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J Gen Physiol
2006 Nov 01;1285:523-33. doi: 10.1085/jgp.200609558.
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Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle.
Csanády L
,
Nairn AC
,
Gadsby DC
.
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CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35 degrees C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(DeltaH(double dagger)) were determined from Eyring plots. DeltaH(double dagger) was 117 +/- 6 and 69 +/- 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 +/- 6 and 73 +/- 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. DeltaH(double dagger) for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 +/- 2, 39 +/- 4, and 37 +/- 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (DeltaS(double dagger)), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. DeltaS(double dagger) appears large for opening but small for normal closure. The large DeltaH(double dagger) and DeltaS(double dagger) (TDeltaS(double dagger) >/= 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small DeltaS(double dagger) for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.
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17043148
???displayArticle.pmcLink???PMC2151586 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. Temperature dependence of gating of partially phosphorylated WT CFTR. (A) Baseline-subtracted current trace of approximately three CFTR channels at â80 mV in 2 mM MgATP (bar, ATP) at 25°C (blue segments) and 31°C (red segment) after activation by 300 nM PKA (bar, PKA). Insets show expanded traces. (B) Temperature near the patch during current recordings in A; boxes identify the analyzed segments of experiment, i.e., segments with temperature 25 ± 1°C (blue box) or 31 ± 1°C (red box). (C) Eyring plot of opening (red symbols and line) and closing (black symbols and line) rates of partially phosphorylated WT CFTR in 2 mM MgATP at 15°C, 31°C, and 35°C, normalized (\documentclass[10pt]{article}
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\begin{equation*}{\hat {k}}\end{equation*}\end{document}) to their average values in bracketing control segments at 25°C; straight lines were fitted to the plots to obtain ÎHâ¡ values shown.
Figure 2. Temperature dependence of gating of highly phosphorylated WT CFTR. Eyring plot of normalized opening (red symbols) and closing (black symbols) rates (\documentclass[10pt]{article}
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\begin{equation*}{\hat {k}}\end{equation*}\end{document} ) of highly phosphorylated WT CFTR channels in 2 mM MgATP plus 300 nM PKA; linear fits yielded ÎHâ¡ values shown.
Figure 3. Temperature dependence of gating of partially phosphorylated K1250R CFTR. (A) Macroscopic current trace (top) from â¼2,000 K1250R CFTR channels at â20 mV, with simultaneously recorded temperature (bottom). Prephosphorylated channels were repeatedly opened by brief exposures to 2 mM MgATP, while bath temperature was toggled between 25°C (red box) and 15°C (blue box). Single exponentials (colored smooth lines) fitted to all current decay time courses at 25°C (red curves) and 15°C (blue curves) yielded time constants (Ï) shown. PKA was reapplied after the first bracketed experiment to recover channel activity lost due to slow dephosphorylation; note minimal influence on decay time constants of the presence of PKA during channel activation (magenta fit lines). (B) Eyring plot of normalized closing rates (\documentclass[10pt]{article}
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\begin{equation*}{\hat {k}}\end{equation*}\end{document} ) of K1250R CFTR channels upon ATP removal, fitted by a straight line to obtain ÎHâ¡ value shown; closing rates, obtained as 1/Ï from single-exponential fits, as in A, were normalized to their average values in bracketing control segments at 25°C.
Figure 4. Temperature dependence of gating of partially phosphorylated K1250A CFTR. (A) Macroscopic current recording (top) from K1250A CFTR channels with simultaneously recorded temperature (bottom). Prephosphorylated channels were repeatedly opened by brief exposures to 10 mM MgATP, while bath temperature was toggled between 25°C (red box) and 15°C (blue box). Single exponentials (colored smooth lines) fitted to all current decay time courses at 25°C (red curves) and 15°C (blue curve) yielded time constants (Ï) shown. (B) Eyring plot of normalized closing rates (\documentclass[10pt]{article}
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\begin{equation*}{\hat {k}}\end{equation*}\end{document} ) of K1250A CFTR channels upon ATP removal, fitted by a straight line to obtain ÎHâ¡; closing rates, obtained as 1/Ï from single-exponential fits, as in A, were normalized to their average values in bracketing control segments at 25°C.
Figure 5. Temperature dependence of unlocking rate of WT CFTR channels locked open by ATP+AMPPNP. (A) Macroscopic current trace (top) from â¼800 prephosphorylated WT CFTR channels, with simultaneously recorded temperature (bottom). Channels were repeatedly opened by brief exposures to 2 mM MgATP alone (bar, ATP) or with PKA (to reactivate dephosphorylated channels; bars, ATP+PKA), or to 0.1 mM MgATP+1 mM MgAMPPNP (bars, T+M), while bath temperature was toggled between 25°C (red box) and 16°C (blue box). Two consecutive decay time courses after removal of ATP+AMPPNP, in both test and bracketing segments, were summed to produce quasi-macroscopic time courses (insets) that were fit by double exponentials (colored smooth lines). (B) Unlocking rates of WT CFTR were obtained as the reciprocals of the slow time constants (as in A, insets, Ï2), and normalized to their average values in bracketing segments at 25°C; the resulting \documentclass[10pt]{article}
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\begin{equation*}{\hat {k}}\end{equation*}\end{document} were used to construct the Eyring plot shown.
Figure 6. Energetic profile of gating of partially phosphorylated CFTR channels. (A) Profile of measured ÎH (red line), and computed ÎGmax (blue line), and TÎSmin (green line) for a partially phosphorylated CFTR channel as it transits from an initial ATP-bound closed state (Cα) through a transition state (\documentclass[10pt]{article}
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\begin{equation*}T_{{\mathrm{C}}_{{\alpha}},{\mathrm{O}}}\end{equation*}\end{document} ) to an open-burst state (O), and then through a different transition state (\documentclass[10pt]{article}
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\begin{equation*}T_{{\mathrm{O,C}}_{{\omega}}}\end{equation*}\end{document} ) to a final closed state (CÏ, with ADP at the binding site of the hydrolyzed ATP). Forward (left to right) ÎHâ¡ values were obtained from slopes of Eyring plots for WT opening and closing rates (Fig. 1 C), ÎHâ¡ for the reversal of opening reflects the slope of the Eyring plot for K1250R closing rate (Fig. 3B). Corresponding \documentclass[10pt]{article}
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\begin{equation*}{\Delta}G_{{\mathrm{max}}}^{{\mathrm{{\ddagger}}}}\end{equation*}\end{document} values were computed as RTln(kBT/(kh)), by substituting the rates of WT opening (0.3 sâ1) and closure (3.9 sâ1), and of K1250R closure (0.2 sâ1), for k. \documentclass[10pt]{article}
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\begin{equation*}T{\Delta}S_{{\mathrm{min}}}^{{\mathrm{{\ddagger}}}}\end{equation*}\end{document} was obtained as ÎHâ¡ â \documentclass[10pt]{article}
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\begin{equation*}{\Delta}G_{{\mathrm{max}}}^{{\mathrm{{\ddagger}}}}\end{equation*}\end{document} . Downward blue and upward green âsmearâ on the ÎGmax and TÎSmin lines, respectively, indicate the direction of possible error in those estimates; the breaks in those lines indicate uncertainty due to possible asymmetry in transmission coefficient κ (see text). Barrier heights from CÏ back to O could not be measured (dotted lines); ÎG for CÏ was tentatively drawn at â¼â40 kJ/mol relative to Cα to represent hydrolysis of ATP at physiologically relevant concentrations of ATP, ADP, and Pi. (B) Cartoon illustrating mechanistic interpretation of the thermodynamic profile. Opening of the pore (cyan vertical ovals in gray horizontal membrane) is a consequence of formation of an NBD1(green)/NBD2(blue) tight dimer, with two ATP molecules (yellow circles) sandwiched in the interface. Channel closure normally follows hydrolysis of the ATP at the composite NBD2 catalytic site (lower site) to ADP (red) + Pi, which causes disruption of the dimer. The transition state for opening represents a strained channel molecule in which the NBD dimer is already formed but the transmembrane pore (cyan) is still closed. The transition state for normal closure represents the transition state for hydrolysis of the β-γ bond of the ATP (broken yellow circle).
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