Weinreich F , Riordan JR , Nagel G .

DK51619 NIDDK NIH HHS , R01 DK051619 NIDDK NIH HHS

	Figure 1. Calibration of the solution exchange rate by chloride concentration jumps and photolysis of caged ATP. (A) Current through endogenous calcium-activated chloride channels in the presence of 500 μM free calcium and at two different chloride concentrations in the bath. Chloride concentration in the pipette is 150 mM, the holding potential is set to 0 mV. A slow inactivation of the calcium-induced current is seen that is negligible on the time scale of the solution exchange. Bars indicate the presence of calcium and the chloride concentration of the bath solution; the arrow indicates zero current. (B) Current steps in response to a chloride jump shown at a higher time resolution. The traces are from the recording shown in A at the position marked by the asterisk. The broken lines represent fitted functions simulating mixing of the two chloride concentrations by continuous dilution of a fixed solution volume. Time constants for the solution mixing are 16 ms for the jump from 120 to 10 mM chloride and 37 ms for the jump back to 120 mM. (C) Photolysis of 450 μM NPE-ATP with a 10-ns laser pulse of 275 mJ/cm2 and 308 nm wavelength generates an exponentially rising current. In this example, the exponential time constant is 1,280 ms. The vertical arrow signifies the application of the UV light pulse, which is also visible from the artifact induced by the discharge of the excimer laser, the horizontal arrow indicates zero current.
	Figure 2. Time-dependent changes in chloride current amplitude and relaxation kinetics. (A) A representative trace of a complete patch clamp experiment is shown, with initial activation by PKA catalytic subunit (100 U/ml) and repeated ATP (500 μM) pulses generated by rapid solution exchange. Chloride concentrations in the pipette and bath were 150 and 4 mM, respectively. Holding potential was set to 0 mV. Parts of the trace were left out for greater clarity. At the positions indicated by the lowercase letters, the ATP-induced current rise is shown at a higher time resolution as an insert. The ATP-induced steady state current exhibits a time-dependent rundown. Furthermore, a gradual change in the overall shape of the current trace is seen, with the relaxation time course changing from biexponential to monoexponential. The dotted lines represent monoexponential fits to the initial part of the traces shown in the inserts with the following time constants: 1,660, 1,130, and 890 ms at positions a, b, and c. The fractional amplitude of this fast component in relation to the steady state current was 68, 75, and 93% at positions a, b, and c. The bars below the trace identify additions to the bath solution, the arrow indicates zero current. (B) The histogram shows the fraction of steady state current fitted by the fast exponential component after ATP addition in relation to the time interval after PKA removal. The fractional amplitude of the slow component diminishes from 33% shortly after PKA removal to <10% after 1,400 s. The values are taken from 54 relaxation experiments, including the data shown in A with 4–12 data points per bin. Bin size is 200 s, the error bars represent the SD of the means.
	Figure 3. Comparison of ATP concentration jumps by rapid solution exchange and by photolysis of NPE-ATP. (A) The trace shows four ATP pulses by solution exchange and by photolytic cleavage of NPE-ATP using the 325-nm light of a He/Cd laser. As the pulse length is increased, the steady state current generated by photolysis of NPE-ATP (450 μM) approaches the current seen with ATP (500 μM). The power density of the UV radiation leaving the light guide was 25 W cm−2. Note that the signal induced by photolytic cleavage of the NPE-ATP is not stable, because the ATP released at the patch membrane is slowly diluted with unphotolyzed NPE-ATP from the surrounding solution. Bars identify additions to the bath solution, the hatched areas indicate interruption of the otherwise continuous perfusion. Arrows below the trace identify laser pulses with pulse lengths indicated next to the arrows. The horizontal arrow denotes zero current. (B) Comparison of an ATP jump by photolysis of 450 μM NPE-ATP with a 50-ms laser pulse with an ATP jump from 0 to 500 μM ATP by rapid solution exchange. The two ATP jumps are from the experiment shown in A at the positions identified by the lowercase letters. The traces were fitted with an exponential function that yielded relaxation rates of 1.3 s−1 for the photolysis jump (a) and 1.4 s−1 for the solution exchange jump (b). Fits are superimposed on the traces (broken lines), the arrow indicates the laser pulse.
	Figure 4. Effects of NPE-ATP on the relaxation current after an ATP jump. (A) The trace shows four consecutive ATP jumps, one of which is an NPE-ATP to ATP jump and one is by photolysis of NPE-ATP. Nucleotide concentrations are 500 μM for both ATP and NPE-ATP. Bars identify additions to the bath solution, the hatched area indicates interruption of the otherwise continuous perfusion. The arrow below the trace identifies the laser pulse used to photolyze the NPE-ATP (325 nm, 25 W cm−2). The horizontal arrow denotes zero current. (B) Comparison of a jump from 0 to 500 μM ATP with two NPE-ATP to ATP jumps, one of which is by rapid solution exchange and one by photolytic cleavage of the NPE-ATP. The ATP jumps are taken from the experiment shown in A at the positions identified by the lowercase letters. The relaxation currents in a and c could be fitted with monoexponential functions with rates of 1.9 s−1 for the solution exchange jump and 2.2 s−1 for the photolysis jump. The relaxation current in b necessitated a fit with two exponentials of approximately equal amplitude, a fast relaxing current with a rate of 2.1 s−1, and a slowly relaxing current with a rate of 0.11 s−1. Experiments were performed ∼800 s after washout of PKA. Similar results were obtained in 12 more solution changes (from nine patches) at 570 ± 150 s after washout of PKA.
	Figure 5. Effects of AMP-PNP on the relaxation current after an ATP jump. The trace shows three consecutive ATP pulses, one of which represents a jump from AMP-PNP to ATP and back to AMP-PNP. The presence of AMP-PNP profoundly alters the relaxation kinetics of both ATP addition and ATP removal. Nucleotide concentrations are 500 μM for ATP and for AMP-PNP. Bars indicate additions to the bath solution. The change from AMP-PNP to ATP was 300 s after washout of PKA.
	Figure 7. Nucleotide dependence of the ATP-induced stationary current. (A) Dependence of the chloride current on the ATP concentration. Data are shown as means ± SD of at least three determinations. The values could be fitted with a Michaelis-Menten function yielding a Km for ATP of 84 ± 9 μM (broken line). (B) Inhibition of the current induced by 500 μM ATP as a function of ADP concentration. (IATP − IADP)/IATP is plotted versus [ADP], where IATP represents the current in the presence of 500 μM ATP, and IADP represents the current in the presence of 500 μM ATP and the indicated amount of ADP. Data are shown as means ± SD of at least three determinations. The broken line shows a fit to the data, yielding an apparent inhibition constant for ADP, Kapp, of 215 μM. In the case of competitive inhibition, this corresponds to a KI of 31 ± 2 μM, according to the equation \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}K_{{\mathrm{app}}}\;=\;K_{{\mathrm{I}}}\;*\;(1\;+\;[{\mathrm{ATP}}]/{\mathrm{K}}_{{\mathrm{M}}})\end{equation*}\end{document}.
	Figure 6. ATP dependence of the relaxation rates. The rate constants of the fast relaxing component seen upon addition (▪) and withdrawal (○) of the indicated amount of ATP by rapid solution exchange are plotted against ATP concentration. Data represent the means ± SD of at least three independent measurements. All values are normalized to rates measured with 500 μM ATP in the respective experiment; i.e., giant patch. The average of these standard relaxation rates is 1.2 ± 0.4 s−1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{means}}\;{\pm}\;{\mathrm{SD}},\;n\;=\;189)\end{equation*}\end{document} for the current rise after ATP addition and 0.8 ± 0.4 s−1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}({\mathrm{means}}\;{\pm}\;{\mathrm{SD}},\;n\;=\;183)\end{equation*}\end{document} for the current decay after ATP removal. Also included are two different fits to the rates upon ATP addition according to the equation (broken line): \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k\;=\;k_{{\mathrm{max}}}\;*\;[{\mathrm{ATP}}]/([{\mathrm{ATP}}]\;+\;K_{1/2})\;{\mathrm{with}}\;k_{{\mathrm{max}}}\;=\;1.24\;({\mathrm{normalized}})\;{\mathrm{or}}\;1.62\;{\mathrm{s}}^{-}1,\;{\mathrm{and}}\;K_{1/2}\;=\;105\;{\mathrm{{\mu}M,\;or\;the\;equation}}\;({\mathrm{solid\;line}}):\;k\;=\;k_{0}\;+\;k_{{\mathrm{max}}}\;*\;[{\mathrm{ATP}}]/([{\mathrm{ATP}}]\;+\;K_{1/2})\;{\mathrm{with}}\;k_{0}\;=\;0.18\;({\mathrm{normalized}})\;{\mathrm{or}}\;0.22\;{\mathrm{s}}^{-}1,\;k_{{\mathrm{max}}}\;=\;1.13\;({\mathrm{normalized}})\;{\mathrm{or}}\;1.35\;{\mathrm{s}}^{-}1,\;{\mathrm{and}}\;K_{1/2}\;=\;190\;{\mathrm{{\mu}M}}\end{equation*}\end{document}.
	Figure 9. Relaxation kinetics of ATP jumps in the presence of ADP. The relaxation current after ATP addition (left) and removal (right) is shown for three ATP pulses taken from the same experiment. The ATP pulse shown in b was applied in the continued presence of ADP. Note the lower steady state current due to the inhibition by ADP. Nucleotides were used at a concentration of 500 μM. Relaxation rates for ATP addition were 0.96, 0.25, and 1.03 s−1, and for ATP removal were 0.44, 0.71, and 0.45 s−1 for pulses a, b, and c, respectively. The arrows indicate zero current.
	Figure 8. Relaxation kinetics of jumps between ADP and ATP. (A) The trace shows three ATP pulses, one of which represents a jump from ADP to ATP and back to ADP. Nucleotides were used at a concentration of 500 μM. Bars indicate additions to the bath solution, the arrow indicates zero current. (B) The current response to ATP addition (left) and removal (right) is shown at a higher time resolution for the three pulses depicted in A, as identified by the lowercase letters. Relaxation rates for ATP addition were 1.3, 0.41, and 1.4 s−1, and for ATP removal were 0.54, 1.39, and 0.78 s−1 for pulses a, b, and c, respectively. The arrows indicate zero current.
	Figure 10. Changes in the relaxation rates induced by ADP. (A) Relaxation rates for ATP addition under different nucleotide conditions as indicated below the diagram. AMP represents an AMP to ATP jump, ADP represents an ADP to ATP jump, and ATP+ADP represents a jump from ADP to ADP+ATP. All nucleotides were used at a concentration of 500 μM. Relaxation rates are referenced to standard conditions (jump from 0 to 500 μM ATP), error bars show the SD of the data. The individual values for the three types of jumps are 1.0 ± 0.1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;7)\end{equation*}\end{document}, 0.43 ± 0.08 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;8)\end{equation*}\end{document}, and 0.25 ± 0.04 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;9)\end{equation*}\end{document}, respectively. Concentration jumps were performed 350–940 s after PKA washout without observing significant differences in the deceleration factor. (B) Relaxation rates for ATP removal under different nucleotide conditions as indicated below the diagram. AMP represents an ATP to AMP jump, ADP represents an ATP to ADP jump and ATP+ADP represents a jump from ATP+ADP to ADP. All nucleotides were used at a concentration of 500 μM. Relaxation rates are referenced to standard conditions (jump from 500 to 0 μM ATP), error bars show the SD of the data. The individual values for the three types of jumps are 1.03 ± 0.09 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;6)\end{equation*}\end{document}, 1.6 ± 0.4 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;8)\end{equation*}\end{document}, and 1.7 ± 0.3 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(n\;=\;9)\end{equation*}\end{document}, respectively. Concentration jumps were performed 390–990 s after PKA washout without observing significant differences in the acceleration factor.
	Figure 11. Models of CFTR gating involving only one nucleotide binding domain. (A) General gating cycle including ATP hydrolysis. C1 to C3 are closed states of the channels, X1 and X2 may be closed or open states or fluctuate between open and closed. Y and Z denote either of the two ATP hydrolysis products ADP and Pi. (B) Proposed gating cycle based on the relaxation measurements. A1 and A2 are activated states of the channel with open probability >0 (see main text for details). The A2 to C1 transition was made irreversible because in our experiments \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}[{\mathrm{P}}_{{\mathrm{i}}}]\;=\;0\end{equation*}\end{document}.
	Figure 12. Model of CFTR regulation involving both nucleotide binding domains (modified after Gadsby and Nairn 1999). The two NBDs are represented as adjoining squares with NBD-A on the left. Only five major states are given that are important to explain the action of ADP on the activated channel. In this model, NBD-B becomes accessible to nucleotides after the channel has been activated; i.e., in state S3. If ATP is bound to NBD-B, the channel enters a long-lived activated state (S4) that is terminated by hydrolysis of the ATP to ADP. If ADP is bound instead, this long-lived activated state is bypassed and the channel enters a short-lived activated state (S5). If neither ATP nor ADP is bound to NBD-B, the channel may still inactivate from state S3, but not as fast as from the short-lived activated state S5. Note that in this model Pi is always the first ATP hydrolysis product to be released. Only transitions where Pi is released were shown irreversible as in our experiments the concentration of Pi was always zero. See text for further explanation.

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