Csanády L , Töröcsik B .

R01 DK051767 NIDDK NIH HHS , R01-DK051767 NIDDK NIH HHS , Howard Hughes Medical Institute

	Figure 1. Voltage and dose dependence of pore block by NPPB and MOPS−. (A and B) Unitary WT CFTR currents in symmetrical ∼140-mM Cl− with and without cytosolically applied blockers, displayed at two bandwidths (fc, filter corner frequency). We could not reliably discern two distinct conductance levels in MOPS− (Gunderson and Kopito, 1995). (C) Apparent unitary amplitudes at 10 Hz, with and without blockers, plotted against voltage. (D) Fractional unitary current in 210 µM NPPB and 20 mM MOPS− (symbols) and Boltzmann fits (solid lines); midpoint voltages (V1/2) and apparent valences (z) were V1/2 = −85 ± 3 mV and z = −0.45 ± 0.03 for MOPS− and V1/2 = 24 ± 2 mV and z = −0.51 ± 0.03 for NPPB. (E, G, I, and K) Slowly decaying macroscopic ”locked-open” currents of E1371S CFTR in the absence of bath ATP; brief applications of 210 µM NPPB or 20 mM MOPS− at various voltages (E and G) or of various blocker concentrations at −120 mV (I and K). Dotted lines show zero-current levels estimated from final segments; in E and G, small (<1 pA/40 mV) linear seal currents were subtracted. (F and H) Voltage dependence of macroscopic current block by (F) 210 µM NPPB and (H) 20 mM MOPS− (closed symbols) and Boltzmann fits (solid lines); V1/2 = −87 ± 3 mV and z = −0.54 ± 0.07 for MOPS− and V1/2 = 7 ± 3 mV and z = −0.48 ± 0.03 for NPPB; open symbols and dotted lines replotted from panel D. (J and L) Dose dependence of macroscopic current block by (J) NPPB and (L) MOPS− at −120 mV (closed symbols) and Michaelis–Menten fits (solid lines). Open symbol in L: block by 40 mM of total MOPS (MOPS-H plus MOPS−) at pH 6.2; calculated [MOPS−] = 3.6 mM: fractional block by MOPS depends on [MOPS−], not total [MOPS], confirming anionic MOPS− to be responsible for pore block (Ishihara and Welsh, 1997).
	Figure 2. Discrepancy between macroscopic and unitary effects on WT CFTR quantifies gating stimulation. (A and C) Dose-dependent inhibition by cytosolic NPPB (A) and MOPS− (C) of steady-state macroscopic WT CFTR currents in 2 mM ATP at −120 mV. (B and D) Dose dependence of fractional currents in (B) NPPB and (D) MOPS− at −120 mV (closed symbols), and Hill fits (solid lines); open symbols and dotted lines show dose dependence of pore block, replotted from Fig. 1 (J and L). (E) Gating stimulation by NPPB and MOPS− at −120 mV; Po/Po; control was calculated as the ratio (I/Icontrol):(i/icontrol).
	Figure 3. Effects of NPPB and MOPS− on WT CFTR opening and closing rate. (A and B) Macroscopic WT CFTR currents at −120 mV elicited by brief exposures to 2 mM ATP in the absence or presence of (A) 210 µM NPPB or (B) 20 mM MOPS−; colored lines, single-exponential fits (τ, time constants). (C) Macroscopic closing rates (bars, rOC = 1/τ) in the absence (red) and presence of NPPB (blue) or MOPS− (green). (D and E) Macroscopic WT CFTR currents in 2 mM ATP at −120 mV, and brief exposure to (D) 210 µM NPPB or (E) 20 mM MOPS−. Colored lines in D, single-exponential fits. (F) Sums of opening and closing rates in the absence and presence of NPPB (1/τ from D); opening rates (rCO, bottom vertical arrows) are estimated by subtraction of closing rates (rOC, top vertical arrows; see C) from the sums. Cartoons in C and F (also in Figs. 4, B and E, and 5, B and E) depict simplified cyclic CFTR gating model: cyan, TMDs; green, NBD1; blue, NBD2; yellow, ATP; red, ADP. Purple arrows highlight the pathway under study.
	Figure 4. Effects of NPPB and MOPS− on gating rates under nonhydrolytic conditions. (A) Macroscopic K1250A CFTR current at −120 mV elicited by exposures to 10 mM ATP in the absence or presence of blockers. Colored lines, single-exponential fits (τ, time constants). (B) Macroscopic closing rates (bars, 1/τ) in the absence (red) and presence of NPPB (blue) or MOPS− (green) quantify effects on rate k-1 (cartoon, purple arrow). The K1250A mutation (B and E, cartoons, red stars) disrupts ATP hydrolysis in site 2 (red cross). (C) Macroscopic K1250A CFTR current elicited by 10 mM ATP at −40 mV, prolonged exposure to 210 µM NPPB of channels gating at steady state, and brief exposure to NPPB of surviving locked-open channels after ATP removal (10-s yellow box, expanded in inset). Bracketing brief applications of NPPB to small residual CFTR currents were used to estimate zero-current level (dotted line). (D) Fractional K1250A CFTR currents at −40 mV in 210 µM NPPB applied during steady-state gating (red bar) or in the locked-open state (yellow bar). (E) Effect of NPPB on the closed–open equilibrium (cartoon, purple double arrow). Fractional effect on Po for K1250A CFTR (blue bar) was calculated as in Fig. 2 E.
	Figure 5. Effects of NPPB and MOPS− on WT CFTR microscopic steady-state gating parameters. (A) Currents from single WT CFTR channels at −120 mV in 2 mM ATP ± blockers; bandwidth, 100 Hz. (B) Mean burst (τb) and interburst (τib) durations (bars) in 2 mM ATP (red), 2 mM ATP plus 20 µM NPPB (blue), and 2 mM ATP plus 20 mM MOPS− (green); closing rate (cartoon, purple arrow) is 1/τb. Bars were generated from data originating from 15, 10, and 9 patches, respectively, for the control, NPPB, and MOPS conditions; error bars represent SEM. (C and D) Dwell-time histograms of burst durations of WT CFTR channels at −120 mV in 2 mM ATP (C; 2,999 bursts, pooled from 15 patches) or 2 mM ATP plus 20 µM NPPB (D; 2,069 bursts, pooled from 10 patches) and maximum likelihood fits (solid lines) to the scheme cartooned in E (with k-1 fixed to zero). (E) Estimates of rates k1 (step O1→O2) and k2 (step O2→C2) in ATP and ATP plus 20 µM NPPB. Asymmetric error bars represent 0.5-unit log-likelihood intervals.
	Figure 6. Gating stimulation by NPPB is largely voltage independent. (A) Dose-dependent stimulation by NPPB of steady-state macroscopic WT CFTR currents in 2 mM ATP at +60 mV. (B) Dose–response curves for fractional effects of NPPB on steady-state macroscopic current I (open squares; obtained from A), on unitary current i (open diamonds; obtained as in Fig. 1I), and on Po (closed circles; obtained as in Fig. 2 E). (C; left) Macroscopic WT CFTR current at +60 mV elicited by brief exposures to 2 mM ATP in the absence or presence of 210 µM NPPB; colored lines, single-exponential fits (τ, time constants). (Right) Macroscopic WT closing rates (bars, 1/τ) in the absence (red) and presence of NPPB (blue). (D; left) Macroscopic WT CFTR current in 2 mM ATP at +60 mV and brief exposure to 210 µM NPPB; colored lines, single-exponential fits (τ, time constants). (Right) Sums of opening and closing rates (bars, 1/τ) in the absence and presence of NPPB; opening rates (rCO, bottom vertical arrows) are estimated by subtraction of closing rates (rOC, top vertical arrows) from the sums. (E; left) Macroscopic K1250A CFTR current at +60 mV elicited by exposures to 10 mM ATP in the absence or presence of 210 µM NPPB; colored lines, single-exponential fits (τ, time constants). (Right) Macroscopic K1250A closing rates (bars, 1/τ) in the absence (red) and presence (blue) of NPPB. (F; top) Currents from a single WT CFTR channel gating at +60 mV in 2 mM ATP before (top trace) and during (bottom trace) exposure to 210 µM NPPB; bandwidth, 100 Hz. (Bottom) Mean burst (left) and interburst (right) durations (bars) of WT CFTR in 2 mM ATP (red) and 2 mM ATP plus 210 µM NPPB (blue) at +60 mV. Bars were generated from 16 segments of record in ATP and 15 segments in ATP plus NPPB originating from nine patches; error bars represent SEM.
	Figure 7. Stimulation of ΔF508 CFTR gating by NPPB. (A and B) Quasi-macroscopic currents of low temperature–rescued, prephosphorylated ΔF508 CFTR channels in 2 mM ATP at −120 mV (A) and +60 mV (B), and brief exposures to 210 µM NPPB. Note overall current stimulation by NPPB, and extreme current overshoot upon its removal, at −120 mV. (C and D) Fractional effects of 210 µM NPPB on (C) steady-state macroscopic currents and (D) Po of ΔF508 CFTR at −120 mV (yellow bars) and +60 mV (cyan bars).
	Figure 8. Phosphorylation dependence of NPPB stimulation of WT CFTR reflects nonlinear dependence of Po on opening and closing rates. (A) Macroscopic WT CFTR currents in 2 mM ATP at +60 mV, and applications of 210 µM NPPB before exposure to (current scale magnified in inset), in the presence of, and after removal of 300 nM PKA catalytic subunit. (B and C) Fractional effects of 210 µM NPPB on (B) steady-state macroscopic currents and (C) Po of unphosphorylated (pre-PKA), fully phosphorylated (in PKA), and partially dephosphorylated (post-PKA) WT CFTR channels at +60 mV. (D) Table summarizing expected changes in gating parameters assuming a constant (phosphorylation-independent) fourfold increase in τb and fourfold decrease in τib in the presence of 210 µM NPPB, as measured in this study for the post-PKA condition at −120 mV (Fig. 3; slightly smaller, approximately threefold, effects were measured at +60 mV; Fig. 6). Control parameters (left column) reflect typical values measured for WT CFTR in this and previous studies (e.g., Csanády et al., 2005; Szollosi et al., 2010); the post-PKA values in NPPB (right column, bottom row) reflect the values measured in Fig. 3.
	Figure 9. Anionic form of NPPB is responsible for the observed gating effects. (A) Titration curve of NPPB; 10 ml 100 µM NPPB (in H2O) was titrated with 10-µl aliquots of either 10 mM NaOH (closed symbols; n = 3) or 10 mM HCl (open symbols; n = 2, plotted on negative side of abscissa). Solid line is a fit to the titration curve of a monoprotic weak acid, corrected for the presence of 10 mM CO2 (see Materials and methods); fitted pKa is plotted. (B) Structures of protonated (zwitterionic) and deprotonated (anionic) forms of NPPB; the proton released with a pKa of ∼5.6 is highlighted in red; dotted line depicts suggested hydrogen bond. (C) Segments of unitary current at +60 mV from a single locked-open E1371S CFTR channel in symmetrical ∼140-mM Cl− at three different cytosolic pH values, with and without cytosolically applied 210 µM NPPB, displayed at two bandwidths (fc, filter corner frequency). Horizontal lines depict closed current level and average current through a bursting channel. (D) Apparent unitary amplitudes (of heavily filtered currents), normalized to those obtained in the absence of NPPB at the respective pH (bars). Table lists concentrations of anionic and uncharged NPPB for the tested conditions, calculated using a pKa of 5.6. (E and F) Stimulation at +60 mV of steady-state macroscopic WT CFTR currents in 2 mM ATP (red bars) by the application of (E and F) 210 µM NPPB (blue bars) at bath pH values of 6.1, 7.1, and 8.1 (gray bars), or of (F) 21 µM NPPB at a bath pH value of 6.1. (G) pH dependence of fractional effects of NPPB on steady-state macroscopic current I (blue bars; obtained from E and F), and on Po (dark blue bars; Po/Po; control was calculated as the ratio (I/Icontrol):(i/icontrol)). (Table) Concentrations of anionic and uncharged NPPB for the tested conditions, assuming pKa = 5.6.
	Figure 10. Dose dependence of NPPB gating effects. Macroscopic closing rates of WT (closed circles) and K1250A (closed squares) CFTR in the presence of various cytosolic [NPPB], measured at −120 mV using the protocols shown in Figs. 3 A and 4 A, respectively; leftmost data points represent control values in the absence of NPPB. The 50- and 100-µM data points for K1250A were measured at −40 mV. Solid lines are fits to the equation rOC([NPPB]) = r0 + (r∞ − r0)([NPPB]/([NPPB] + K1/2)), with r∞ fixed to zero for WT but left free for K1250A; K1/2 values are plotted. Open diamonds and dotted line depict fractional pore block at −120 mV, replotted from Fig. 1 J.
	Figure 11. Catalyst effect as a unique strategy to enhance Po of a nonequilibrium ion channel. (A) Simple equilibrium scheme (left) and cyclic nonequilibrium scheme (right; compare to cartoons in Figs. 3–5), with transition rates in the absence (red) and presence (blue) of a catalyst for step C↔O (left) or C1↔O1 (right). (B) Free energy profiles (not drawn to scale) for the schemes in A, in the absence (red) and presence (blue) of the same catalyst. (C) Illustration of channel gating patterns, and calculated Po, for the schemes in A, in the absence (red) and presence (blue) of the catalyst.
	Figure 12. Full kinetic model of NPPB effects on CFTR gating and permeation. (A; left) Two-tiered kinetic model of NPPB gating effects (cube). States C1, O1, O2, and C2 (red) correspond to the four states depicted in the cartoons in Figs. 3–5 and represent states in which NPPB is not bound at its “gating site.” The C2↔C1 transition, which reflects exchange of ADP for ATP at site 2, is modeled as a single step, with a Kd of 50 µM for ATP. For simplicity, a single voltage-independent Kd of 80 µM is used to characterize rapid binding/unbinding of NPPB to the gating site in all four conformational states (vertical transitions; gray arrows). States C1*, O1*, O2*, and C2* (blue) are conformational states analogous to C1, O1, O2, and C2, but with NPPB bound at the gating site. In 0 or 210 µM NPPB, the full model (cube) reduces to the four-state models to its left (red) and right (blue), respectively; states X^ in the blue reduced model are compound states (C^1 = {C1; C1*}, O^1 = {O1; O1*}, etc.), and printed rates are apparent rates of transition between them. (Right) Voltage and dose dependence of pore block by NPPB is modeled as an instantaneous effect on apparent unitary conductance (g) and is approximated by the Boltzmann equation with parameters printed (g0, control unitary conductance; T, temperature in Kelvin; R = 8.31 J · mol−1 · K−1; F = 96,500 C · mol−1). (B–L) Predictions of the model in A for the experimental protocols and analysis results obtained in this study. Macroscopic current time courses for the experimental protocols in B–E and I–L were calculated, whereas single-channel traces for F–H were simulated using standard Q-matrix techniques (Colquhoun and Sigworth, 1995). Rate O1*→O2* was tentatively set to zero. Gating effects of the K1250A mutation were modeled by setting rate O1→O2 to zero while increasing the Kd for ATP to 5 mM (Vergani et al., 2003), those of the ΔF508 mutation were modeled by decreasing rate C1→O1 30-fold while increasing rate O1→C1 threefold (Miki et al., 2010; Jih et al., 2011). Analysis of macroscopic currents was done as described in Figs. 2–4, 6, 7, and 10. For F and H, five independent events lists, containing 100 open events each, were simulated for each condition; bar charts show mean ± SEM of obtained mean open and closed times. Open duration histograms in G were created from 3,000 simulated open events for both conditions and fitted by maximum likelihood as described for Fig. 5 (C–E).

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