Bao L , Cox DH .

R01 HL 64831 NHLBI NIH HHS , R01 HL064831 NHLBI NIH HHS

	Figure 1. β1 increases the Ca2+ sensitivity of the BKCa channel. (A and B) Macroscopic currents recorded from BKα channels (A) and BKα+β1 channels (B). Currents are from inside-out Xenopus oocyte macropatches exposed to 10 μM internal Ca2+. (C and D) G–V relations determined at the following Ca2+ concentrations: 0.003, 1, 10, and 100 μM for the BKα channel (C) and BKα+β1 channel (D). Each curve represents the average of between 4 and 22 individual curves. Error bars indicate SEM. The solid curves are Boltzmann fits with the following parameters: BKα, 3 nM Ca2+: Q = 0.93 e, V1/2 = 200.3 mV; 1 μM Ca2+: Q = 1.36 e, V1/2 = 120.6 mV; 10 μM Ca2+: Q = 1.18 e, V1/2 = 32.8 mV; 100 μM Ca2+: Q = 1.15 e, V1/2 = −2.4 mV. BKα1β1, 3 nM Ca2+: Q = 0.62 e, V1/2 = 213.1 mV; 1 μM Ca2+: Q = 0.94 e, V1/2 = 82.1 mV; 10 μM Ca2+: Q = 1.02 e, V1/2 = −59.5 mV; 100 μM Ca2+: Q = 0.96 e, V1/2 = −101 mV. (E) Plots of half-maximal activation voltage (V1/2) vs. Ca2+ concentration. The V1/2 values are from Table I. Error bars represent SEM.
	Figure 2. The effects of β1 are both Ca2+ and voltage dependent. (A–C) Ca2+ dose–response curves determined for the BKα and BKα+β1 channel at (A) −40, (B) 0, and (C) +40 mV. Curves are fitted with the hill equation:\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{{\mathit{G}}}{{\mathit{G}}_{max}}\end{equation*}\end{document}=\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}{1+ \left \left({{\mathit{Kd}}}/{ \left \left[{\mathit{Ca}}\right] \right }\right) \right ^{{\mathit{n}}}}\end{equation*}\end{document}.Fit parameters are as follows: −40 mV, BKα (Kd = 36.5 μM, n = 1.24); BKα+β1(Kd = 6.89 μM, n = 2.7); 0 mV, BKα (Kd = 22.1 μM, n = 1.5); BKα+β1 (Kd = 3.4 μM, n = 1.9); +40 mV, BKα (Kd = 7.25 μM, n = 1.6); BKα+β1(Kd = 3.68 μM, n = 1.5). (D–G) G–V relations are shown for the BKα and the BKα+β1 channel at (D) 100 μM Ca2+, (E) 10 μM Ca2+, (F) 1 μM Ca2+, and (G) 3 nM Ca2+. The curves are fitted with Boltzmann functions as described in the legend to Fig. 1.
	Figure 3. BKCa gating currents. (Top traces) Gating current families recorded from BKα (A) and BKα+β1 (B) channels with 0.5 nM internal Ca2+. The second and third traces in A and B demonstrate that gating currents are not observed in patches from oocytes that were not injected with BKCa cRNA (second) or with hyperpolarizing voltage steps (third). The lowest traces in A and B are gating currents recorded with pulses to +160 mV. Repolarizations are to −80 mV. Exponential fits to the on and off currents are indicated with dashed line. (C and D) Comparisons of on-gating current (Ig) and potassium current (IK) from BKα (C) and BKα+β1 (D) channels. Pulses were to +160 mV. Ca2+ = 0.5 nM. The gating and ionic currents compared in C and D are from different patches.
	Figure 4. The β1 subunit shifts the closed channel's charge–voltage (Q–V) relation leftward without changing its shape. (A) On and Off Q–V relations determined from a single BKα channel patch. (B) On and Off Q–V relations determined from a single BKα+β1 channel patch. (C) Normalized averaged Qon–V relations for the BKα (11 curves averaged) and BKα+β1 channels (14 curves averaged). Each curve in C is fitted with a Boltzmann function. The fit parameters are as follows: BKα: zJ = 0.577 ± 0.023e, Vhc = 151 ± 1.9 mV; BKα+β1: zJ = 0.571 ± 0.025 e, Vhc = 80 ± 2.4 mV. The error bars in C represent SEM.
	Figure 5. The allosteric model of BKCa voltage-dependent gating of Horrigan et al. (1999). Horizontal transitions represent voltage senor movement. Vertical transitions represent channel opening. For details of the model see RESULTS and MATERIALS AND METHODS.
	Figure 6. Estimating L and zL from Popen at negative voltages. Unitary currents were recorded with 3 nM internal Ca2+ from BKα (A) and BKα+β1(B) channels. The patch in A contained 2,859 channels. The patch in B contained 3,270 channels. Mean log(Popen)–voltage relations determined from patches like that in A (12 patches) and B (15 patches) are shown in C for the BKα channel and D for the BKα+β1 channel. In C, the bottom of the curve is fitted with the following function: log(Popen) = log(L) + 0.4342zLFV/RT. The resulting parameters were log(L) = −5.66 ± 0.13, zL = 0.410 ± 0.065. In D, no submaximal limiting slope was identified.
	Figure 7. 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.
	Figure 8. Estimating L and Vho from Popen–V curve fits. (A) The Po–V relation of the BKα channel at 3 nM Ca2+ is shown over a wide range of voltages. At +60 mV and below, the data points are from unitary current measurements; above +60 mV, the data are from macroscopic current measurements. The curve displayed represents the average of 35 experiments, and error bars represent SEM. The data are fitted with Eq. 9 as described in RESULTS. Free parameters: D = 16.8 ± 0.28. (B) The Po–V relation of the BKα+β1 channel. The curve displayed represents the average of 43 experiments, and error bars represent SEM. As in A, the data are fitted with Eq. 9. Free parameters: D = 12.8 ± 0.55, L = 2.53 × 10−6 ± 0.55 × 10−6. The dashed red curve is the fit from A placed here for ease of comparison. (C) Simulated Q–V relations for the open BKα and BKα+β1 channels demonstrating the −61 mV shift induced by β1 that the fits in A and B predict. The functions displayed are\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{{\mathit{Q}}}{{\mathit{Q}}_{max}}\end{equation*}\end{document}=\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}{1+{\mathit{e}}^{{\mathit{z}}_{{\mathit{J}}}{\mathit{F}}{ \left \left({\mathit{V}}_{{\mathit{ho}}}-V\right) \right }/{{\mathit{RT}}}}}\end{equation*}\end{document},with Vho equal to 27 mV (BKα) and −34 mV (BKα+β1) and zJ = 0.58 (BKα), zJ = 0.57 (BKα+β1).
	Figure 9. β1 also affects Ca2+ binding. (A) BKα G–V relations at a series of Ca2+ concentrations fitted simultaneously (solid curves) with Eq. 14. Only KC and KO were allowed to vary. The fit yielded KC = 3.71 μM and KO = 0.88 μM. The other parameters of the fit were determined from experiments performed with nominally 0 Ca2+ (3 nM) (Vhc = 151 mV, L = 2.2 × 10−6, Vho = 27 mV, zJ = 0.58, zL = 0.41). (B) The fit from A is superimposed on a series of BKα+β1 G–V curves. (C) The voltage-sensing parameters of the model were altered to reflect the changes that occur as β1 binds, Vho = (27→−34 mV), Vhc = (151→80 mV), L = (2.2 × 10−6→2.5 × 10−6). (D) With BKα+β1 voltage-sensing parameters KC and KO were allowed to vary freely, yielding the fit shown and KC = 4.72 μM, KO = 0.82 μM. (E) Here, the BKα+β1 voltage-sensing parameters were used for the fit, and β1 was allowed to influence only half of the channels' eight Ca2+-binding sites. The data are fit with Eq. 13. KC1 and KO1 were held at 3.71 μM and 0.88 μM, respectively. KC2 and KO2 were allowed to vary freely, yielding KC2 = 5.78 μM, KO2 = 0.73 μM. (F) The data are again fit with Eq. 13 but now KO2 was held at 0.88 μM and only KC2 was allowed to vary. This yielded KC2 = 7.14 μM.
	Figure 10. Examining the effects of β1 in energetic terms. (A) Plot of the sum of terms 3 and 4 from Eq. 15. (B) Plots of term 2 from Eq. 15 using either the BKα (red) or BKα+β1 (blue) voltage-sensing parameters (Table II). (C) Plots for both channels of terms 2, 3, and 4 of Eq. 15 combined. In the inset are drawn simulated Popen–V curves for the BKα and BKα+β1 channels based on the parameters in Table II and assuming 0 Ca2+. The boxed region indicates the energy range over which Popen moves from 0.05 (top of box) to 0.95 (bottom of box). A full Popen vs. ΔGO−C is shown in D. In E, a plot is shown like that in C except that Vho for the BKα+β1 channel has been set to −44 mV such that Vhc and Vho shift equally upon β1 coexpression. (F) Here Vho for the BKα+β1 channel has been set to −20 mV to mimic a larger change in the coupling factor D upon β1 coexpression.
	Figure 11. Ca2+ shifts the ΔGO−C–V relations to lower energies. (A) Plot of ΔGO−C vs. Ca2+ concentration for the BKα (red) and BKα+β1(blue) channels using the Ca2+ binding parameters determined from Fig. 11 (A and D). The plots were calculated using term 1 of Eq. 15. (B) As in Fig. 10 C, plotted here are the two model channel's ΔGO−C–V relations in the absence of Ca2+. (C) The same relations now with 1 μM Ca2+, (D) 10 μM Ca2+, and (E) 100 μM Ca2+. The dashed lines in C, D, and E indicate the BKα+β1 curve that would be observed, if there were no changes in Ca2+ bindng upon β1 coexpression. (F) Plotted are the total V1/2 shifts predicted by the BKα and BKα+β1 model channels upon β1 coexpression at 0, 1, 10, and 100 μM Ca2+. In green is the percent of the total shift due to changes in voltage sensing, and in yellow is the percentage due to changes in Ca2+ binding.

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