hslo mutant G-V relations at 41 µM Ca²⁺ (hslo_α_D434G: n = 10; hslo_α_D434G β4: n = 11; mslo_α_D369G: n = 18; mslo_α_D369G β4: n = 15). Symbols represent mean G/G_max data, curves represent fits to the Boltzmann function, and error bars represent SEM. (C) Effects of β4 on steady-state gating of mutant channels. Mean V_1/2 (top) and mean effective gating charge (Q) values (bottom) plotted as a function of Ca²⁺. Error bars represent SEM. (D) Effects of β4 on steady-state gating of wild-type channels. Mean V_1/2 (top) and mean effective gating charge (Q) values (bottom) plotted as a function of Ca²⁺. Error bars represent SEM.

Figure 6.

Effects of β4 on D369G BK channel gating kinetics. (A; left) Compare activation kinetics. α_D369G and α_D369Gβ4 currents at 2.1 µM Ca²⁺. Patches were held at −80 mV and stepped to +80 mV for 200 ms. Superimposed on the current traces are the single-exponential fits to the activation time courses (α_D369G: τ = 4.7 ms; α_D369Gβ4: τ = 40.3 ms). (Right) Compare deactivation kinetics. α_D369G and α_D369Gβ4 currents at 2.1 µM Ca²⁺. Channels were activated at +50 mV before membrane was stepped to −80 mV for 100 ms. Superimposed on the current traces are the single-exponential fits to the deactivation time courses (α_D369G: τ = 1.1ms; α_D369Gβ4: τ = 11.7 ms). (B) Comparison of α_WT and α_WTβ4 channel kinetics at 41 µM Ca²⁺ (α_WT activation: n = 8–26; α_WT deactivation: n = 12; α_WTβ4 activation: n = 13–35; α_WTβ4 deactivation: n = 16–21). (C) Comparison of α_WT and α_WTβ4 channel kinetics at 2.1 µM Ca²⁺ (α_WT activation: n = 17; α_WT deactivation: n = 6–30; α_WTβ4 activation: n = 7–22; α_WTβ4 deactivation: n = 11–12). (D) Comparison of α_D369G and α_D369Gβ4 channel kinetics at 41 µM Ca²⁺ (α_D369G activation: n = 5–18; α_D369G deactivation: n = 13–14; α_D369Gβ4 activation: n = 5–16; α_D369Gβ4 deactivation: n = 13). (E) Comparison of α_D369G and α_D369Gβ4 channel kinetics at 2.1 µM Ca²⁺ (α_D369G activation: n = 5–23; α_D369G deactivation: n = 13–19; α_D369Gβ4 activation: n = 6–34; α_D369Gβ4 deactivation: n = 8–20). (F) Comparison of α_D434G and α_D434Gβ4 channel kinetics at 41 µM Ca²⁺ (α_D434G activation: n = 11; α_D434G deactivation: n = 7; α_D434Gβ4 activation: n = 11; α_D434Gβ4 deactivation: n = 10). (G) Comparison of α_D434G and α_D434Gβ4 channel kinetics at 2.1 µM Ca²⁺ (α_D434G activation: n = 7; α_D434G deactivation: n = 5; α_D434Gβ4 activation: n = 9; α_D434Gβ4 deactivation: n = 9). Filled symbols represent measurements obtained from tail currents (deactivation time constant), and empty symbols represent measurements obtained from activation time constant.

Figure 7.

Ca²⁺-dependent effects of the mutation on BK channel recruitment by spike-shaped depolarization. (A) Voltage command of the spike-shaped depolarization (dashed line) approximating average DG granule cell action potentials (trace). (B) Representative patch showing α_D369Gβ4 current evoked by spike depolarization and various intracellular Ca²⁺. (C) Average BK current for different channels at 41 µM Ca²⁺ (α_WT: n = 19; α_WTβ4: n = 16; α_D369G: n = 22; α_D369Gβ4: n = 18), 7.3 µM Ca²⁺ (α_WT: n = 10; α_WTβ4: n = 11; α_D369G: n = 16; α_D369Gβ4: n = 9), and 3.4 µM Ca²⁺ (α_WT: n = 9; α_WTβ4: n = 8; α_D369G: n = 12; α_D369Gβ4: n = 7). Currents in B and C were normalized to maximal current size obtained from 0 mV tail current (0 mV) at saturating (1 mM) Ca²⁺. (D) Average current integral as a function of intracellular Ca²⁺ concentration. Error bars represent SEM. Curves represent fits to Hill equations (α_WT: K_1/2 = 13.7, n = 1.6; α_WTβ4: K_1/2 = 24.8, n = 1.9; α_D369G: K_1/2 = 6.3, n = 0.9; α_D369Gβ4: K_1/2 = 15.0, n = 1.5). (E) Fold increase in current resulting from the D369G mutation measured from ratio values (from D) of α_D369G/α_WT (red) and α_D369Gβ4/ α_WTβ4 (green). (F) Fold increase in current resulting from channels lacking β4 measured from ratio values (from D) of α/α_WTβ4 (green) and α_D369G/α_D369Gβ4 (blue).

Figure 8.

Ca²⁺-dependent effects of the mutation on BK channel gating in “physiological” solutions. (A; top) Average G-V relationship at 7.3 µM Ca²⁺ (α_WT: n = 18; α_WTβ4: n = 10; α_D369G: n = 8; α_D369Gβ4: n = 10). (Bottom) Average activation time constants at 7.3 µM Ca²⁺ (α_WT: n = 15; α_WTβ4: n = 10; α_D369G: n = 8; α_D369Gβ4: n = 9). Boxed regions indicate values at +50 mV. (B; top) G/G_max versus Ca²⁺ concentration at +50 mV. (Bottom) Average activation time constants at +50 mV as a function of Ca²⁺ concentration.