Figure 1.
Molecular structure of the lipids used in this study.
The left panel shows lipids having identical acyl chains but with different polar headgroups. The right panel shows lipids having identical polar headgroups but different acyl chain length.
Figure 2.
β1 creates a leftward shift in the G-V relationship of BK channels.
A) The macroscopic currents recorded from HEK 293 cells stably transected with hSlo α with 5 uM Ca2+ inside the pipette and in the bath. The holding potential is −60 mV, traces from 0 to 220 mV with delta 20 mV. B) G-V relations determined at 1, 5, and 10 µM Ca2+ concentrations. C) The macroscopic currents recorded for hSlo αβ1 at 5 µM Ca2+. The holding potential is −60 mV, and traces from −60 to 160 mV with delta 20 mV. D) G-V relations determined at 1, 5, and 10 µM Ca2+ concentrations for hSlo αβ1. Each curve in B and D represents the average of between 3 and 7 individual curves. Error bars indicate SEM. E) Plots of half-maximal activation voltage (V1/2) vs. Ca2+ concentration. V1/2 were statistically different between hSlo and hSlo αβ1 for every calcium concentration tested. Error bars represent SEM, n = 4–7.
Figure 3.
β1 increases the apparent Ca2+ sensitivity of the BK channel in a POPE/POPS bilayer.
A, Single channel current traces of hSlo α and hSlo αβ1 recorded in low [Ca2+] of 6.2 µM under varied holding potentials from −40 mV to +40 mV, show an increase in channel open probability (Po) through an increase in channel open durations after co-expression of αβ1 subunit. B, Single channel currents of hSlo α and hSlo αβ1 recorded at high [Ca2+] of 22.5 µM under varied holding potentials from −40 mV to +40 mV, show a decrease of voltage sensitivity after co-expression of αβ1 subunit (reduced change of Po per unit change of voltage; i.e., the slope of Po-V plot). C, plots of log Po vs V of BK α (open symbols) and BK αβ1 (filled symbols) recorded at three different [Ca2+] concentrations: 6.2 µM (triangles), 15.6 µM (squares) and 22.5 µM (circles). Error bars represent SEM, n = 5–9.
Figure 4.
The open and closed time distribution of BK hSlo α and hSlo αβ1 recorded at free [Ca2+] of 6. 2 µM.
Dwell-time data were plotted with a logarithmic time axis along the abscissa and a square-root ordinate exhibiting the number of events in each bin. The bin density is 20 bins per decade. A lower limit of 0.6 ms was set for the dwell time distribution of open and closed intervals, consistent with the time resolution of sampling and filtering. The time constants for each fit of the open and closed time distribution are listed in the figure, and the fractional contribution of each particular component to the composite fit is given in parentheses.
Figure 5.
β1 has little effect on the apparent Ca2+ sensitivity of BK in the neutral POPE/POPC bilayer, but dramatically increases the apparent Ca2+ in negatively charged lipid bilayers of POPE/POPA, POPE/POPS, and POPE/POPG.
A, Sample currents from a single channel of hSlo α and hSlo αβ1 recorded in POPE/POPC bilayer at low [Ca2+] of 6.2 µM with holding potentials from −40 mV to +40 mV. B, Sample currents of a single channel of hSlo α and hSlo αβ1 recorded in POPE/POPC bilayer at high [Ca2+] of 22.5 µM with varied holding potentials from −40 mV to +40 mV. C, plots of log Po vs V of BK α (open symbols) and BK αβ1 (filled symbols) recorded at three different [Ca2+] concentrations of 6.2 µM (triangles), 15.6 µM (squares) and 22.5 µM (circles) in POPE/POPC bilayers. These data demonstrate that β1 has little effect on the apparent Ca2+ sensitivity of the BK channel in POPE/POPC bilayers. D, Plots of log Po – V of BK α (open symbols) and BK αβ1 (closed symbols) in bilayers of POPE/POPC, POPE/POPG, POPE/POPS and POPE/POPA at [Ca2+] = 15.6 µM. Error bars represent SEM, n = 3–7.
Figure 6.
β1 dramatically affects the gating of BK in thin bilayers of DOPE/PC 14∶1 and thick bilayer of DOPE/SPM, but has little effect on gating of channel in bilayers of DOPE/PC 18∶1, DOPE/PC22∶1 and DOPE/PC24∶1.
A. Sample traces of single channel currents of BK α and BK αβ1 in each bilayer. B. Plots of log Po – V for BK α (open symbols) and BK αβ1 (closed symbols) in bilayers of DOPE/PC 14∶1, DOPE/PC 18∶1 and DOPE/SPM. Error bars represent SEM, n = 3–5. C & D. The single channel slope conductance and open probability (Po) for BK α and BK αβ1 in bilayers of POPE/POPS, DOPE/PC14∶1, DOPE/PC18∶1, DOPE/PC 22∶1, DOPE/PC24∶1, and DOPE/SPM. The numbers of experiments are indicated in each column. All data are means ± S.E. *(p<0.05); ** (p<0.01); *** (p<0.001) indicate BK hSlo αβ1 different from hSlo α.