R treatment with 10 mM DTT (pH 7.5) for 5 min, and after subsequent treatment with 40 mM QPD (pH 7.5) for 2 min. Recordings were from outside-out macropatches with 10 mM Ca2+ inside the pipette. At each potential, the mean relative conductance 22948146 averaged from several cells is plotted. The mean G-V curve for pWT1 a is shown as a dashed-line. (C) Mean V50 6 SD of the V50s from the individual fits of the Boltzmann equation to the currents from each cell. The V50s were determined after endogenous disulfide crosslinking (black bars), after subsequent DTT (red bars), and finally after 40 mM QPD (green bars). The mean V50 for pWT1 a is shown as a dashed-line. In C, the macropatches were held at 2100 mV (closed state) during the QPDinduced reoxidation. N = 3?1. (D) As C, except that the patches were held at +80 mV (open state) during the application of QPD. The mean V50 for pWT1 a is shown as a dashed-line. * P,0.05, **P,0.01, *** P,0.001, **** P, 0.0001 by one-way Anova followed by IQ-1 site Tukey’s post-hoc analysis for multiple comparisons between brackets. Without Docosahexaenoyl ethanolamide brackets, comparison to pWT1 aby one-way Anova followed by Tukey’s post-hoc analysis. doi:10.1371/journal.pone.0058335.gOrientations and Proximities of BK a S0 and SFigure 3. Kinetics of reformation of disulfide bond between W22C and W203C in the closed state (A) and in the open state (B). Outside-out patches were bathed in 10 mM DTT (pH 7.5) for 5 min. During the subsequent application of 40 mM QPD, membrane potential was held for 1890 ms at either 2100 mV (A) or +80 mV (B). After 50 ms at 2120 mV, the patch was depolarized to +20 mV for 30 ms and hyperpolarized to 2120 mV for 30 ms, during which the tail current was recorded. This cycle, represented in the insets, was repeated every 2 s. The peak amplitudes of the tail currents are plotted against elapsed time. The data were fit with a single exponential function. The means of the rate constants from the least-squares fits of 5 independent experiments are given under the curves. The pipette solution contained 10 mM Ca2+. N = 4 for closed state and n = 5 for open state. P = not significant by unpaired Student’s t-test. doi:10.1371/journal.pone.0058335.gsively in the presence of L157C (Fig. 6B, lane 1) as in its absence (Fig. 6C, lane 1). Reduction of this crosslink with DTT and its reoxidation by QPD also proceed nearly to the same extent in the presence of L157C (Fig. 6B, lanes 2 and 3) as in its absence (Fig. 6C, lanes 2 and 3). This last result argues against the possibility that the preference of W22C for W203C compared to L157C is due to this crosslinking taking place in the ER before b1 containing L157C associates with a containing W22C and W203C. The QPD result shows that W22C prefers W203C even on the cell surface in a complex with b1 L157C (Fig. 1C). It is possible that the juxtaposition of W22C and W203C is enhanced by the docking of b1 between S0 of 
one VSD and S1 and S2 of the adjacent VSD. After reduction of the W22C-W203C disulfide by DTT, its reoxidation by QPD is on average 26 greater in the presence of b1 than in its absence (Fig. 6C; P = 0.01). Consistent with b1 acting in part to stabilize a particular interaction of S0 with S4 is 
the result that the effect on V50 of crosslinking W22C and W203C is similar to the effect of incorporating b1 in the channel complex (Fig. 7A,B; Fig. S1). In the first case, the V50 of the channel formed by a W22C/W203C alone is shifted negatively about 20 mV (at 10 mM Ca2+), whereas b1 shifts t.R treatment with 10 mM DTT (pH 7.5) for 5 min, and after subsequent treatment with 40 mM QPD (pH 7.5) for 2 min. Recordings were from outside-out macropatches with 10 mM Ca2+ inside the pipette. At each potential, the mean relative conductance 22948146 averaged from several cells is plotted. The mean G-V curve for pWT1 a is shown as a dashed-line. (C) Mean V50 6 SD of the V50s from the individual fits of the Boltzmann equation to the currents from each cell. The V50s were determined after endogenous disulfide crosslinking (black bars), after subsequent DTT (red bars), and finally after 40 mM QPD (green bars). The mean V50 for pWT1 a is shown as a dashed-line. In C, the macropatches were held at 2100 mV (closed state) during the QPDinduced reoxidation. N = 3?1. (D) As C, except that the patches were held at +80 mV (open state) during the application of QPD. The mean V50 for pWT1 a is shown as a dashed-line. * P,0.05, **P,0.01, *** P,0.001, **** P, 0.0001 by one-way Anova followed by Tukey’s post-hoc analysis for multiple comparisons between brackets. Without brackets, comparison to pWT1 aby one-way Anova followed by Tukey’s post-hoc analysis. doi:10.1371/journal.pone.0058335.gOrientations and Proximities of BK a S0 and SFigure 3. Kinetics of reformation of disulfide bond between W22C and W203C in the closed state (A) and in the open state (B). Outside-out patches were bathed in 10 mM DTT (pH 7.5) for 5 min. During the subsequent application of 40 mM QPD, membrane potential was held for 1890 ms at either 2100 mV (A) or +80 mV (B). After 50 ms at 2120 mV, the patch was depolarized to +20 mV for 30 ms and hyperpolarized to 2120 mV for 30 ms, during which the tail current was recorded. This cycle, represented in the insets, was repeated every 2 s. The peak amplitudes of the tail currents are plotted against elapsed time. The data were fit with a single exponential function. The means of the rate constants from the least-squares fits of 5 independent experiments are given under the curves. The pipette solution contained 10 mM Ca2+. N = 4 for closed state and n = 5 for open state. P = not significant by unpaired Student’s t-test. doi:10.1371/journal.pone.0058335.gsively in the presence of L157C (Fig. 6B, lane 1) as in its absence (Fig. 6C, lane 1). Reduction of this crosslink with DTT and its reoxidation by QPD also proceed nearly to the same extent in the presence of L157C (Fig. 6B, lanes 2 and 3) as in its absence (Fig. 6C, lanes 2 and 3). This last result argues against the possibility that the preference of W22C for W203C compared to L157C is due to this crosslinking taking place in the ER before b1 containing L157C associates with a containing W22C and W203C. The QPD result shows that W22C prefers W203C even on the cell surface in a complex with b1 L157C (Fig. 1C). It is possible that the juxtaposition of W22C and W203C is enhanced by the docking of b1 between S0 of one VSD and S1 and S2 of the adjacent VSD. After reduction of the W22C-W203C disulfide by DTT, its reoxidation by QPD is on average 26 greater in the presence of b1 than in its absence (Fig. 6C; P = 0.01). Consistent with b1 acting in part to stabilize a particular interaction of S0 with S4 is the result that the effect on V50 of crosslinking W22C and W203C is similar to the effect of incorporating b1 in the channel complex (Fig. 7A,B; Fig. S1). In the first case, the V50 of the channel formed by a W22C/W203C alone is shifted negatively about 20 mV (at 10 mM Ca2+), whereas b1 shifts t.
