Limits...
Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit.

Bao L, Cox DH - J. Gen. Physiol. (2005)

Bottom Line: Our results may be summarized as follows.The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present.The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology Research Institute, New England Medical Center, Boston, MA 02111, USA.

ABSTRACT
Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are regulated by the tissue-specific expression of auxiliary beta subunits. Beta1 is predominantly expressed in smooth muscle, where it greatly enhances the BK(Ca) channel's Ca(2+) sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present. Furthermore, beta1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by approximately 24% (16.8-->12.8). The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca(2+) affinity brought about by beta1 at negative voltages, our studies suggest that beta1 also decreases the true Ca(2+) affinity of the closed channel, increasing its Ca(2+) dissociation constant from approximately 3.7 microM to between 4.7 and 7.1 microM, depending on how many binding sites are affected.

Show MeSH

Related in: MedlinePlus

The β1 subunit does not alter the voltage dependence of the closed-to-open conformational change. Macroscopic ionic currents were recorded from BKα (A) and BKα+β1 (B) channels in excised oocyte macropatches. Voltage steps were varied as indicated, and the time courses of relaxation were fitted with single-exponential functions. Time constants (τ) from the fits are plotted in C. For voltages of +100 mV and greater, activation time constants are plotted. Otherwise deactivation time courses are plotted. The τ–V curves in C were fitted (solid lines) to a function that approximates the kinetics of Scheme I in the limit that voltage sensor movement is fast relative to channel opening and closing (see MATERIALS AND METHODS). The fit parameters were as follows: BKα: held Vhc=151 mV, L = 2.2 × 10−6, zJ = 0.58 e, zL = 0.41 e, fitting yielded D = 12.6 ± 0.43, zγ = 0.10 ± 0.002 e, γ0(0) = 7452.3 s−1, γ1(0) = 4121.4 s−1, γ2(0) = 5645.8 s−1, γ3(0) = 851 s−1, γ4(0) = 1025 s−1, δ0(0) = 0.016 s−1, δ1(0) = 0.114 s−1, δ2(0) = 1.98 s−1, δ3(0) = 3.76 s−1, δ4(0) = 57.12 s−1; BKα+β1: held Vhc = 80 mV, zJ = 0.57 e, fitting yielded L = 3.3 × 10−6 ± 6.4 × 10−6, zL = 0.46 e, D = 10.4 ± 6.4, zγ = 0.17 e, γ0(0) = 931.7 s−1, γ1(0) = 213.2 s−1, γ2(0) = 547.8 s−1, γ3(0) = 333.5 s−1, γ4(0) = 126.7 s−1; δ0(0) = 0.003 s−1, δ1(0) = 0.007 s−1, δ2(0) = 0.198 s−1, δ3(0) = 1.251 s−1, δ4(0) = 4.934 s−1. Both curves are also fitted (dashed lines) at far negative voltages with the following function:τ=\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\frac{1}{{\gamma} \left \left(0\right) \right {\mathit{e}}^{-{\mathit{z}}_{{\gamma}}{{\mathit{FV}}}/{{\mathit{RT}}}}}\end{equation*}\end{document}.Fit parameters are as follows: BKα (voltages <−180 mV) γ(0) = 6940.8 s−1, z = 0.11 e; BKα+β1 (voltages <−280 mV) γ(0) = 1808.7 s−1, zγ = 0.11 e.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2266624&req=5

fig7: The β1 subunit does not alter the voltage dependence of the closed-to-open conformational change. Macroscopic ionic currents were recorded from BKα (A) and BKα+β1 (B) channels in excised oocyte macropatches. Voltage steps were varied as indicated, and the time courses of relaxation were fitted with single-exponential functions. Time constants (τ) from the fits are plotted in C. For voltages of +100 mV and greater, activation time constants are plotted. Otherwise deactivation time courses are plotted. The τ–V curves in C were fitted (solid lines) to a function that approximates the kinetics of Scheme I in the limit that voltage sensor movement is fast relative to channel opening and closing (see MATERIALS AND METHODS). The fit parameters were as follows: BKα: held Vhc=151 mV, L = 2.2 × 10−6, zJ = 0.58 e, zL = 0.41 e, fitting yielded D = 12.6 ± 0.43, zγ = 0.10 ± 0.002 e, γ0(0) = 7452.3 s−1, γ1(0) = 4121.4 s−1, γ2(0) = 5645.8 s−1, γ3(0) = 851 s−1, γ4(0) = 1025 s−1, δ0(0) = 0.016 s−1, δ1(0) = 0.114 s−1, δ2(0) = 1.98 s−1, δ3(0) = 3.76 s−1, δ4(0) = 57.12 s−1; BKα+β1: held Vhc = 80 mV, zJ = 0.57 e, fitting yielded L = 3.3 × 10−6 ± 6.4 × 10−6, zL = 0.46 e, D = 10.4 ± 6.4, zγ = 0.17 e, γ0(0) = 931.7 s−1, γ1(0) = 213.2 s−1, γ2(0) = 547.8 s−1, γ3(0) = 333.5 s−1, γ4(0) = 126.7 s−1; δ0(0) = 0.003 s−1, δ1(0) = 0.007 s−1, δ2(0) = 0.198 s−1, δ3(0) = 1.251 s−1, δ4(0) = 4.934 s−1. Both curves are also fitted (dashed lines) at far negative voltages with the following function:τ=\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\frac{1}{{\gamma} \left \left(0\right) \right {\mathit{e}}^{-{\mathit{z}}_{{\gamma}}{{\mathit{FV}}}/{{\mathit{RT}}}}}\end{equation*}\end{document}.Fit parameters are as follows: BKα (voltages <−180 mV) γ(0) = 6940.8 s−1, z = 0.11 e; BKα+β1 (voltages <−280 mV) γ(0) = 1808.7 s−1, zγ = 0.11 e.

Mentions: Thus, to fit the τ–V curves in Fig. 7, 11 independent parameters were required: L, Vhc, D, zJ, zL, zγ, γ0(0), γ1(0), γ2(0), γ3(0), and γ4(0). For definitions of L, Vhc, D, zJ, and zL see RESULTS.


Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit.

Bao L, Cox DH - J. Gen. Physiol. (2005)

The β1 subunit does not alter the voltage dependence of the closed-to-open conformational change. Macroscopic ionic currents were recorded from BKα (A) and BKα+β1 (B) channels in excised oocyte macropatches. Voltage steps were varied as indicated, and the time courses of relaxation were fitted with single-exponential functions. Time constants (τ) from the fits are plotted in C. For voltages of +100 mV and greater, activation time constants are plotted. Otherwise deactivation time courses are plotted. The τ–V curves in C were fitted (solid lines) to a function that approximates the kinetics of Scheme I in the limit that voltage sensor movement is fast relative to channel opening and closing (see MATERIALS AND METHODS). The fit parameters were as follows: BKα: held Vhc=151 mV, L = 2.2 × 10−6, zJ = 0.58 e, zL = 0.41 e, fitting yielded D = 12.6 ± 0.43, zγ = 0.10 ± 0.002 e, γ0(0) = 7452.3 s−1, γ1(0) = 4121.4 s−1, γ2(0) = 5645.8 s−1, γ3(0) = 851 s−1, γ4(0) = 1025 s−1, δ0(0) = 0.016 s−1, δ1(0) = 0.114 s−1, δ2(0) = 1.98 s−1, δ3(0) = 3.76 s−1, δ4(0) = 57.12 s−1; BKα+β1: held Vhc = 80 mV, zJ = 0.57 e, fitting yielded L = 3.3 × 10−6 ± 6.4 × 10−6, zL = 0.46 e, D = 10.4 ± 6.4, zγ = 0.17 e, γ0(0) = 931.7 s−1, γ1(0) = 213.2 s−1, γ2(0) = 547.8 s−1, γ3(0) = 333.5 s−1, γ4(0) = 126.7 s−1; δ0(0) = 0.003 s−1, δ1(0) = 0.007 s−1, δ2(0) = 0.198 s−1, δ3(0) = 1.251 s−1, δ4(0) = 4.934 s−1. Both curves are also fitted (dashed lines) at far negative voltages with the following function:τ=\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\frac{1}{{\gamma} \left \left(0\right) \right {\mathit{e}}^{-{\mathit{z}}_{{\gamma}}{{\mathit{FV}}}/{{\mathit{RT}}}}}\end{equation*}\end{document}.Fit parameters are as follows: BKα (voltages <−180 mV) γ(0) = 6940.8 s−1, z = 0.11 e; BKα+β1 (voltages <−280 mV) γ(0) = 1808.7 s−1, zγ = 0.11 e.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266624&req=5

fig7: The β1 subunit does not alter the voltage dependence of the closed-to-open conformational change. Macroscopic ionic currents were recorded from BKα (A) and BKα+β1 (B) channels in excised oocyte macropatches. Voltage steps were varied as indicated, and the time courses of relaxation were fitted with single-exponential functions. Time constants (τ) from the fits are plotted in C. For voltages of +100 mV and greater, activation time constants are plotted. Otherwise deactivation time courses are plotted. The τ–V curves in C were fitted (solid lines) to a function that approximates the kinetics of Scheme I in the limit that voltage sensor movement is fast relative to channel opening and closing (see MATERIALS AND METHODS). The fit parameters were as follows: BKα: held Vhc=151 mV, L = 2.2 × 10−6, zJ = 0.58 e, zL = 0.41 e, fitting yielded D = 12.6 ± 0.43, zγ = 0.10 ± 0.002 e, γ0(0) = 7452.3 s−1, γ1(0) = 4121.4 s−1, γ2(0) = 5645.8 s−1, γ3(0) = 851 s−1, γ4(0) = 1025 s−1, δ0(0) = 0.016 s−1, δ1(0) = 0.114 s−1, δ2(0) = 1.98 s−1, δ3(0) = 3.76 s−1, δ4(0) = 57.12 s−1; BKα+β1: held Vhc = 80 mV, zJ = 0.57 e, fitting yielded L = 3.3 × 10−6 ± 6.4 × 10−6, zL = 0.46 e, D = 10.4 ± 6.4, zγ = 0.17 e, γ0(0) = 931.7 s−1, γ1(0) = 213.2 s−1, γ2(0) = 547.8 s−1, γ3(0) = 333.5 s−1, γ4(0) = 126.7 s−1; δ0(0) = 0.003 s−1, δ1(0) = 0.007 s−1, δ2(0) = 0.198 s−1, δ3(0) = 1.251 s−1, δ4(0) = 4.934 s−1. Both curves are also fitted (dashed lines) at far negative voltages with the following function:τ=\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\frac{1}{{\gamma} \left \left(0\right) \right {\mathit{e}}^{-{\mathit{z}}_{{\gamma}}{{\mathit{FV}}}/{{\mathit{RT}}}}}\end{equation*}\end{document}.Fit parameters are as follows: BKα (voltages <−180 mV) γ(0) = 6940.8 s−1, z = 0.11 e; BKα+β1 (voltages <−280 mV) γ(0) = 1808.7 s−1, zγ = 0.11 e.
Mentions: Thus, to fit the τ–V curves in Fig. 7, 11 independent parameters were required: L, Vhc, D, zJ, zL, zγ, γ0(0), γ1(0), γ2(0), γ3(0), and γ4(0). For definitions of L, Vhc, D, zJ, and zL see RESULTS.

Bottom Line: Our results may be summarized as follows.The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present.The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology Research Institute, New England Medical Center, Boston, MA 02111, USA.

ABSTRACT
Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are regulated by the tissue-specific expression of auxiliary beta subunits. Beta1 is predominantly expressed in smooth muscle, where it greatly enhances the BK(Ca) channel's Ca(2+) sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present. Furthermore, beta1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by approximately 24% (16.8-->12.8). The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca(2+) affinity brought about by beta1 at negative voltages, our studies suggest that beta1 also decreases the true Ca(2+) affinity of the closed channel, increasing its Ca(2+) dissociation constant from approximately 3.7 microM to between 4.7 and 7.1 microM, depending on how many binding sites are affected.

Show MeSH
Related in: MedlinePlus