Chen RS, Geng Y, Magleby KL.

AR32805 NIAMS NIH HHS , R01 AR032805 NIAMS NIH HHS

	Figure 1. Schematic diagram of a voltage-sensing domain (membrane segments S0-S4) and cytosolic RCK1 domain from a BK channel showing the proposed low-affinity Mg2+ binding site (Yang et al., 2007; Horrigan and Ma, 2008; Cui, 2010). For this proposed model, each of the four low-affinity Mg2+ sites on the BK channel is formed by E374/E399 on the upper surface of an intracellular RCK1 domain together with D99/N172 on the lower surface of a voltage sensor transmembrane domain from an adjacent subunit. (A) The activation of BK channels by Mg2+ involves electrostatic interactions of Mg2+ located at the low-affinity binding sites with S4 in the voltage sensor domain (Yang et al., 2007; Horrigan and Ma, 2008). Depicted is the interaction of Mg2+ with an elevated S4 due to depolarization, but the interactions are likely to be more complicated than in this simple diagram (Horrigan and Ma, 2008). (B) Hypothetical diagram depicting the possibility that an S4 voltage sensor in the down position due to extensive hyperpolarization might displace Mg2+ from the binding site.
	Figure 2. Determining a critical gap to separate bursts. Mean first burst duration is plotted as a function of the duration of the gap (closed interval) used to define the end of the burst. Results are with the same channel analyzed for Fig. 3, where the experimental details are presented. The critical gap was selected as 10 ms for both 0 and 10 mM Mg2+. This selection falls on the part of the curves where burst duration is less dependent on gap duration for both 0 and 10 mM Mg2+. Selecting the critical gap in this manner provides a functional definition of bursts, as shown in the diagram, and allows the use of the same critical gap for both 0 and 10 mM Mg2+, facilitating direct comparison of gating parameters because the same identical analysis was used for each.
	Figure 3. Mg2+ shortens the mean latency to the first opening and lengthens mean burst duration of BK channels. For this and all experiments in the paper, the two high-affinity Ca2+ binding sites have been disabled by mutation (see Materials and methods). (A and B) Representative traces from a single BK channel in a patch showing examples of sweeps after voltage steps from −100 mV to +100 mV for 400 ms. The channel in this patch was relatively active compared with a typical channel. The sweeps were selected (except for the second plotted sweep) to have at least one opening. The voltage protocol was 600 ms at the prepulse holding potential of −100 mV and then 400 ms at +100 mV, applied once per second. Latencies to opening are marked by double-ended arrows and first burst durations are marked by thick horizontal bars, which appear as dots for short bursts. 10 mM Mg2+ shortened mean latency and lengthened mean burst duration. Capacitive transients at the beginning and end of the sweeps have been removed. (C and E) Distributions of latency to first opening. For sweeps without openings, one count was added to the bin at 400 ms, where intervals were counted as ≥400 ms for the fitting. The distributions of latencies to first opening were described with sums of exponentials. For 0 Mg2+: τ1 = 56 ms (area = 0.27) and τ2 = 575 ms (area = 0.73). For 10 mM Mg2+: τ1 = 31 ms (area = 0.63) and τ2 = 228 ms (area = 0.37). The distribution for 0 Mg2+ (broken line) is overlaid on the distribution for 10 mM Mg2+. 10 mM Mg2+ shifted the distribution toward shorter latency. (D and F) Distributions of burst duration are plotted. For 0 Mg2+: τ1 = 5.9 ms (area = 1.0). For 10 mM Mg2+: τ1 = 0.04 ms (area = 0.27) and τ2 = 53 ms (area = 0.73). 10 mM Mg2+ shifted the distribution for burst duration to a longer duration. Distributions are scaled to the same number of events to facilitate visual comparisons.
	Figure 4. 10 mM Mg2+ alters the single-channel gating kinetics after a step to +100 mV with little effect of prepulse holding potentials. The mean values with and without 10 mM Mg2+ are plotted against prepulse holding potential for the measured parameters. The error bars plot the SEM.. The voltage protocol was the same as in Fig. 3 (A and B), but with data collected at additional prepulse holding potentials. Of the regression fits to the 16 sets of data over the voltage range (not depicted), only the regression line for 10 mM Mg2+ data in E had a slope significantly different from 0 (not depicted), which suggests that the prepulse holding potential has little consistent effect on the action of Mg2+ in these experiments. In contrast, based on the response over the examined range of holding potentials with and without Mg2+, 10 mM Mg2+ significantly: increased the fraction of sweeps with at least one opening(A); decreased the mean latency to first opening (B); increased the mean number of openings in the first burst (C); increased first mean burst duration (D); increased mean open interval duration (E); decreased mean closed interval duration (F); decreased mean gap duration (interburst duration) between bursts (G); and increased Po (H). The number of single-channel patches (separate experiments) studied for each plotted point ranged from 6 to 8. Data were typically obtained at both 0 and 10 mM Mg2+ from the same patch, and data from all four holding potentials was obtained from about half of the patches. The points plotted in parts B, F, G, and H were calculated assuming that open and closed intervals at the end of the 400 ms sweep terminated at the end of the sweep.
	Figure 5. Rapid equilibrium of Mg2+ action on channel gating. The first six successive mean open intervals and the first seven successive mean closed interval durations are plotted separately against successive open and closed interval numbers after a voltage step from negative holding potentials to +100 mV for 0 and 10 mM Mg2+. Data from the different prepulse holding potentials (−150, −100, and −50) were combined because there were no significant differences for the different holding potentials. The plotted points and error bars are the mean ± SEM for data at the four different prepulse holding potentials. The experimental protocol was the same as in Fig. 3, but with additional holding potentials. (A and B) Mg2+ increased the duration of the first six open intervals with no significant difference for successive interval number. (C and D) Mg2+ decreased the durations of mean closed intervals with no significant differences for successive intervals after the first. The slight trend toward briefer successive closed intervals after the first closed interval was not significant for any combination of paired intervals, but may reflect a greater likelihood for inclusion of a closed interval artificially truncated by the end of the 400-ms pulse as interval number increased. The observations in A–D suggest that Mg2+ action reaches a rapid equilibrium with the first opening. The number of single-channel patches contributing to each plotted data point ranged from 19 to 28.
	Figure 6. Using macropatches to explore the mechanism of activation of BK channels by Mg2+. (A) Representative current traces from macropatches containing hundreds of channels held at a constant −50 mV for 0 Mg2+ (left) and 10 mM Mg2+ (right). Because the Po of each individual channel is so low, typically only one channel is open at any time. The indicated part of the upper recordings is expanded tenfold three successive times to show the data on faster time bases. Most openings in 0 Mg2+ are unit bursts consisting of single openings. Mg2+ increased open interval duration. Mg2+ also increased the frequency of openings by increasing the number of openings per burst and decreasing the duration of closed intervals (gaps) between bursts. (B) When the voltage sensors were held deactivated by the R167E mutation, 10 mM Mg2+ no longer enhanced channel activation. (C) When the voltage sensors were held constitutively activated by the R210C mutation, Mg2+ still activated the channels at −200 mV.
	Figure 7. Voltage sensor deactivation prevents increases in Po and modulation of open and closed interval durations by Mg2+. Plotted data points are mean ± SEM obtained from macropatch experiments like those shown in Fig. 6. (A1–A3 and B1–B3) Plots of the indicated parameters versus the steady membrane potential used to collect the data. The data in B1–B3 replot the same data used for A1–A3, but normalized for each macropatch before averaging by dividing the values in 10 mM Mg2+ by the values in 0 Mg2+. The error bars for the normalized data were calculated using the SEM formula, but error would not be normally distributed because of the division. Mg2+ had little effect on Po and mean open and closed interval durations at −150 mV, when voltage sensors would be deactivated (down). Progressively activating voltage sensors by depolarizing to −100 mV and then to −50 mV progressively increased Po and mean open interval duration, and decreased mean closed interval duration. (C1–C3) Holding the voltage sensors down with the mutation R167E prevents Mg2+ action on Po and mean interval durations. (D1–D3) Holding voltage sensors activated (up) with the mutation R210C allows Mg2+ to increase Po through increases in mean open interval durations and decreases in mean closed interval durations at −200 mV. Data are from 6–14 macropatches for plots in A and B, from 6 macropatches for C, and from 4 macropatches for D. Data points that overlap have been shifted slightly laterally so that both points can be seen.

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