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Figure 1. . Unitary currents were measured with a custom program using histogram analysis. (A) A representative single-channel current is shown as it appears on the screen of the program interface. The membrane potential was jumped from the holding potential of 0 to +150 mV for 42 ms and than jumped back to 0 mV. (B) The same recording as in A but with the data at the ends of the recording removed. The peaks of the current histograms correspond to the closed (C) and open (O1âO3 for one to three open channels) current levels. [K+i] was 3.4 M. Effective filtering was 33 kHz.
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Figure 2. . Current/voltage plots of unitary currents are sublinear at high voltages, and the Ca2+ and pHi buffers used in the solutions are not the cause of the sublinearity. (A) Representative single-channel currents recorded from BK channels expressed in oocytes with symmetrical 150 mM KCl. Effective filtering: 4 kHz. (B) Plot of the outward unitary currents versus voltage obtained with different solutions. Pipette solution (mM): filled circles, filled squares, open circles, open diamonds, and for the rest of the figures in the paper, 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, 60 μM GdCl3, pH 7.0; open squares, 140 KMeSO3, 20 HEPES, 2 KCl, 2 MgCl2, pH 7.2. Intracellular solution (mM): filled squares, 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, pH 7.0; filled circles, 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, 50 μM crown ether, pH 7.0; open circles, only 150 KCl; open diamonds, 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, 100 μM added Ba2+, pH 7.0; open squares, 140 KMeSO3, 20 HEPES, 2 KCl, 1 HEDTA, 0.84 μM free Ca2+, pH 7.2. Some of the symbols are shifted to the left or right by 5 mV so they can be seen. The slope of the straight line corresponds to the average chord conductance of 326 pS at +100 mV for the filled circles.
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Figure 3. . Intracellular protons decrease unitary current amplitudes of BK channels. (A) Representative single-channel currents from BK channels with symmetrical 150 mM KCl at the indicated pHi. Intracellular solution (mM): 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, and 50 μM crown ether. Membrane voltage: +250 mV. Effective filtering: 4 kHz. (B) Semilogarithmic plot of the dose response curves for unitary current amplitudes obtained at +100, +200, and +250 mV versus pHi. The lines represent simultaneous fits of all the data with Eq. 1, with ki = 8.7 μM (pKa = 5.1) and n = 0.48. Intracellular solution (mM): open squares, open circles, and filled circles, 150 KCl, 5 TES, 1 EGTA, 1 HEDTA, and 50 μM crown ether; filled diamonds, 150 KCl, 10 propionic acid, 10 MES, 10 TES, 10 TABS, 1 EGTA, 1 HEDTA, and 50 μM crown ether. (C) Single-channel currents from a BK channel at the indicated pHi. Intracellular solution (mM): 150 KCl, 10 propionic acid, 10 MES, 10 TES, 10 TABS, 1 EGTA, 1 HEDTA, and 50 μM crown ether. Membrane voltage: +150 mV. Effective filtering: 4 kHz. (D) Open-channel current traces at pHi 5.0 and pHi 9.0 with all points histograms from the same patch as in C. For the open-channel current trace at pHi 9.0, two brief closings were replaced by a horizontal line to exclude kinetics from the noise histogram. Effective filtering: 33 kHz.
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Figure 4. . Proton block of BK channels is concentration and voltage dependent. (A) Plots of unitary current amplitudes versus voltage at the indicated pHi. The slope of the straight line corresponds to the average chord conductance of 340 pS at +100 mV for the data at pHi 9.0. (B) Plots of the ratio of the unitary current amplitudes at the indicated pHi to the amplitudes at pHi 9.0 versus voltage, where X can be 5.0, 6.0, or 7.0, and is indicated for each curve. The dashed lines are polynomial fits. (C) Plots of unitary current amplitudes versus voltage at the indicated pHi with four pH buffers in the intracellular solutions. Symmetrical 150 mM [K+] for AâC. The dashed lines in A and C are polynomial fits to the data in A.
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Figure 5. . Comparison of the Woodhull model and competitive inhibition models for proton block of BK channels. (A) Predictions of the Woodhull model (Eq. 5, dashed lines) fitting data for each pHi separately. Different kd and d parameters where found for each pHi. The values of the kds were 20, 8.7, and 2.1 μM for pHis of 5.0, 6.0, and 7.0, respectively, and the values of d were 0.08, 0.12, and 0.13 for pHis of 5.0, 6.0, and 7.0, respectively. (B) Predictions of the Woodhull model (dashed lines) for simultaneously fitting with Eq. 5 for data at different pHi with single values for Kd and d of 20 μM and 0.15. (C) Predictions of the competitive inhibition model for simultaneously fitting the data at different pHi with Eq. 4 when d is the same for both K+ and H+: d = 0.2, kdK = 500 mM, kdH = 10 μM, n = 1 for the dashed lines, and n = 0.57 for the continuous lines. (D) Same as C except that d can be different for K+ and H+. Dashed lines: dK = 0, dH = 0.13, kdK = 500 mM, kdH = 10 μM, and n = 1. Continuous lines: dK = 0, dH = 0.2, kdK = 203 mM, kdH = 10 μM, and n = 0.46. The parameters were poorly defined in the fits for C and D, but to describe the data it was necessary for n to be â¼0.5 and for the electrical distance from the inside for dH to be â¼0.2 greater than for dK. The single-channel current at pHi 8.0 was used as the i0 current in the absence of protons.
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Figure 6. . Proton block of BK channels is relieved by K+i. (A) Plots of the unitary current amplitudes versus voltage for the indicated [K+] i and pHi. Dotted lines are predictions of the competitive inhibition model for simultaneously fitting the data at different pHi and [K+]i with Eq. 4 when dK = 0.2, dH = 0.21, kdK = 200 mM, kdH = 10 μM, and n = 1.6. The parameters were poorly defined, but to describe the data it was necessary for n to be â¥1.0 and <1.7 and for the electrical distance from the inside for dH to be equal to 1 to 1.1 times dK. The single-channel current at pHi 9.0 was used as the i0 current in the absence of protons. (B) Semilogarithmic plot of the ratio (in %) of the unitary current amplitudes at pHi 5.0 to the unitary current amplitudes at pHi 9.0 versus [K+]i. Dashed lines in A and B are polynomial fits.
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Figure 7. . Proton block of BK channels is consistent with competitive inhibition of H+i on K+i. (A) Double reciprocal plots of theoretical unitary current amplitudes versus [K+]i to illustrate competitive and noncompetitive block. (B) Double reciprocal plots of unitary current amplitudes versus [K+]i at pHi 5.0 (high concentration of the H+i blocker) and at pHi 9.0 (no blocker).
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Figure 8. . Proton block is still present after the removal of the ring of charge. (A) Plots of unitary current amplitudes versus voltage for WT channels at the indicated pHi (from Fig. 4). The dashed lines for pHi 5.0 and 7.0 are the same as the continuous lines in Fig. 5 D. The dashed line for pHi 9.0 is a polynomial fit. (B) Plots of unitary current amplitudes versus voltage for E321N/E324N mutant channels at the indicated pHi. The dashed lines for pHi 5.0 and 7.0 were drawn with Eq. 4 with dK = 0.0, dH = 0.15, kdK = 200 mM, kdH = 2.2 μM, and n = 0.23, using the data at pH 9.0 for i0. The dashed line for pHi 9.0 is a polynomial fit. (C) The data for WT channels from A, and the data for E321N/E324N channels from B, normalized by multiplying the data in (B) by 1.77. Some of the symbols at pHi 9.0 are shifted by 4 mV so they can be seen. (D) Semilogarithmic plot of the dose response curves for unitary current amplitudes for E321N/E324N mutant channels at +80, +150, and +200 mV versus pHi. The solid lines are fits of the data with Eq. 1 with ki = 12.6 μM (pKa = 4.90) and n = 0.29. Symmetrical 150 mM [K+]i for AâD.
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