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Figure 1. Structures of NPPB, 3NB, and 3PP. NPPB structure and its division into head (3NB, blue) and tail (3PP, red) parts.
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Figure 2. MOPSâ competes with NPPB for binding in the pore but not at the gating site. (A) Macroscopic locked-open E1371S CFTR current at â120 mV after removal of ATP from the bath; exposures to various NPPB concentrations (brown bars) in the continued presence of 80 mM MOPSâ (green bar; magnified in yellow inset). Dotted line marks zero-current level. (B) Doseâresponse curves for NPPB block at â120 mV in the absence (open symbols; replotted from Csanády and Töröcsik [2014]) and presence (green-filled symbols) of 80 mM MOPSâ. Leftmost symbols in these and all other doseâresponse plots represent zero drug concentration. Fits to the Michaelis-Menten equation (dotted and solid line) reveal an approximate ninefold increase in the apparent KI of NPPB in the presence of 80 mM MOPSâ (KI = 8.3 mM for MOPSâ; compare with Csanády and Töröcsik [2014]). (C) Macroscopic WT CFTR currents at â120 mV elicited by brief exposures to 2 mM ATP in the absence or presence of blockers. Current relaxations after ATP removal were fitted by single exponentials (colored lines), and colored numbers are time constants (in milliseconds). (D) Macroscopic closing rates of WT CFTR in the absence of blocker (gray) and in the presence of 100 µM NPPB (brown), 80 mM MOPSâ (green), or 100 µM NPPB + 80 mM MOPSâ (striped). (E) Macroscopic K1250A CFTR currents at â40 mV elicited by brief exposures to 10 mM ATP in the absence or presence of blockers. Current relaxations after ATP removal were fitted by single exponentials with time constants indicated. (F) Macroscopic K1250A closing rates in the absence of blocker (gray) and in the presence of 100 µM NPPB (brown), 80 mM MOPSâ (green), or 100 µM NPPB + 80 mM MOPSâ (striped). Mean ± SEM is shown.
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Figure 3. Effects of 3NB, 3PP, and sulfate on CFTR permeation. (A, B, F, G, I, and J) Decaying macroscopic currents of locked-open K1250A CFTR channels after removal of ATP, recorded at membrane potentials of â80 (A, F, and I) or 60 mV (B, G, and J) and responses to brief applications of various concentrations of 3NB (A and B, blue bars), 3PP-sulfate (F and G, red bars), or sulfate (I and J, green bars). Zero-current levels (dotted lines) were estimated from the responses of final current segments to application of 32 mM 3NB (blue bars in all panels). (C and H) Doseâresponse curves at â80 (cyan-filled symbols) and 60 mV (yellow-filled symbols) of fractional currents as a function of [3NB] (C), [3PP-sulfate] (H, red), and sulfate (H, green). The two alternative abscissae in H were scaled to reflect the presence of 0.45 mol/mol sulfate ions in 3PP-sulfate; i.e., the green abscissa correctly reflects sulfate concentrations for all four plots. The plots in C were fitted by the Michaelis-Menten and the yellowâred plot in H to the Hill equation, and midpoints are printed in the panels. (D) Responses of decaying macroscopic locked-open K1250A CFTR current to brief applications of 32 mM 3NB (blue bars) at various membrane potentials. Zero-current level (dotted line) was estimated from the final current segment after subtraction of the small (â¼2 pA/60 mV) linear seal current. (E) Voltage dependence of macroscopic current block by 32 mM 3NB (symbols) and Boltzmann fit (solid line) yielding parameters z = 0.49 ± 0.02 mV and V1/2 = 41 ± 2 mV (calculated Kd(0) = 14.7 mM). Mean ± SEM is shown.
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Figure 4. Competition for CFTR pore block between NPPB, 3NB, and 3PP. (AâF) Responses of decaying macroscopic locked-open K1250A CFTR currents, recorded at membrane potentials of â80 (A, C, and E) or 60 mV (B, D, and F), to brief exposures to the following drug combinations: (A and B) 32 mM 3NB (blue bars) and/or 210 µM NPPB (brown bars), (C and D) 20 mM 3PP (red bars) and/or 210 µM NPPB (brown bars), and (E and F) 32 mM 3NB (blue bars) and/or 20 mM 3PP (red bars). Zero-current levels (dotted lines) were estimated from the responses of final current segments to application of either 32 mM 3NB or 210 µM NPPB. (G and H) Fractional currents, normalized to control (gray bars), recorded at â80 mV (G) or 60 mV (H) membrane potentials in the presence of 210 µM NPPB (brown bars), 32 mM 3NB (blue bars), 20 mM 3PP (red bars), or their combinations (striped bars, same color coding). Horizontal lines illustrate fractional currents predicted for mixtures (see Materials and methods), assuming either completely independent (yellow lines) or mutually exclusive (green lines) binding of the two drugs in the pore. Mean ± SEM is shown.
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Figure 5. Voltage-independent stimulation of CFTR open probability by both 3NB and 3PP. (A and E) Macroscopic WT CFTR currents in 2 mM ATP (gray bars) at â80 (left traces) and 60 mV (right traces) and brief exposures to 32 mM 3NB (A, blue bars) or 20 mM 3PP (E, red bars). (B) Responses of macroscopic WT CFTR currents, elicited at â80 mV by repeated exposures to 2 mM ATP (gray bars), to applications of increasing concentrations of 3NB (blue bars). (F) Macroscopic WT CFTR currents elicited at â80 mV by exposures to 2 mM ATP (gray bars) either in the absence or in the presence (red bars) of various concentrations of 3PP. (C and G) Dose dependence of fractional currents on 3NB (C) and 3PP (G) concentrations at â80 mV (closed symbols) and Hill fits (solid lines; midpoints printed in the panels); open symbols and dotted lines show dose dependence of pore block, replotted from Fig. 3 (C and H). (D and H) Gating stimulation by 3NB (D) and 3PP (H) at â80 mV; Po/Po;control was calculated as the ratio (I/Icontrol)/(i/icontrol). Control Po (dotted lines), estimated for single WT channels under identical conditions (see Fig. 7 C), is â¼0.2. Mean ± SEM is shown.
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Figure 6. Effects of 3NB and 3PP on macroscopic closing rate of WT CFTR. (A and B) Macroscopic WT CFTR currents at â80 mV, elicited by brief applications of 2 mM ATP (gray bars) in the absence of drug or in the presence of either 32 mM 3NB (A, blue bar) or 20 mM 3PP (B, red bar). All four current decay time courses after ATP removal were fitted by single exponentials (colored lines), and time constants are indicated. (C) Macroscopic closing rates, obtained as the inverses of fitted time constants (see Materials and methods), under control conditions (gray bar) or in the presence of either 32 mM 3NB (blue bar) or 20 mM 3PP (red bar). Mean ± SEM is shown. Cartoon in C (also in Fig. 7 B; Fig. 8, C and G; and Fig. 10) depicts simplified cyclic CFTR gating model: cyan, TMDs; green, NBD1; blue, NBD2; yellow, ATP; red, ADP. Purple arrow highlights the pathway under study.
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Figure 7. Effects of NPPB, 3NB, and 3PP on WT CFTR microscopic steady-state gating parameters. (A) Currents from single WT CFTR channels at â80 mV in 2 mM ATP (top), 2 mM ATP + 32 mM 3NB (middle), and 2 mM ATP + 20 mM 3PP (bottom); bandwidth, 50 Hz. (B) Cartoon gating model; opening rate (top purple arrow) and closing rate (bottom purple arrow) are given by 1/Ïib and 1/Ïb, respectively. (CâE) Open probabilities (C) and mean burst (Ïb; D) and mean interburst (Ïib; E) durations in 2 mM ATP (gray), 2 mM ATP + 210 µM NPPB (brown), 2 mM ATP + 32 mM 3NB (blue), and 2 mM ATP + 20 mM 3PP (red). The data for NPPB (brown bars) are replotted from Csanády and Töröcsik (2014) and reflect values measured at 60 mV. Mean ± SEM is shown.
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Figure 8. Effects of 3NB and 3PP on gating rates under nonhydrolytic conditions. (A and B) Macroscopic K1250A CFTR currents at â20 mV, elicited by exposures to 10 mM ATP (gray bars) in the absence of drug or in the presence of either 32 mM 3NB (A, blue bar) or 20 mM 3PP (B, red bar). All current decay time courses after ATP removal were fitted by single exponentials (colored lines), and time constants are indicated. (C) Macroscopic closing rates (bars; 1/Ï; see Materials and methods) in the absence (gray bar) or presence of 32 mM 3NB (blue bar) or 20 mM 3PP (red bar) quantify effects on rate kâ1 (cartoon, purple arrow). The K1250A mutation (cartoon, red stars) disrupts ATP hydrolysis in site 2 (red cross). (D and E) Macroscopic K1250A CFTR currents elicited by 10 mM ATP at â20 mV and prolonged exposures to 32 mM 3NB (D, blue bars) or 20 mM 3PP (E, red bars) of channels gating at steady-state. Zero-current levels (dotted lines) were estimated from final segments. In D, brief exposure to 3NB of surviving locked-open channels after ATP removal (20-s yellow box, expanded in inset) measures fractional pore block (see Fig. 3). (F) Fractional K1250A CFTR currents at â20 mV in 32 mM 3NB (left pair of bars) or 20 mM 3PP (right pair of bars) applied during steady-state gating (gray bars) or in the locked-open state (yellow bars). (G) Effects of 3NB (blue bar) and 3PP (red bar) on the closed-open equilibrium (cartoon, purple double arrow). Fractional effects on Po for K1250A CFTR were calculated as in Fig. 5 (D and H). Mean ± SEM is shown.
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Figure 9. Competition between NPPB, 3NB, and 3PP for affecting hydrolytic closing rate. (AâC) Macroscopic WT CFTR currents at â80 mV, elicited by brief applications of 2 mM ATP (gray bars) in the absence of drug, or in the presence of either 210 µM NPPB (A, brown bar) or the following drug combinations: (A) 20 mM 3PP (red bar) plus 210 µM NPPB (brown bar), (B) 32 mM 3NB (blue bar) plus 210 µM NPPB (brown bar), and (C) 32 mM 3NB (blue bar) plus 20 mM 3PP (red bar). Current decay time courses after ATP removal were fitted by single exponentials (colored lines), and time constants are indicated. (D) Macroscopic closing rates, obtained as the inverses of fitted time constants (see Materials and methods), under control conditions (gray bar) or in the presence of 210 µM NPPB (brown bar), 32 mM 3NB (blue bar), or 20 mM 3PP (red bar), or their combinations (striped bars, same color coding). Horizontal lines illustrate the predicted closing rates in mixtures (see Materials and methods), assuming either completely independent (yellow lines) or mutually exclusive (green lines) binding of the coapplied drugs at their respective gating sites. Blue and red bars were replotted from Fig. 6 C. Mean ± SEM is shown.
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Figure 10. Cartoon summary of 3NB and 3PP gating effects. 3NB acts as a pure catalyst for the C1âO1 transition (blue arrow), thereby increasing channel opening rate and nonhydrolytic closing rate. 3PP acts to inhibit the ATP hydrolysis step (red line). Both effects independently stimulate Po for WT CFTR, but neither effect changes Po for a nonhydrolytic mutant. NPPB exerts both effects at the same time, resulting in extremely efficacious stimulation of Po for WT (and ÎF508) CFTR (Csanády and Töröcsik, 2014).
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