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Figure 1. Characterization of BK F380 mutant ionic currents. (A) BK-WT currents (IK) evoked by voltage steps (−100 to 300 mV; in 10-mV steps of 10 ms) at two different [Ca2+]i (0 and 100 µM). (B) BK-F380A macroscopic currents recorded at 0 and 100 µM (−100 to 350 mV; in 10-mV steps of 2 ms). Note the difference in time scale because of the fast activation in the case of BK-F380A. (C) BK-F380W currents (IK) evoked by voltage steps (−220 to 300 mV; in 10-mV steps of 10 ms) in two different [Ca2+]i (0 and 100 µM). (D–F) Relative conductance versus voltage ((G/GMAX) − V) curves were obtained from tail current measurements from experiments as those shown in A–C. Data were fitted using a Boltzmann function (solid lines). For the BK-WT channel, parameters were (mean ± SEM) Vh = 176 ± 8 mV, zG = 0.96 ± 0.08 e0 at [Ca+2]i = 0 µM, n = 5; Vh = 120 ± 10 mV, zG = 1.30 ± 0.10 e0 at [Ca+2]i = 0.1 µM, n = 5; Vh = 110 ± 8 mV, zG = 1.40 ± 0.10 e0 at [Ca+2]i = 0.2 µM, n = 6; Vh = 81 ± 5 mV, zG = 1.33 ± 0.12 e0 at [Ca+2]i = 0.5 µM, n = 5; Vh = 52 ± 8 mV, zG = 1.40 ± 0.06 e0 at [Ca+2]i = 1.5 µM, n = 5; Vh = 20 ± 6 mV, zG = 1.34 ± 0.11 e0 at [Ca+2]i = 3 µM, n = 6; Vh = −43.7 ± 11 mV, zG = 1.12 ± 0.04 e0 at [Ca+2]i = 100 µM, n = 5. BK-F380A: Vh = 620 ± 34 mV, zG = 0.32 ± 0.03 e0 at [Ca+2]i = 0.2 µM, n = 4; Vh = 492 ± 19 mV, zG = 0.43 ± 0.02 e0 at [Ca+2]i = 0.5 µM, n = 4; Vh = 349.8 ± 11.7 mV, zG = 0.42 ± 0.01 e0 at [Ca+2]i = 1.5 µM, n = 5; Vh = 321 ± 9 mV, zG = 0.45 ± 0.01 e0 at [Ca+2]i = 3 µM, n = 4; Vh = 232 ± 25 mV, zG = 0.48 ± 0.05 e0 at [Ca+2]i = 100 µM, n = 5; BK-F380W: Vh = 133 ± 3 mV, zG = 0.73 ± 0.03 e0 at [Ca+2]i = 0 µM, n = 17; Vh = 102 ± 6 mV, zG = 0.52 ± 0.02 e0 at [Ca+2]i = 0.1 µM, n = 6; Vh = 92 ± 7 mV, zG = 0.52 ± 0.01 e0 at [Ca+2]i = 0.2 µM, n = 7; Vh = 80 ± 5 mV, zG = 0.55 ± 0.03 e0 at [Ca+2]i = 0.5 µM, n = 7; Vh = 26 ± 5 mV, zG = 0.70 ± 0.01 e0 at [Ca+2]i = 3 µM, n = 7; Vh = −58 ± 3 mV, zG = 0.52 ± 0.03 e0 at [Ca+2]i = 100 µM, n = 14. (G and H) BK-F380I and BK-F380L currents (IK) evoked by voltage steps (−100 to 350 mV; in 10-mV steps of 2 ms) at [Ca2+]i = 100 µM, respectively. (I) Relative conductance versus voltage ((G/GMAX) − V) curves were obtained from tail current measurements from the experiments shown in G and H. Data were fitted using a Boltzmann function (solid lines). For the BK-F380I channel, parameters were (mean ± SEM) Vh = 261 ± 6 mV, zG = 0.72 ± 0.08 e0 at [Ca+2]i = 100 µM, n = 4. BK-F380L: Vh = 267 ± 6 mV, zG = 0.58 ± 0.04 e0 at [Ca+2]i = 100 µM, n = 7. Black and red lines are the G/GMAX − V curves and Boltzmann fitting of the BK channel WT and F380A mutant at [Ca+2]i = 100 µM (as is seen in D and E), respectively.
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Figure 2. Characterization of BK F380A mutant. (A) IbTx blocks the BK-F380A mutant channel. Currents elicited by a 150-mV pulse were recorded in outside-out patches and symmetrical K+ 110 mM. The bath solution contained 100 nM IbTx, [Ca2+]i = 100 µM. Currents were recorded immediately before (t1), 1 min after (t2), and 5 min after IbTx addition (t3). (B) Ig and leak currents were recorded in symmetrical 110 mM K+ and internal 0 Ca2+, and the pipette solution contained 500 nM IbTx. Currents were recorded immediately after gigaseal formation (t1), after 1 min (t2), and after 5 min (t3). (C) Single-channel recordings of WT BK, F380A, and F380W in the absence of internal Ca2+ and upon depolarization at 50 mV. To elicit F380A mutant current, we used a huge pipette, ∼30–40 µm. (D–F) Nonstationary noise analysis of BK-WT, BK-F380A, and BK-F380W. Inside-out patches were held to 0 mV and pulsed 200 times from −100 to 100 mV at steps of 2-ms, 10-ms, and 10-ms duration for F380A, F380W, and WT, respectively. [Ca2+]i = 100 µM. (G) Voltage dependence of deactivation time constants (mean ± SEM) for the WT and F380A channels. Voltage dependence of deactivation rates were calculated from the slope of the straight lines (solid lines) at very negative potentials: zγ = 0.159 for WT; zγ = 0.138 for F380A. Deactivation rates at 0 mV were estimated by extrapolating the straight lines: γ0 = 1,494/s for WT; γ0 = 7,243/s for F380A.
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Figure 3. Gating currents of BK-F380A mutant. (A) Fast ON gating charge versus voltage curves obtained in the presence of symmetrical 110 mM KMES (closed circles) and in the absence of permeant ions (open circles), the inset shows the gating and macroscopic currents in the presence of permeant ions (KMES). Ig BK-F380A was recorded in inside-out macropatches at 0 [Ca+2]i (inset). (B) Open probability versus voltage curve (mean ± SEM) was obtained from F380A mutant current record shown in the inset of A and calculated as was described in Materials and methods. The best fitting of this data to a monoexponential curve was L*(V)=L0*ezLFVRT=L0D4ezLFVRT=6.59*10−5e16.5V. (C and D) Ig of BK and BK-F380A was recorded from inside-out macropatches at 0 and 100 µM of [Ca+2]i. To isolate Ig of all ionic currents and Ileak, our internal and external solutions contained nonpermeant ions (see Materials and methods). Ig was evoked by 2-ms voltage steps of 10 mV, going from −90 to 350 mV. (E) Qc(V) relationships of BK and BK-F380A were obtained by integrating the fast component for each ON Ig trace. Boltzmann fitting to the experimental data (mean ± SEM) is indicated by solid lines (V0 = 169 ± 3 mV, zQ = 0.63 ± 0.02 e0 in BK at [Ca+2]i = 0 µM, n = 14; V0 = 29 ± 8 mV, zQ = 0.71 ± 0.07 e0 in BK at [Ca+2]i = 100 µM, n = 3; V0 = 149 ± 3 mV, zQ = 0.59 ± 0.01 e0 in BK-F380A at [Ca+2]i = 0 µM, n = 9; V0 = 72 ± 3 mV, zQ = 0.87 ± 0.05 e0 in BK-F380A at [Ca+2]i = 100 µM, n = 3).
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Figure 4. Gating currents of BK-F380 mutants in internal calcium absence. (A–C, insets) Ig of mutants (F380I, F380L, and F380W) was recorded in inside-out macropatches at 0 µM [Ca+2]i. Ig was evoked by 2-ms voltage steps of 10 mV going from −90 to 350 mV. (A–C) Qc(V) relationships of BK and BK-F380 mutants were obtained by integrating each Ig trace. Boltzmann fitting to the experimental data (mean ± SEM) is indicated by solid lines (V0 = 182 ± 9 mV, zQ = 0.56 ± 0.01 e0 in BK-F380I, n = 5; V0 = 173 ± 4 mV, zQ = 0.55 ± 0.01 e0 in BK-F380L, n = 7; V0 = 153 ± 6 mV, zQ = 0.56 ± 0.02 e0 in BK-F380W, n = 4). Dashed lines represent the Boltzmann fitting to the WT Qc(V) obtained in the absence of intracellular Ca2+ (Fig. 3 E). (D) V0 of Qc(V) of mutants (F380I, F380L, and F380W) compared with Qc(V) of WT and F380A. Mean ± SEM.
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Figure 5. Slow gating charge recovery of WT and F380A. (A) Representative recording of gating currents evoked by 200-mV pulses with different durations from 0.04 to 120 ms. (B and C) Slow gating charge kinetics of BK and BK-F380A. The OFF charge (QOFF; filled circles; mean ± SEM) was integrated in a range of 1 ms, normalized to the QMAX for each patch, and plotted against the pulse duration (time; logarithmic scale) for different voltage steps (40, 80, 120, 160, and 200 mV). The QOFF/QMAX − t relations were fitted with a two-component exponential function (solid line) with time constant and amplitude of the fast component constrained to the exponential fit of IgON at these voltages. (D and E) 10-state allosteric model proposed for WT and mutant F380A channel, respectively. Darkness of the arrows indicates the probability of transitions.
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Figure 6. Model fitting. (A) BK WT data (mean ± SEM) were fitted with the HA kinetic model (Horrigan and Aldrich, 2002) as is described in Materials and methods (final parameter values were L0 = 5.5 × 10−6, zL = 0.33 e0, J0 = 0.021, zJ = 0.58 e0, kd = 9.9 µM, C = 5.6, D = 19.6, E = 24.2). (B) F380A data (mean ± SEM) were fitted with the HA kinetic model. Effects of mutation on the gating can be explained by a decrease in the allosteric parameter between calcium binding and opening (C = 5.6; P < 0.01), the allosteric parameter between the voltage sensor activation and opening (D = 11.7; P < 0.01), the allosteric parameter between the calcium binding and the voltage sensor activation (E = 5.8; P < 0.01), and a large decrease in the open–closed intrinsic equilibrium constant (L0 = 3.3 × 10−9; P < 0.01). Those parameters whose change was significant are colored in red. (C) F380W data (mean ± SEM) were fitted with the HA kinetic model. Effects of mutation on the gating can be explained by a decrease in the allosteric parameter between calcium binding and opening (C = 2.6; P < 0.01), the allosteric parameter between the voltage sensor activation and opening (D = 2.3; P < 0.01), and a large increase in the open–closed intrinsic equilibrium constant (L0 = 4.2 × 10−2; P < 0.01). Those parameters whose change was significant are colored in blue. (D) Experimental Vh (mean ± SEM) of WT BK, F380A, and F380W versus the predicted Vh to different internal Ca2+ concentration using the HA allosteric model with the parameters obtained in A–C.
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Figure 7. Molecular BK model and MD analysis. (A) Model built for open BK WT channel pore region (green balls are K+ and blue balls are Cl−). (B) Side view of the hydrophobic ring formed by the F380 and L377 residues in BK WT (one subunit is transparent to better view). (C–E) Top view of the hydrophobic ring formed by the 380 and 377 residues in BK WT, F380A, and F380W, respectively. (F) Interaction energy between L377 and 380 residues (WT is considered a reference).
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