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

Wang B, Rothberg BS, Brenner R - J. Gen. Physiol. (2009)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.

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.

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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 αD369Gβ4 current evoked by spike depolarization and various intracellular Ca2+. (C) Average BK current for different channels at 41 µM Ca2+ (αWT: n = 19; αWTβ4: n = 16; αD369G: n = 22; αD369Gβ4: n = 18), 7.3 µM Ca2+ (αWT: n = 10; αWTβ4: n = 11; αD369G: n = 16; αD369Gβ4: n = 9), and 3.4 µM Ca2+ (αWT: n = 9; αWTβ4: n = 8; αD369G: n = 12; αD369Gβ4: 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 (αWT: K1/2 = 13.7, n = 1.6; αWTβ4: K1/2 = 24.8, n = 1.9; αD369G: K1/2 = 6.3, n = 0.9; αD369Gβ4: K1/2 = 15.0, n = 1.5). (E) Fold increase in current resulting from the D369G mutation measured from ratio values (from D) of αD369G/αWT (red) and αD369Gβ4/ αWTβ4 (green). (F) Fold increase in current resulting from channels lacking β4 measured from ratio values (from D) of α/αWTβ4 (green) and αD369G/αD369Gβ4 (blue).
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fig7: 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 αD369Gβ4 current evoked by spike depolarization and various intracellular Ca2+. (C) Average BK current for different channels at 41 µM Ca2+ (αWT: n = 19; αWTβ4: n = 16; αD369G: n = 22; αD369Gβ4: n = 18), 7.3 µM Ca2+ (αWT: n = 10; αWTβ4: n = 11; αD369G: n = 16; αD369Gβ4: n = 9), and 3.4 µM Ca2+ (αWT: n = 9; αWTβ4: n = 8; αD369G: n = 12; αD369Gβ4: 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 (αWT: K1/2 = 13.7, n = 1.6; αWTβ4: K1/2 = 24.8, n = 1.9; αD369G: K1/2 = 6.3, n = 0.9; αD369Gβ4: K1/2 = 15.0, n = 1.5). (E) Fold increase in current resulting from the D369G mutation measured from ratio values (from D) of αD369G/αWT (red) and αD369Gβ4/ αWTβ4 (green). (F) Fold increase in current resulting from channels lacking β4 measured from ratio values (from D) of α/αWTβ4 (green) and αD369G/αD369Gβ4 (blue).

Mentions: The “symmetrical” external recording solution (electrode solution for Figs. 1–6) was composed of the following (in mM): 20 HEPES, 140 KMeSO3, 2 KCl, and 2 MgCl2, pH 7.2. The “physiological” external recording solution (electrode solution for Figs. 7 and 8) was composed of the following (in mM): 10 HEPES, 145 NaCl, 5 KCl, 1 MgCl2, and 2 CaCl2, pH 7.2. Internal solutions were composed of a pH 7.2 solution of the following (in mM): 20 HEPES, 140 KMeSO3, and 2 KCl. Intracellular Ca2+ was buffered with 5 mM EGTA (0.073 and 0.006 µM), HEDTA (2.1 and 0.363 µM) or NTA (41 µM), and free [Ca2+] was measured using a Ca2+-sensitive electrode (Orion Research, Inc.). In low Ca2+ solutions (0.073 and 0.006 µM), Ba2+ was chelated with 40 µM (+)-18-crown-6-tetracarboxylic acid (Cox et al., 1997).


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

Wang B, Rothberg BS, Brenner R - J. Gen. Physiol. (2009)

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 αD369Gβ4 current evoked by spike depolarization and various intracellular Ca2+. (C) Average BK current for different channels at 41 µM Ca2+ (αWT: n = 19; αWTβ4: n = 16; αD369G: n = 22; αD369Gβ4: n = 18), 7.3 µM Ca2+ (αWT: n = 10; αWTβ4: n = 11; αD369G: n = 16; αD369Gβ4: n = 9), and 3.4 µM Ca2+ (αWT: n = 9; αWTβ4: n = 8; αD369G: n = 12; αD369Gβ4: 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 (αWT: K1/2 = 13.7, n = 1.6; αWTβ4: K1/2 = 24.8, n = 1.9; αD369G: K1/2 = 6.3, n = 0.9; αD369Gβ4: K1/2 = 15.0, n = 1.5). (E) Fold increase in current resulting from the D369G mutation measured from ratio values (from D) of αD369G/αWT (red) and αD369Gβ4/ αWTβ4 (green). (F) Fold increase in current resulting from channels lacking β4 measured from ratio values (from D) of α/αWTβ4 (green) and αD369G/αD369Gβ4 (blue).
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Related In: Results  -  Collection

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fig7: 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 αD369Gβ4 current evoked by spike depolarization and various intracellular Ca2+. (C) Average BK current for different channels at 41 µM Ca2+ (αWT: n = 19; αWTβ4: n = 16; αD369G: n = 22; αD369Gβ4: n = 18), 7.3 µM Ca2+ (αWT: n = 10; αWTβ4: n = 11; αD369G: n = 16; αD369Gβ4: n = 9), and 3.4 µM Ca2+ (αWT: n = 9; αWTβ4: n = 8; αD369G: n = 12; αD369Gβ4: 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 (αWT: K1/2 = 13.7, n = 1.6; αWTβ4: K1/2 = 24.8, n = 1.9; αD369G: K1/2 = 6.3, n = 0.9; αD369Gβ4: K1/2 = 15.0, n = 1.5). (E) Fold increase in current resulting from the D369G mutation measured from ratio values (from D) of αD369G/αWT (red) and αD369Gβ4/ αWTβ4 (green). (F) Fold increase in current resulting from channels lacking β4 measured from ratio values (from D) of α/αWTβ4 (green) and αD369G/αD369Gβ4 (blue).
Mentions: The “symmetrical” external recording solution (electrode solution for Figs. 1–6) was composed of the following (in mM): 20 HEPES, 140 KMeSO3, 2 KCl, and 2 MgCl2, pH 7.2. The “physiological” external recording solution (electrode solution for Figs. 7 and 8) was composed of the following (in mM): 10 HEPES, 145 NaCl, 5 KCl, 1 MgCl2, and 2 CaCl2, pH 7.2. Internal solutions were composed of a pH 7.2 solution of the following (in mM): 20 HEPES, 140 KMeSO3, and 2 KCl. Intracellular Ca2+ was buffered with 5 mM EGTA (0.073 and 0.006 µM), HEDTA (2.1 and 0.363 µM) or NTA (41 µM), and free [Ca2+] was measured using a Ca2+-sensitive electrode (Orion Research, Inc.). In low Ca2+ solutions (0.073 and 0.006 µM), Ba2+ was chelated with 40 µM (+)-18-crown-6-tetracarboxylic acid (Cox et al., 1997).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.

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.

Show MeSH
Related in: MedlinePlus