(A) E65K genotype distribution by DBP. P values for genotype distributions at each level of diastolic hypertension are shown (with the number of subjects in parentheses). (B) Age- and sex-adjusted ORs at each level of diastolic hypertension are presented for K-carriers versus EE genotype. The adjusted ORs for K-carriers of DBP ≥ 105 mmHg and DBP ≥ 110 mmHg were 0.39 (95% confidence interval, 0.17–0.93, P = 0.034) and 0.12 (95% confidence interval, 0.02–0.90, P = 0.039), respectively. *Subjects under hypertension treatment with DBP < 90 mmHg were also included in this group.

BK channel currents obtained from different combinations of α and β₁ BK channel subunits. Currents were recorded from excised inside-out macropatches obtained from HEK-293 cells expressing the BK α subunit (A), α+β_1WT (B), α+β_1E65K (C), or α+β_1WT+β_1E65K (D). Currents were recorded at 0.1 μM (left panels), 1.6 μM (middle panels), and 10 μM cytosolic Ca²⁺ (right panels). The voltage protocol was as described in Methods.

Plots of conductance versus voltage for BK channel currents at different Ca²⁺ concentrations. BK channel currents were recorded from patches excised from cells expressing α (A), α+β_1WT (B), α+β_1E65K (C), and α+β_1WT+β_1E65K (D) channel subunits. Normalized conductance was obtained for each test potential from the peak tail current at –80 mV measured at 0 (circles), 100 nM (squares), 500 nM (triangles), and 10 μM (inverted triangles) Ca²⁺. Solid curves represent fits to the Boltzmann equation. The data at 0 and 100 nM Ca²⁺ were normalized using the value obtained at 500 nM as maximum tail current. To obtain an average G-V curve for each case, individual curves were displaced along the voltage axis by ΔV = (<V_1/2> – V_1/2) (where <V_1/2> denotes the average V_1/2 for each transfection group). The 300–360 resulting points were then reduced to 61 with the smoothing function of SigmaPlot (SPSS Inc., Richmond, California, USA).

Plot of V_1/2 versus Ca²⁺ concentration. (A) V_1/2 versus Ca²⁺ concentration obtained for α+β_1WT (n = 13) and α+β_1E65K (n = 15). (B) V_1/2 versus Ca²⁺ concentration for α+β_1WT and α+β_1WT+β_1E65K (n = 6). (C) V_1/2 versus Ca²⁺ concentration for α (n = 3). Data are shown as mean ± SEM. Differences in V_1/2 curves between different β₁ subunits were tested by ANOVA with repeated measurements. P = 0.002 for β_1WT vs. β_1E65K, and P = 0.005 for β_1WT vs. α+β_1WT+β_1E65K.

Effect of β_1E65K on BK channel activation and deactivation kinetics. (A) Current traces normalized to peak current were obtained with a pulse from 0 mV to +200 mV in the presence of 1.6 μM Ca²⁺. (B) Activation-time constants (at 1.6 μM Ca²⁺) were fitted with a single exponential function and plotted versus the pulse potential for α (open triangles, n = 4), α+β_1WT (open circles, n = 13), α+β_1E65K (filled circles, n = 15), and α+β_1WT+β_1E65K currents (open squares, n = 6). (C) Plot of activation-time constants versus Ca²⁺ concentrations measured with a pulse to +200 mV. (D) Families of tail currents recorded at 1.6 μM Ca²⁺ under the four conditions. (E and F) Deactivation-time constants were obtained by fitting of the tail currents with a single exponential function and plotted versus the pulse potential (E) or versus the Ca²⁺ concentration at –80 mV (F).

Fitting of the experimental data to an allosteric model of BK channel gating. (A and B) G-V plots for α+β_1WT (A) and α+β_1E65K (B) currents measured at 0 (circles), 100 nM (squares), 500 nM (triangles), and 10 μM (inverted triangles) Ca²⁺. Solid curves represent fits to Equation 1 (see Methods) with parameters restricted as described in the text. (C) G-V plots for α+β_1WT (solid line) and α+β_1E65K (dashed line) channels as predicted by the model. (D) V_1/2-versus-Ca²⁺ plots obtained from the G-V curves presented in C. (E) Allosteric kinetic scheme proposed for the BK channel by Horrigan and Aldrich (33, 34). The C-O transition corresponds to the closed-open equilibrium where L = L₀ exp(z_L × V / kT). The R-A transition corresponds to the resting-active equilibrium of a single voltage sensor where J = J₀ exp(z_J × V / kT). The X·Ca²⁺ transition is calcium binding to a single calcium sensor, with equilibrium constant K = [Ca²⁺] / K_d. These three equilibriums are related to each other by the allosteric factors C, D, and E, as shown. When there are n voltage sensors active, the C-O equilibrium constant is LDⁿ. Conversely, when the channel is open, the R-A equilibrium constant is JD. The same applies for the allosteric factors C and E.

Proposed effect of BK-β_1E65K channels in VSMCs. (A) Absence of the β₁ subunit shifts the basal tone toward a more contracted state. Ca²⁺ sparks (a1) caused by the opening ryanodine receptors (RR) in the sarcoplasmic reticulum (SR) activate BK channels (a2); however, because of the absence of the β₁ subunit, there is a decreased coupling of Ca²⁺ sparks to BK channel activation, resulting in low BK channel activity, membrane depolarization, and higher Ca²⁺ entry via depolarization-activated Ca²⁺ channels (a3). (B) Ca²⁺ sparks (b1) in the presence of the β₁ subunit induced a larger activation of BK-β₁ channels (b2), leading to an average 20-mV hyperpolarization and thus reducing the entry of Ca²⁺ (b3). (C) The mutant BK-β_1E65K channels (c2) show increased activity (compared with BK-β_1WT channels) at the Ca²⁺ concentrations produced within a single spark (c1), resulting in increased hyperpolarization and reduced activation of voltage-gated Ca²⁺ channels (c3), which further reduces Ca²⁺ entry (c3), offering a more efficient negative-feedback mechanism and, hence, vasodilation.