Correspondence to: Georg Nagel, Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt/M., Germany., nagel{at}biophys.mpg.de (E-mail), Fax: 49-69-6303-305; (fax)
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Abstract |
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The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.
Key Words: ATP-binding cassette transporters, protein phosphorylation, caged ATP, cystic fibrosis transmembrane conductance regulator gating, ATP hydrolysis
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Introduction |
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10 yr ago, the gene that is mutated in the hereditary disease cystic fibrosis was cloned and sequenced and named cystic fibrosis transmembrane conductance regulator (CFTR)1 (
Early on, it was recognized (
Although it is undisputed that nucleoside triphosphates are necessary to open CFTR channels after phosphorylation by PKA, the mechanism of action of ATP is the topic of some controversy. In some studies, it was suggested that ATP is hydrolyzed during CFTR gating, based on the inability of the nonhydrolyzable ATP-analogue adenylylimidodiphosphate (AMP-PNP) to open CFTR channels (
The stimulatory effect of AMP-PNP on CFTR channels opened by ATP was confirmed later (
Most studies of CFTR gating have investigated single-channel or multichannel nucleotide-dependent gating under steady state conditions. Under these conditions, it may be difficult to separate the effects of a nucleotide interacting in more than one way with the two NBDs of the CFTR. Therefore, we designed experiments under presteady state conditions and followed the relaxation of large populations of CFTR channels to a new steady state. To achieve fast access to the cytosolic membrane surface, and to improve the signal-to-noise ratio, we used the giant patch technique (
We employed flash photolysis of ATP-P3-[1-(2-nitrophenyl)ethyl]ester (NPE-ATP, or caged ATP;
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Materials and Methods |
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Expression of CFTR in Xenopus Oocytes
cRNA was prepared from the plasmid, pCFTR(SP), containing the human CFTR cDNA as described (
Giant Excised Patch Clamping
Oocytes were shrunken by brief immersion in a hypertonic solution containing 200 mM potassium aspartate, 20 mM KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, pH 7.4. Thereafter, the vitelline membrane could be removed using sharpened watchmaker's forceps. The oocyte was placed in a small petri dish (35 mm) filled with 60 mM N-methyl-glucamine-Cl, 40 mM NaCl, 20 mM tetraethylammonium-Cl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, pH 7.4, which was mounted on the stage of an inverted microscope (Zeiss Telaval 31; Carl Zeiss Jena). Seals were formed using borosilicate glass pipettes with tip openings of 1824 µm.
The pipette solution contained 150 mM N-methyl-glucamine (NMG)-Cl, 2 mM BaCl2, 2 mM MgCl2, 0.5 mM CdCl2, 10 mM HEPES, pH 7.4. To accelerate seal formation, slight suction was applied to the pipette. Withdrawal of the pipette yielded large inside-out membrane patches having seal resistances of 220 G. The excised patch was then transferred to a temperature controlled chamber (
To perform time-resolved relaxation measurements after a concentration jump, the system time constant for the solution exchange in the measuring chamber had to be determined. To do this, the chloride current through the oocyte's endogenous calcium-activated chloride channels (
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Measurements of nucleotide-induced relaxations were rejected for kinetic analysis if the observed relaxation time constants were not significantly (i.e., by a factor of five or more) larger than the solution exchange time constant. The experimental conditions also necessitated a delay (system delay or solution exchange delay) between the switching of the valve and the arrival of the solution at the pipette tip. This solution exchange delay could be measured as the time between valve switching and the onset of the current response. It was determined to 0.5 ± 0.1 s for the chloride concentration changes and 0.7 ± 0.2 s for changes from 0 to 500 µM ATP, whereas it was 1.6 ± 0.5 s for changes from 500 to 0 µM ATP (n = 11). We therefore concluded that the delay observed with ATP addition is not significantly different from the "solution exchange delay" (see also caged ATP photolysis experiments, below), whereas the delay observed with ATP removal is significantly longer. Although this delay time is considered to reveal a valuable parameter of CFTR gating, it was not included in the analysis of this study.
Under the conditions used to measure CFTR currents, the calcium-activated chloride channels did not contribute to the current signal, as intracellular calcium was kept low by the addition of the chelator EGTA. The chamber was further equipped with a duct through which a quartz fibre serving as a light guide could be positioned in close proximity (100200 µm) to the pipette tip (see
An example of a current induced by incomplete photolysis of 450 µM NPE-ATP is shown in Figure 1 C. The rise in current resulting from the ATP liberation was not instantaneous, but showed a short delay of several milliseconds that may be attributed to the time required for the photolysis reaction to generate ATP. More importantly, the signal rise time was rather slow with time constants of 5001,500 ms if an exponentially rising signal was assumed. As was obvious from the slow rise times seen with a 10-ns pulse, the pulse duration could safely be extended to several milliseconds using a low-power continuous wave He/Cd laser of 325-nm wavelength (Kimmon Electric Co., Ltd.) without compromising the time resolution of the current signal. The 325-nm continuous laser beam was pulsed by means of an electrical shutter device (Uniblitz; Vincent Associates) with a response time of ~1 ms.
Pipette current was measured with an EPC-7 patch clamp amplifier (List Medical) at a holding potential of 0 mV. Seal resistance was checked before and after each experiment. All experiments were carried out at 25°C.
Data Acquisition and Analysis
Membrane currents and solution switching pulses were routinely recorded on a chart recorder (Kipp & Zonen). Currents were filtered online at 20 Hz and continuously recorded at 100 Hz on the hard disk of a personal computer using KAN1 software (MFK; Michael Friedrich, Neidernhausen, Germany). In addition, currents filtered at 100 Hz were recorded at 500 Hz on a second personal computer using PCLAMP 6 software (Axon Instruments), which was also employed to trigger the laser pulses and for electric valve switching. Relaxation currents were fitted using either the PCLAMP simplex fitting algorithm or the Levenberg-Marquardt fitting tool from the ORIGIN 5.0 analysis program (Microcal Software).
Drugs and Chemicals
Mg-ATP (from equine muscle), ADP, and AMP (free acid) were from Sigma Chemical Co., NPE-ATP was from Molecular Probes, Inc., AMP-PNP was from Boehringer Mannheim, and PKA catalytic subunit was from Promega Corp. Buffer chemicals were from Merck or Sigma Chemical Co.
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Results |
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Jumps in the ATP Concentration Generate Characteristic Relaxation Currents
When a giant patch was excised from an unstimulated oocyte expressing human CFTR channels, activity in response to the channel opener ATP was negligible, indicating a low basal PKA activity in the resting oocyte. Application of exogenous PKA catalytic subunits in conjunction with ATP gave rise to a substantial chloride current in the range of hundreds of picoamperes to a few nanoamperes (compare Figure 2 A), recorded at a holding potential of 0 mV and with a chloride gradient of 150 mM:4 mM between pipette and bath. Upon removal of the kinase, the signal underwent first a rapid and subsequently a slow rundown that was partly reversible by readdition of the PKA (not shown, but see
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In the absence of PKA and in the presence of millimolar concentrations of magnesium ions, the prephosphorylated channels could be opened by ATP alone. The application of an ATP pulse thus provided a means to analyze the kinetics of channel gating from the multichannel current signal. To judge whether the observed relaxation kinetics were determined by the interaction of ATP with CFTR or by the solution exchange at the patch membrane, the solution exchange speed was always calibrated with chloride concentration jumps under conditions when endogenous chloride channels in the oocyte membrane were open (see the description in METHODS). This provided a CFTR-independent estimation of the solution exchange time and of the delay between valve switching and arrival of the solution at the patch membrane. When ATP was added to closed channels, the current followed an exponential time course in approaching the new steady state level.
Usually, relaxation kinetics could best be approximated by the sum of two exponentials with a fast time constant in the range of several hundreds of milliseconds that accounted for 6095% of the signal amplitude and a slow time constant ~10x greater that was often difficult to determine because of its small amplitude (see Figure 2). The slow time constants, as well as the above mentioned delay times, were not systematically evaluated. In most cases, therefore, our analysis is restricted to the rates of the fast-relaxing current component.
Changes in Kinetics Accompany the Rundown of CFTR Activity
When ATP pulses were applied consecutively, the resulting currents exhibited a time-dependent change in both signal amplitude and relaxation rates. The relative amplitude of the slowly relaxing current always diminished with time and the signal could then be approximated reasonably well by a monoexponential function. This is shown in Figure 2 for the current rise upon ATP addition, but was similarly observed for the current decay upon ATP withdrawal. For the kinetic analysis, the slowly relaxing amplitude of the current signal was neglected and the signal was fitted with a simple monoexponential function using only the initial part of the record. In all cases (68 of 68 experiments) the rundown of the current signal was accompanied by an acceleration of the fast relaxation rate.
Presumably because of differing phosphatase activity in different patches, the relaxation rates exhibited considerable variance between measurements (see below). This necessitated the use of a standard ATP jump (between 0 and 500 µM ATP) as an internal reference, which was applied at regular intervals during the course of an experiment. The average value of all these standard rates was 1.2 ± 0.4 s-1 (means ± SD, n = 189) for the current rise after ATP addition and 0.8 ± 0.4 s-1 (means ± SD, n = 183) for the current decay after ATP removal. Referencing relaxation rates to the standard rate enabled us to compare normalized relaxation rates obtained from different patches. Because ATP jump experiments were performed after much of the rundown had already taken place (~10 min after washout of PKA, see Figure 2 B and figure legends), the change in the kinetics over time was not large.
Flash Photolysis of Caged ATP Confirms the Data Obtained Via Fast Solution Exchange
To confirm the physiological significance of the observed relaxation rates, a second approach was followed to complement the calibration via chloride concentration jumps (see MATERIALS AND METHODS). It has been shown for CFTR reconstituted into a black lipid membrane (
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The relaxation rates after a photolytically engineered ATP jump were compared with the relaxation observed after an ATP jump by rapid solution change and were found to be virtually identical (example in Figure 3 B). Thus, by means of flash photolysis of NPE-ATP, we were able to ascertain that the fastest component in the current rise after an ATP jump had a time constant of several hundred milliseconds and could thus readily be resolved by rapid solution switching. Because removal of ATP from the membrane was only possible by perfusing the bath with an ATP-free solution, a comparison between ATP jumps by solution exchange and by photolysis was limited to ATP addition. Nevertheless, because the kinetics of ATP removal showed relaxation rates in the same time range as those of ATP addition (see the examples in Figure 5, Figure 8, and Figure 9) and because these were slower than the routinely performed chloride concentration jumps (yielding the solution exchange time), we are confident that the rates obtained with rapid solution exchange reflect the true kinetics of the channels.
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The Presence of ATP Analogues Alters the Kinetics
The observed inhibition of the ATP-induced steady state current by NPE-ATP was investigated by means of relaxation experiments. When channels were incubated with NPE-ATP and the NPE-ATP was subsequently replaced by ATP, an additional slow component was introduced into the relaxation kinetics (in 12 of 12 cases, compare the example shown in Figure 4). This slowly relaxing current was not seen in the absence of NPE-ATP or when the NPE-ATP was converted into ATP by photolysis (Figure 4). The rate of the fast relaxing component was not affected by the NPE-ATP nor was the relaxation when ATP was replaced by NPE-ATP. This shows that NPE-ATP may bind to the CFTR in the absence of ATP; i.e., when all channels are closed.
Although the concentration dependence of the effect of NPE-ATP was not investigated, the slowly relaxing component in the current after ATP addition most likely is caused by those channels that have bound NPE-ATP, which prevents ATP binding until it has dissociated. The fast relaxing component then represents those channels without bound NPE-ATP that can open normally once ATP is applied.
As shown for NPE-ATP, the comparison of a standard ATP-induced relaxation current with the relaxation signal after preincubation with other substances allows detection of interactions with the closed channel. When this is done using the nonhydrolyzable ATP analogue AMP-PNP instead of NPE-ATP, a slow component is again introduced into the relaxation after ATP addition (Figure 5), indicating binding of AMP-PNP to the closed channel (at NBD-A) resulting in competitive inhibition of ATP binding. If the presumed competitive binding of AMP-PNP and ATP at NBD-A was the only AMP-PNP effect on CFTR gating, no alteration of the relaxation after ATP removal is to be expected. This is true for NPE-ATP, but not for AMP-PNP, as shown in Figure 5.
When ATP was removed in the presence of 500 µM AMP-PNP, an additional slow component with a relaxation rate of 0.033 ± 0.008 s-1 (n = 4) was observed. The amplitude of this slow component varied and appeared to be higher if AMP-PNP was applied early after phosphorylation; i.e., before most of the rundown had occurred. In all cases, however, more than half of the total amplitude was accounted for by a fast relaxing current with a relaxation rate indistinguishable from the rate in the absence of AMP-PNP, indicating that the greater part of the channels was not affected in its kinetics by AMP-PNP.
The Relaxation Rate of Channel Opening Is a Function of the ATP Concentration
Next, the dependence of the ATP jump relaxation rates on the ATP concentration was determined. There was a pronounced dependence of the relaxation rate after ATP addition (kad) on the ATP concentration in the range from 25 µM to 2.5 mM. The ATP dependence could be reasonably well fitted by a simple saturation function: kad = kmax * [ATP]/(K1/2 + [ATP]), with a K1/2 of ~100 µM ATP (see Figure 6, broken line).
An even better fit could be obtained by a saturation function including a basal rate k0 (Figure 6, solid line): kad = k0 + kmax * [ATP]/(K1/2 + [ATP]), with a K1/2 of 190 µM ATP, a kmax of 1.35 s-1, and a basal rate k0 (at 0 ATP) of 0.22 s-1, leading to a maximal rate (at saturating ATP) of ~1.6 s-1. The fitted basal rate ko seems high. It is difficult to determine it more accurately by further lowering the ATP concentration as the resulting amplitudes are usually very small and lead to a greater error. It is, however, an important observation that this basal rate is significantly lower than the relaxation upon ATP removal. This observation already excludes certain simple gating models similar to ligand-gated channels.
The relaxation rate obtained after ATP removal was not sensitive to the ATP concentration present before ATP removal (Figure 6). This relaxation rate was always the same when normalized to the standard rate (which was itself not a constant but subject to a time-dependent acceleration, as described earlier). Such a dependence is to be expected if only ATP-independent events contribute to the relaxation. The ATP-invariant fast closing rate is therefore an additional proof that the rate of solution exchange is not limiting for the observed relaxation rates. If the solution exchange rate were limiting for the observed current decay, then the removal of high ATP concentrations would result in a slower current response than the removal of low concentrations of ATP.
ADP Decreases the Relaxation upon ATP Addition
Finally, we investigated the interaction of ADP with the CFTR, which is known to inhibit ATP-induced opening (
Given that a competitive binding of ADP to the ATP binding site responsible for channel opening occurs, preincubation of the channels with ADP should result in a delayed opening and therefore a lower relaxation rate. Since no biphasic relaxation was seen, it must be postulated that under the conditions employed here all channels have bound ADP at the time the ATP is applied. This is to be expected with 500 µM ADP and a KI of 31 µM, although further experiments should determine the exact KI of ADP binding to closed channels under these presteady state conditions. The inhibitory effect of ADP on channel opening should be even more pronounced if ADP is present throughout the entire ATP pulse. In this case, ADP that dissociates from the binding site may be replaced by either ATP or ADP, depending on the concentration ratio of the two nucleotides, thereby reducing the rate of ATP binding. This reduced relaxation rate was indeed observed as shown in Figure 9.
When an ATP jump in the presence of 500 µM ADP was performed, the relaxation rate of ATP-induced opening was by a factor of 4.0 lower than in the absence of ADP and by a factor of 1.7 lower than the relaxation rate after an ADP to ATP jump (compare Figure 10). The steady state amplitude of the chloride current was also reduced, as expected from the ADP titration shown in Figure 7 B. When ADP was replaced by AMP, which was reported to have no effect on CFTR gating (
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ADP Accelerates the Relaxation upon ATP Removal
Interestingly, besides reducing the relaxation rate after ATP addition, ADP enhanced the relaxation rate of channel closing by a factor of ~1.7 (compare the examples shown in Figure 8 and Figure 9) without introducing additional components into the current signal. This effect seemed to saturate at high ADP concentrations since a doubling of the ADP concentration from 0.5 to 1.0 mM lead only to a 25% increase in the relative relaxation rate from 1.6 ± 0.4 (n = 8) to 2.0 ± 0.3 (n = 6). Again, equally high concentrations of AMP did not exhibit any effect on the relaxation kinetics. The accelerating effect of ADP on the relaxation rate was the same regardless of whether or not ADP was present before ATP removal (Figure 10), providing further evidence that only channel closing events contribute to the current signal after ATP removal. The significance of this finding, which to our knowledge has not been reported thus far, will be discussed later with respect to a possible CFTR gating cycle.
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Discussion |
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This study introduces a new experimental approach to efforts to gain insight into the complicated regulation of CFTR channel gating by nucleotides. We applied fast changes of nucleotide concentrations and measured the relaxation of many CFTR channels to a new equilibrium. This presteady state experiment is a different and complementary approach to single channel studies that measure rate constants under steady state conditions.
In our discussion, we use the terms NBD-A and NBD-B to differentiate between two nucleotide binding sites as observed in this and previous studies and introduced by
Our experimental approach could only provide meaningful results if the concentration changes are faster than the observed relaxations that were tested by two independent procedures.
Are the Solution Changes Fast Enough?
The time constant of solution exchange was tested for each individual patch and was faster than 150 ms (typically 1530 ms, Figure 1) in all experiments used for analysis. The observed relaxation rates were at least five times slower than this solution exchange rate. An additional independent test of the validity of the relaxation rates obtained by solution exchange was provided by flash photolysis of caged ATP to generate a fast increase in ATP concentration. The observed relaxation was indistinguishable from the one obtained by a fast solution change (Figure 3). Further support for the validity of the observed relaxation rates comes from the [ATP] dependence (see Figure 6 and below) of the relaxation rates upon ATP withdrawal.
Data Analysis and Interpretation
We interpret these data under the assumption that the observed relaxations of the transmembrane chloride current are caused by conformational changes of CFTR from one or several closed states to one or several "activated states" with a certain open probability or vice versa. The open probability of this activated state might be 1 (i.e., the activated state is an open state) or it might be >0 and <1 (i.e., a fluctuation between an open and a closed state). By stepping the ATP concentration from zero to a given value and back to zero again, we were able to generate two different types of relaxation currents. The current recorded after ATP addition is governed by the kinetics of channel opening and closing, since ATP is present to support the complete gating cycle. The current relaxation recorded after sudden ATP withdrawal might only depend on the kinetics of channel closing or, if ATP-bound closed channels open equally fast or faster than ATP dissociates, might again depend on the kinetics of channel opening and closing.
Although determination of rate constants for channel opening and closing from single channel measurements have produced a wealth of valuable information about nucleotide-dependent CFTR gating (for review, see
Gradual Dephosphorylation of CFTR Underlies Rundown of ATP-activated Current
As reported earlier (
The rundown accompanying the change from a two- to a mono-exponential relaxation can be explained on the basis of the postulated (
NPE-ATP and AMP-PNP Bind to the Closed Channel
Early on, it was found that the hydrolysis-resistant ATP-analogue AMP-PNP is unable to open prephosphorylated CFTR (
The observed slowing of ATP-induced opening of CFTR channels when ATP was applied concomitant with AMP-PNP removal is a clear indication of AMP-PNP binding to closed CFTR channels via NBD-A, similar to ADP binding (see below). Although such an inhibitory binding of AMP-PNP to NBD-A should be observed also under steady state conditions, so far it has not been, presumably because of the strong stimulatory effect of AMP-PNP on NBD-B. Such an inhibitory binding of AMP-PNP to NBD-A was postulated by
Similarly, preincubation with NPE-ATP slowed activation by ATP, suggesting binding of NPE-ATP to NBD-A. On the other hand, NPE-ATP does not seem to bind to NBD-B because the current relaxation when switching from ATP to NPE-ATP was very similar to the one observed when switching to no nucleotide (see Figure 4A and Figure B). The slight difference in the observed slow relaxations with small amplitude might either reflect residual ATP in the caged ATP solution (typically 0.5%), or indeed very low affinity binding of NPE-ATP to NBD-B. This seems unlikely, however, because we found inhibition of the steady state ATP-activated current in mixtures with NPE-ATP, in accordance with its competitive binding to NBD-A (data not shown).
AMP-PNP Binds to the Activated Channel and Delays Closing
A stimulating effect of AMP-PNP on CFTR activity under certain conditions was reported for endogenous CFTR in sweat glands (
An important modulator of the action of AMP-PNP on CFTR is the degree of its phosphorylation. It was shown that higher phosphorylation corresponds to a higher open probability of CFTR and is a prerequisite of the "open-locking" action of AMP-PNP (
ATP Dependence of Relaxation Rates
As outlined in METHODS, at least three time constants might be found in the current relaxation upon ATP withdrawal: a delay time constant, a fast relaxation with a large amplitude, and a slow relaxation with a small amplitude. For this study, we analyzed only the fast relaxation. It is interesting to compare this rate of ~0.8 s-1 to closing rates obtained from single channel studies. The closing rate is the inverse of the mean open (burst) time obtained from a single channel experiment. Published mean open burst times range from 200 ms to ~1 s at 25°C or room temperature, corresponding to closing rates in the order of 15 s-1 (for review, see
The relaxation upon ATP addition on the other hand appears to have no delay time constant, as evidenced by experiments with photolysis of caged ATP (NPE-ATP). It can be fitted by a fast relaxation with a large amplitude and a slow relaxation with a small amplitude. Again, we only analyzed the fast relaxation. The inverse time constant of the fast relaxation shows a saturating dependence on the ATP concentration that could be fitted best by a model with a maximal value of ~1.5 s-1, a K1/2 of ~200 µM, and a basal value of ~0.2 s-1. The maximal rate at saturating ATP concentrations corresponds quite well to the opening rate (i.e., the inverse of the long and ATP-dependent mean closed time) derived from single-channel measurements at saturating ATP in several single channel studies (e.g.,
The Relation between the ATP Hydrolysis Cycle and CFTR Gating
A recent publication by
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Possible Mechanisms of Inhibition by ADP
ADP was recognized as an inhibitor of CFTR soon after the activating role of ATP was detected (
Contrary to our expectations, however, we also found a significantly increased relaxation rate upon ATP removal when the ATP-free solution contained ADP. It is obvious that this finding cannot be explained by a competition between ATP and ADP at an empty NBD-A site on the closed (not activated) channel. In the absence of ATP, only interactions with the activated channel can contribute to a change of the relaxation rate because a shift from closed channels to closed channels with bound ADP is electrically "silent" under these conditions. We have to conclude, therefore, that ADP may also bind to the activated channel.
We propose two possible mechanisms for the observed accelerated closing by ADP, depending on whether ADP acts at NBD-A or NBD-B. The two models are distinguished by the order of release of the two hydrolysis products; i.e., ADP or Pi first.
Can ADP Reverse ATP-induced Activation by Binding to NBD-A?
We will first discuss a possible explanation of the ADP effect on the basis of a single hydrolysis cycle (at NBD-A). This requires ADP binding to NBD-A after the channel has been opened by ATP interaction at NBD-A. In this case, ADP may only bind after ADP generated by hydrolysis of ATP has been released from NBD-A. ADP would then act via product inhibition, with the channel driven back to the closed state by reversion of ATP hydrolysis, in this way rebuilding ATP. This is thermodynamically feasible since the process takes place at a very low ATP:ADP ratio (during the relaxation, [ATP] is essentially zero, while [ADP] is 0.5 mM). Also, to explain the observed acceleration of channel closing by ADP, the rate of closing by reversal of activation must be about the same as the rate of closing by completion of the ATP hydrolysis cycle.
This explanation for the effect of ADP on the rate of channel closing requires that during the ATP hydrolysis cycle ADP is released before Pi, because otherwise reversion of ATP hydrolysis is impossible under our conditions where [Pi] = 0. This is illustrated by the gating model shown in Figure 11. In Figure 11 A, a general model of a gating cycle driven by ATP hydrolysis at a single site is presented. The channel is enabled to open simultaneous with or after ATP hydrolysis and loses this ability when the two hydrolysis products, ADP and Pi, are released. This general model does not specify at which step in the hydrolysis cycle the closed-to-open and open-to-closed transitions are located, nor does it specify the order of release of the two hydrolysis products. Included in the model is the competitive binding of ADP to the closed channel (state C1), which explains the slowing of opening upon a change from ADP to ATP, but cannot explain the acceleration by ADP of the relaxation upon ATP withdrawal.
To incorporate the binding of ADP to the activated channel into the model, X2 must be an activated state and must be able to bind ADP. This implies that ADP is released before the phosphate. If one assumes that the closed-to-activated transition is coupled to ATP hydrolysis, rather than to ADP release, X1 is also an activated state. This results in the model in Figure 11 B with two activated states, A1 and A2, and three closed states, C1, C2, and C3. The ratio of A1 to A2 is dependent on the ADP concentration. If [ADP] is low, the channels will close via the A2C1 transition. At high [ADP], however, the A1:A2 ratio will be higher, allowing the channels at low [ATP] to close via the A1C2 transition, which may have a similar or higher rate than the A2C1 transition, resulting in the accelerated relaxation rate observed in our measurements.
Although attractive on the grounds that only one NBD has to be invoked (in accordance with our observation of a single-exponential accelerated decay), this explanation is in contrast to previously published models about the hydrolysis cycle of CFTR (
Can ADP Binding to NBD-B Accelerate the Activated-to-Closed Transition?
Previously, it was shown that binding of nucleotides (specifically AMP-PNP) to a second site, NBD-B, modulates channel closing (
As can be inferred from the only partial effect of AMP-PNP on channel closing, our data were obtained from partly dephosphorylated channels, but show ADP-induced acceleration of closing for all CFTR channels. Assuming binding of ADP to NBD-B, this could indicate that NBD-B is accessible to ADP regardless of the phosphorylation state.
Recently,
In the presence of ATP, if NBD-B is accessible, the gating cycle is driven by ATP hydrolysis at both NBDs. NBD-B becomes accessible to nucleotides only after ATP is bound and hydrolyzed at NBD-A, when the channel is activated.2 In addition to the complete cycle via S4 and S5, which requires ATP binding to NBD-B, two shortcuts in the cycle may occur. One of these bypasses nucleotide binding to NBD-B and allows the channel to close from the S3 state. This corresponds to the closing in the absence of any nucleotide as observed in the relaxation after ATP removal. Although this is the shortest cycle in the scheme (Figure 12), it is not the cycle with the shortest open duration, see below.
In the model proposed by
In the presence of ATP, ATP binding to NBD-B might occur in the state S3 leading to state S4, and the transition rate from S4 to S5, which may be slow, will determine the time the channel remains activated. If this explanation is correct, competition between ATP and ADP not only regulates the likelihood of channel opening, but the rate of channel closure as well.
Based on our relaxation experiments, we cannot yet discriminate between the two possible modes of ADP interaction with the activated channel. It is clear, however, that the inhibitory action of ADP is more complicated than previously thought, since a competition with ATP for binding at the activating site (NBD-A) alone cannot explain all our observations.
Conclusions
By observing the relaxations of large numbers of CFTR channels under presteady state conditions, we have shown that the time course of channel opening and closing is modulated by the presence of different nucleotides. The nonhydrolyzable ATP analogue AMP-PNP delays closing of CFTR channels, previously opened by ATP. This is in agreement with earlier findings and the suggestion that AMP-PNP (like ATP) prevents channel closing by binding to NBD-B. Our new finding of inhibitory binding of AMP-PNP to NBD-A is further strong support for the requirement of ATP hydrolysis at NBD-A in channel opening.
In addition to inhibitory competing with ATP, we found that ADP regulates the closing rate of the channel as well. This constitutes a new twist to the mechanism by which the energy charge of the cell (i.e., the [ADP]:[ATP] ratio) regulates CFTR activity, as proposed by
The results presented in this study were obtained from partly dephosphorylated channels with a lower than maximal open probability. We present evidence that at higher phosphorylation nucleotide-dependent gating is additionally modified, leading to slower relaxations. The characterization of channels with better defined phosphorylation status, as well as of CFTR with mutant NBDs, as discriminating between interactions with either or both NBDs awaits further study.
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Footnotes |
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Dr. Weinreich's present address is Center for Molecular Neurobiology (ZMNH), Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany.
2 The term activated was also used by
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Acknowledgements |
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We thank Doris Ollig for expert technical assistance, Phillip Wood for help in the preparation of the cRNA, Klaus Fendler for discussion of kinetic models, and Ernst Bamberg for constant encouragement. We also thank Tzyh-Chang Hwang and David Gadsby for providing preprints before publication and David Gadsby for reading the manuscript and insightful comments.
This study was supported by the Deutsche Forschungsgemeinschaft (NA 207/4-1 to G. Nagel), Max-Planck-Gesellschaft, the Cystic Fibrosis Foundation, and the National Institutes of Health (DK51619 to J.R. Riordan).
Submitted: December 4, 1998; Revised: April 30, 1999; Accepted: April 30, 1999.
1used in this paper: AMP-PNP, Adenylylimidodiphosphate; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide binding domain; NBF, nucleotide binding fold; NPE-ATP, ATP-P3-[1-(2-nitrophenyl)ethyl]ester (or caged ATP)
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References |
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