Effects of Nucleotide Binding on the Stability of Open States
2 Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211
3 Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211
4 Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
Correspondence to Tzyh-Chang Hwang: hwangt{at}health.missouri.edu
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ABSTRACT |
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Key Words: chloride channel single-channel kinetics ABC transporter energetics macroscopic relaxation
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INTRODUCTION |
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Recently the crystal structures of several NBDs from different prokaryocyte members of the ABC family have been solved (Hung et al., 1998; Diederichs et al., 2000
; Gaudet and Wiley, 2001
; Karpowich et al., 2001
; Yuan et al., 2001
; Smith et al., 2002
; Schmitt et al., 2003
; Verdon et al., 2003
). There is growing evidence that the NBDs form a dimeric structure with the two ATP-binding sites buried in the dimer interface (Smith et al., 2002
; Chen et al., 2003
). Hopfner et al. (2000)
suggested that ATP binding drives the NBD dimerization of the distantly related DNA repair protein Rad50, based on biochemical and structural data. This ATP-driven dimerization of the NBDs was confirmed in bacterial ABC transporters by Moody et al. (2002)
. They showed that MJ0796 and MJ1267, two bacterial ABC transporters' NBDs, can form homodimers in the presence of ATP, when hydrolysis was prevented through the mutation of the Walker B glutamate. Moreover, neither ADP nor AMP-PNP promoted stable dimerization, suggesting that a hydrolyzable gamma phosphate is required for a stable dimer formation. Since dimers were not observed in wild-type (WT) NBDs in the presence of ATP, it was speculated that the free energy from ATP hydrolysis results in dissociation of the WT dimer (Moody et al., 2002
; Smith et al., 2002
).
Lewis et al. (2004) crystallized the NBD1 from mouse CFTR, and found that the structure is very similar to that of NBDs from bacterial ABC transporters, in spite of limited sequence identity. It is interesting to note that purified NBD1 of the mouse CFTR does not hydrolyze ATP presumably because of a lack of Walker B glutamate at NBD1 (Lewis et al., 2004
). Although it is unknown whether CFTR's two NBDs form a heterodimer, it has been hypothesized that ATP binding at both NBDs drives the dimerization reaction, which leads to channel opening, and that ATP hydrolysis at NBD2 breaks the dimer and consequently closes the channel (Vergani et al., 2003
).
Several models have been proposed in the past to explain the gating mechanism of CFTR channels. Although the detailed mechanism of CFTR gating by its two NBDs remains controversial, there is some agreement regarding how ATP binding at the NBDs leads to opening and closing of the channel. Since the mutation of the Walker A lysine K1250 in NBD2, which abolishes hydrolysis, results in channels with prolonged burst durations, it was proposed that hydrolysis at NBD2 leads to channel closing (Carson et al., 1995; Gunderson and Kopito, 1995
; Zeltwanger et al., 1999
; cf. Aleksandrov and Riordan, 1998
; Aleksandrov et al., 2000
). However, the mechanism for channel opening remains unsettled. Previous studies suggested that ATP hydrolysis at NBD1 was involved in the opening of the channel since mutations of the Walker A lysine at NBD1 (e.g., K464A) decrease the channel opening rate (Carson et al., 1995
; Gunderson and Kopito, 1995
; cf. Zeltwanger et al., 1999
). However, Powe et al. (2002)
reexamined the gating kinetics of K464A mutant CFTR and found little difference in the opening rate between this mutant and WT-CFTR. In addition, biochemical studies demonstrate an occlusion of ATP in NBD1 (Szabo et al., 1999
; Basso et al., 2003
), suggesting that associationdissociation of ATP from NBD1 is too slow to account for the opening and closing transitions observed in electrophysiological experiments. It is therefore unclear what role NBD1 is playing in CFTR gating.
A clue to the role of NBD1 was the finding by Vergani et al. (2003) of a rightward shift of the ATP dose response when either one of the two Walker A lysines (K464 or K1250) is altered to alanine. A similar observation was made for the mutation at the Walker B aspartate at NBD2 (D1370). Based on these observations, they proposed a model for CFTR gating in which ATP binding at both NBDs is a requirement for channel opening, and hydrolysis at NBD2 leads to channel closing (i.e., Scheme 2 in the accompanying paper). This latest gating scheme can explain the competitive inhibition of ADP for channel opening. However, it is not immediately clear how the model would explain a decrease of channel opening time by ADP (see accompanying paper for details).
To study in more detail the effect of nucleotide binding on the open time of CFTR, we used both macroscopic current relaxation and single-channel kinetic analysis for CFTR mutants with impaired ATP hydrolysis: specifically, the D1370N and E1371S mutations in NBD2. The D1370 residue coordinates Mg2+, a cofactor for ATP hydrolysis. Mutation of this aspartate to asparagine (D1370N) results in channels that exhibit longer open and closed times (Gunderson and Kopito, 1995; Vergani et al., 2003
), resembling the elusive "slow gating" mode described in our previous paper (Bompadre et al., 2005
). The E1371 residue serves as a catalytic base for ATP hydrolysis. Mutation of this glutamate to serine produces a channel (E1371S) that presents long "locked-open" times (Aleksandrov et al., 2000
; Vergani et al., 2003
). These hydrolysis-impaired mutations were also studied in the
R background, which allows single-channel kinetic analysis in the absence of dephosphorylation-induced rundown of channel activity.
Single-channel dwell-time analysis of R/D1370N-CFTR channels shows unequivocally that the open time of the channel is decreased in the presence of ADP. Relaxation analysis of macroscopic
R/E1371S-CFTR currents upon nucleotide removal shows two different relaxation time constants in the presence of ADP and ATP, indicating that ADP induces a different locked-open state. Moreover, studies of
R/E1371S-CFTR open time in patches containing a single channel at three different ATP concentrations show a change of the relative frequency of the different open times, suggesting the presence of an ATP-binding site, occupancy of which affects the stability of the open state. Similar results were obtained in E1371S mutants constructed in the WT background. Kinetic and structural implications of our results are discussed.
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MATERIALS AND METHODS |
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Cell Culture and Transient Expression of CFTR
Chinese hamster ovary (CHO) cells were grown and transfected with the above-mentioned CFTR mutants as previously described (Bompadre et al., 2005).
Reagents
Mg-ATP purchased from Sigma-Aldrich, was stored in water at 20°C. PKA was purchased from Promega. ADP was purchased from Calbiochem and stored in water at 20°C.
Single-channel Experiments
Single-channel experiments were as described in Bompadre et al. (2005).
Data Analysis
Single-channel dwell-time analysis is described in Bompadre et al. (2005). Fitting of the time course of current relaxation upon removal of nucleotides with single or double exponentials was performed with Igor. For dwell-time analysis of
R/E1371S-CFTR data, we pooled the current records obtained from several patches containing only one channel. No cutoff was used for the construction of the closed time histograms and therefore the histogram is constrained by the bandwidth of the recording. The open time histograms were reconstructed by setting a cutoff time of 500 ms to exclude the ATP-independent closings (see RESULTS for details). The resulting open time histograms show at least two locked-open states (see Fig. 6). To quantify the kinetic parameters within the locked-open events, we used 50 s as a cutoff to separate short-lived and long-lived locked-open events. Least square estimation with sums of exponential components was performed to obtain open and closed time constants. Events shorter than dead times were excluded from the fitting.
Kinetics of spontaneous openings in the absence of ATP was assessed manually with Igor. Since the spontaneous opening rate is extremely low, only single-channel openings were seen, even though most of the patches contained multiple channels. Therefore, the measured open times are fairly accurate. Since the number of events was very small, the lengths of the openings measured in different patches were pooled together and a survivor plot was constructed as in Zeltwanger et al. (1999). The data was fitted with an exponential function using Igor. To calculate the opening rate, the closed time before each opening was also measured manually using Igor, and the average opening rate (reciprocal of the averaged closed time) was calculated. Then, to calculate the spontaneous opening rate per channel, we divided the calculated opening rate by the number of channels in the patch.
Online Supplemental Material
The online supplemental material for this paper consists of one figure (available at http://www.jgp.org/cgi/content/full/jgp.200409228/DC1). Fig. S1 shows the ATP dose response for the D1370N mutant.
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RESULTS |
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To study the effect of ADP on single-channel kinetics, we introduced the D1370N mutation into the R-CFTR background because
R-CFTR is insensitive to dephosphorylation-induced rundown, and we can more easily obtain patches with fewer channels for kinetic analysis (Bompadre et al., 2005
). In excised inside-out patches,
R/D1370N-CFTR channels were opened with 1 mM ATP, and subsequent application of 1 mM ATP plus 1 mM ADP inhibited the currents by an average of 63 ± 5% (n = 5). Bracketing ADP experiments with 1 mM ATP controls ensured us that little rundown was present (Fig. 1 A). Kinetic analysis for patches containing up to four channels was performed as described (see MATERIALS AND METHODS). As expected, the mean closed time increases from 1387 ± 201 ms to 3383 ± 645 ms in the presence of ADP. In all five patches, the open time constant is decreased significantly (P < 0.01). This decrease of the open time is readily apparent for patches containing only one channel as shown in Fig. 1 A. The average decrease of the open time constant was 43 ± 11%, from 976 ± 126 to 558 ± 84 ms (Fig. 1 B). These results confirm that ADP does indeed shorten the channel open time. Although we speculate that this decrease of the open time is caused by the presence of another open state when ADP, not ATP, is bound (see DISCUSSION for details), the model used (Scheme 1 in Bompadre et al., 2005
) for data analysis does not allow us to separate two different open states. Furthermore, the open time constant for D1370N-CFTR may still be too short for isolating the putative ADP-bound open state. We therefore constructed another mutant, E1371S, which has an open time on the order of tens or hundreds of seconds because the ATP hydrolysis is abolished at NBD2 (Aleksandrov et al., 2000
; Vergani et al., 2003
).
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ATP Concentration Dependence of R/E1371S Current Relaxation
Our experiments with ADP suggest the presence of a nucleotide binding site, occupancy of which by ATP or ADP can affect the stability of the locked-open state. Based on this idea, one will predict that lowering [ATP] alone could affect the probability of occupancy of this binding site that affects the channel's locked-open time. If ADP, instead of ATP, binding at this site affects the relaxation, a lack of ATP binding (i.e., unoccupied binding site) may also affect the locked-open time constant. We thus performed similar relaxation experiments using a lower ATP concentration.
Fig. 3 A shows a representative relaxation experiments for R/E1371S-CFTR channels opened with 10 µM ATP. Like the macroscopic current in the presence of ATP plus ADP, the macroscopic current induced by 10 µM ATP exhibits much larger fluctuations. Upon nucleotide removal, the current decay is fast at first and then becomes slower, indicating the presence of multiple exponential components reflecting different populations of locked-open states. Ensemble current was generated by pooling data from 22 different patches (Fig. 3 B). The relaxation curve shows a very fast component at the beginning of the washout (Fig. 3 B, inset), then the major relaxation takes place within the first 30 s (a time much shorter than the relaxation time constant for 1 mM ATP), and a final tail that can last for several minutes The current relaxation at 1 mM ATP (from Fig. 2 B) is plotted for comparison (Fig. 3 B, dash line). Unfortunately our solution change is not fast enough to resolve well the fastest component, and the longest component apparently only takes a minor fraction of the total current decay, thus we cannot do a multiple exponential fit to the relaxation time course. Nevertheless, since the relaxation experiments were performed by completely removing ATP, the channels, once opened by ATP, can only close without the possibility of reopening by ATP again. Three different relaxation time courses must reflect three different ATP-induced open states. Even without quantitative curve fitting, a faster current decay upon removal of 10 µM ATP, compared with that with 1 mM ATP, corroborate with the idea of the presence of an ATP binding site, occupancy of which stabilizes the locked-open state.
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We then analyzed the 1 mM ATP data in the same way. Even with an overall recording of 100 min in duration, the closed time histogram was dominated by short closed events with very few long closings. Nevertheless, the histogram can be better fitted with a triple exponential function. In addition to the very brief flickery closings, we also observe intermediate closed events with a time constant close to 100 ms, and a longer closed time of
500 ms (Fig. 6 C). Compared with the closed time histograms from recordings with 10 µM (Fig. 6 A) or 3 µM ATP (Fig. 6 B), it appears that the two shorter closed time constants remain little affected with an increase of [ATP], again supporting the notion that these two short-lived closed states are ATP independent.
It is very difficult to choose a proper cutoff to analyze the open time because there are very few ATP-dependent closed events and they somewhat overlap with the ATP-independent ones. We used the same cutoff time of 500 ms to construct the open time histogram (Fig. 6 F). Since the channel is locked open most of the time, the number of events is not large enough for a more rigorous quantitative analysis even with 100 min of single-channel recording. Nevertheless, the distribution appears to have three components with roughly similar time constants as those obtained at 10 µM ATP (compare Fig. 6 D). Yet, the proportion of these three open events is very different from that at 10 µM ATP. While, at 10 µM ATP, most of the locked-open state is of the short one (LOS), at 1 mM ATP, the longer locked-open state (LOL) contributes >50% of the events. This result shows that an increase in [ATP] shifts the relative distribution of these two locked-open states, supporting the idea that occupancy of an ATP binding site stabilizes the locked-open state. It should be noted that, even disregarding the fitted curves, we can still visualize a rightward shift of the open time distribution at 1 mM ATP (Fig. 6, compare D and E).
To further elaborate the kinetic mechanism of the two locked-open states (LOS and LOL), we analyzed dwell-time distributions within the locked-open bursts that were collected at 10 µM ATP. We used 50 s as the cutoff value to differentiate these two locked-open states. Both types of bursts show similar features. There are two types of closings within the burst, the flickers (CF closed state) with a characteristic time constant of 2030 ms, and longer closings (CL closed state) with a time constant of 100 ms (Fig. 7, A and C). Although the physical meaning of the longer closings (CL closed state) within a locked-open burst is unclear, the flickery closings (CF closed state) are likely due to a blockade of the channel (Zhou et al., 2001
). To obtain the transition rates in and out of the CL closed state, we used 50 ms as a cutoff to analyze the open time distributions within the short and long locked-open bursts. The resulting open time constants are not very different for both types of bursts (Fig. 7, B and D).
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When performing the relaxation experiments in E1371S-CFTR channels, after several minutes of washout, we can still observe a single channel that remains open (Fig. 9). Within this long burst after ATP is removed, we can distinguish the brief flickery closings and also some slightly longer closings, similar to those with the intermediate closed time constant described above (Fig. 6). We pooled four single-channel locked-open bursts (the last channel still open in the relaxation experiments) and analyzed the closed time distribution within the burst. We found that there are two characteristic closed events with time constants of 20 and
80 ms, both very similar to the values described above. Since these two closed events are observed in the complete absence of ATP, we conclude that neither of them is ATP dependent.
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Since the locked-open time is much shorter when ADP is bound than that with ATP bound, we speculate that the binding energy of ATP (or ADP) contributes to the overall energetics of the locked-open state conformation. This hypothesis predicts that a mutation at NBD1 that lowers the binding strength of ATP will have a shorter locked-open time. The crystal structure of CFTR's NBD1 reveals that the Walker A lysine, K464, coordinates the ß- and -phosphates of the bound ATP (Lewis et al., 2004
). Mutation of this lysine has been shown to lower the ATP binding affinity at NBD1 (Basso et al., 2003
). Since the K464 mutation has a mild trafficking defect (Cheng et al., 1990
; unpublished data), and
R-CFTR already suffers from low expression, we decided to make the K464A/E1371S double mutant construct in the WT background.
Relaxation experiments were performed in excised inside-out patches as shown in Fig. 10 A. The current decay of the K464A/E1371S-CFTR channel currents is indeed faster than that of E1371S-CFTR, resulting in a shorter relaxation time constant (19.60 ± 0.01 s) upon washout of 1 mM ATP (Fig. 10 B). This relaxation time constant is even shorter when the K464A/E1371S-CFTR channel is opened with 10 µM ATP (13.95 ± 0.02 s). Since the number of K464A/E1371S-CFTR channels is relatively low due to a moderate trafficking defect, it is easier to observe microscopic channel behavior at 10 µM ATP (Fig. 10 C). As shown previously for R/E1371S-CFTR (Fig. 5), the current trace reveals that K464A/E1371S-CFTR channels also exhibit numerous brief openings that last for tens to hundreds of milliseconds in the presence of 10 µM ATP. Thus, these brief openings for a mutant CFTR with defective ATP hydrolysis are not due to the removal of the R domain, but are true characteristics of the hydrolysis-deficient mutant CFTR.
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We chose the R background because of its resistance to rundown. The fact that removal of the R domain renders the channel phosphorylation independent also helps to avoid a potential technical problem of ATP contamination in the recording system. Excised inside-out patches from cells expressing
R/E1371S-CFTR channels were exposed to ATP-free perfusion solution for >5 min to determine the opening rate of spontaneous opening events. The patches were then exposed to 1 mM ATP to determine the number of channels present. Fig. 11 A shows a representative experiment. Although this patch contains many channels, there are only
20 opening events in the
5-min period without ATP. The calculated opening rate is 0.005 s1. We used survival plot analysis to obtain the mean open time for these spontaneous opening events. The distribution can be fitted well with a single exponential function with a mean open time of
430 ± 19 ms (Fig. 11 C). We repeated the same experiment with
R-CFTR channels (Fig. 11 B). We measured the open time of the spontaneous openings from six patches and constructed a survivor plot to obtain the mean open time. Note that despite 38 min of recording, the number of events is still very small. A fit with an exponential function gives a mean open time of 485 ± 24 ms (Fig. 11 D). The calculated spontaneous opening rate is 0.006 s1. Thus, mutating the E1371 residue has little effect on the spontaneous opening rate. Although the E1371S mutation increases the lifetime of ATP-opened channel by >100-fold, it only minimally affects the open time constant for these spontaneous opening events.
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DISCUSSION |
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We used macroscopic current relaxation upon nucleotide removal to quantify the locked-open times for E1371S mutant CFTR. Although our solution change has a deadtime of 3 s, most of the relaxation time constants for this mutant can be measured accurately because of the long locked-open time on the order of tens to hundreds of seconds. The measured relaxation time constants reflect only the closing rate of the locked-open channel since, in the absence of ATP, the channel seldom opens, and even when it opens, it does not sojourn to the locked-open state (Fig. 11). Multiple components of the current relaxation represent the presence of multiple locked-open states. The most attractive feature of this type of analysis is that no model or artificial cutoffs need to be set. The raw data are fitted with no a priori assumptions. The main disadvantage is that this approach requires very large currents, especially when trying to discern multiple relaxation time constants that differ by less than an order of magnitude.
Macroscopic current relaxations were also used to demonstrate the existence of a second locked-open state in the presence of ADP. From single-channel analysis, all we could observe was a shortening of the mean open time for R- (Bompadre et al., 2005
) or
R/D1370N-CFTR (Fig. 1). In contrast, the macroscopic current relaxation of
R/E1371S-CFTR clearly shows the presence of two components in the relaxation once the channels are opened by ATP plus ADP (Fig. 2), strongly suggesting the presence of two different locked-open states. Furthermore, the relaxation of E1371S-CFTR channel currents in the presence of different ATP concentrations reveals the presence of two locked-open states, and an [ATP]-dependent shift in the distribution of each state (confirmed by
R/E1371S single-channel data). These results, without assuming any kinetic model, indicate that nucleotide binding affects the stability of the locked-open state.
Perhaps because of a much smaller current amplitude, we did not resolve two relaxation time constants in the presence of 1 mM ATP for R/E1371S-CFTR channels. If we accept the fact that multiple locked-open states do exist for
R/E1371S mutants at 1 mM ATP as shown by the single-channel analysis (Fig. 6), how can we ascertain that ADP induces another locked-open state rather than simply increases the relative occupancy of the short-lived locked-open state? In theory, we cannot differentiate these two possibilities unless a triple exponential current decay is observed with the presence of ADP. As long as the lifetime of the ADP-bound locked-open state is not very different from the short locked-open state, it will be very difficult, if not impossible, to dissect these two time constants. It should be noted that the fraction of short-lived locked-open state (LOS) is small at 1 mM ATP (<30%, Figs. 6 and 8). Nevertheless, in the presence of ADP, the short-lived component upon current relaxation is responsible for 50% of the entire current relaxation, indicating that binding of ADP does affect the lifetime of the locked-open state.
Microscopic kinetic analysis, on the other hand, offers the advantage that individual opening and closing events can be analyzed over a long period of time to extract individual time constants. Unfortunately, the gating of CFTR channels is slow, and for hydrolysis-deficient mutants, it is even slower. Thus, very long recordings are needed to collect enough number of events to perform rigorous kinetic analysis. Although the R construct provides a unique advantage of being dephosphorylation resistant, pooling data from different patches is still an inevitable solution. Cutoffs or predetermined gating models need to be used while analyzing the data, especially for recordings from patches containing multiple channels. The imperfect solution of assuming a gating model for data analysis (e.g., Csanády, 2000
) probably explains why the effects of ADP on the open time constant are not detected in multichannel analysis (Fig. 7 C in Bompadre et al., 2005
). Another technical hurdle for single-channel kinetic analysis is the small conductance of CFTR. Heavy filtering of the data is often necessary to secure a reasonable signal/noise ratio. Although this has not been a problem for quantifying ATP-dependent gating since the relevant events are on the order of hundreds of milliseconds to seconds, heavy filtering inevitably results in a poor resolution of the brief ATP-independent events.
By collecting single-channel events from several patches containing R-CFTR, we observed the effect of ADP on the mean open time (Fig. 8 in Bompadre et al., 2005
). Using similar dwell time analysis, we were able to detect multiple components in open time histograms (Fig. 6) for
R/E1371S mutants. An ATP-dependent shift of the distribution of these openings corroborates with the results of macroscopic current relaxations. Therefore, despite all the limitations discussed above, integrating both macroscopic and microscopic analyses of our data, we reach an unremitting conclusion that nucleotide binding affects the relative distribution of different (locked) open states.
Multiple Open States
The results presented in the current study indicate the presence of multiple open states. The distribution of these opens states changes as [ATP] is changed. The [ATP]-dependent shift of the distribution of two locked-open states can be better explained by the idea, as proposed above, that ATP binding at NBD1 affects the stability of the locked-open state. As [ATP] is increased, the fraction of channels with NBD1 occupied by ATP will increase. We propose that channels with both NBDs occupied with ATP will exhibit a longer locked-open time than the singly occupied channels to explain how changing [ATP] shifts the distribution of these two different locked-open states. This same idea can also explain ADP's effects on the current relaxation of R/E1371S mutants (Fig. 2). If the locked-open state with ADP binding at NBD1 is less stable than in the case of ATP binding, we will then expect the presence of a shorter locked-open time with ADP. Although we were not able to resolve two open time constants for
R-CFTR in the presence of ADP (Fig. 8 in Bompadre et al., 2005
), it seems reasonable to speculate that the same mechanism may apply in WT-CFTR when ATP hydrolysis is intact.
In addition to two locked-open states, single-channel recordings also show a brief opening that lasts for 300 ms. A similar short-lived open state was reported previously for K1250A-CFTR (
o =
250 ms), another hydrolysis-deficient mutant (Zeltwanger et al., 1999
). Vergani et al. (2003)
proposed that this unstable open state represents spontaneous opening in the absence of ATP. Indeed, the mean lifetime of the spontaneous openings for
R/E1371S is
400 ms. However, at 10 µM ATP, the frequency of these short-lived events is higher than in the absence of ATP. ATP binding at NBD(s) must also contribute to the appearance of these events in the presence of 10 µM ATP.
Distinct Roles of NBDs in CFTR Gating
Although the data shown in the current report were mostly obtained from studies of CFTR mutants, we will extend these results to get a glimpse of how normal gating occurs. First, we propose that ATP binding at NBD2 plays a major role in channel opening. This idea is based on the observation that mutating the Walker A lysine, K464, at NBD1 does not affect the opening rate (Powe et al., 2002), although this residue is involved in coordinating bound ATP (Lewis et al., 2004
) and the same mutation lowers the ATP binding affinity at NBD1 (Basso et al., 2003
). Second, hydrolysis of bound ATP at NBD2 and subsequent dissociation of ADP and Pi drives channel closing. This idea is supported by the demonstration that WT CFTR gating shows kinetics that demands an input of free energy (Zeltwanger et al., 1999
). Furthermore, mutations that abolish ATP hydrolysis (e.g., K1250A and E1371S) dramatically prolong the open state (Gunderson and Kopito, 1995
; Zeltwanger et al., 1999
; Powe et al., 2002
; Vergani et al., 2003
). Third, ligand binding at NBD1 can modulate the stability of the open state. Powe et al. (2002)
showed that the K464A mutation shortens the open time by 40% at high [ATP]. Interestingly, introducing the K464A mutations into the K1250A construct significantly decreases the locked-open time (Powe et al., 2002
; Vergani et al., 2003
). An equivalent observation is also made in the current report for K464A/E1371S mutants. Fourth, contrary to the model proposed by Vergani et al. (2003)
, ATP binding at NBD1 is not required for channel opening. The fact that the channel can open in the complete absence of ATP already indicates that ATP binding at neither NBD is absolutely required for channel opening. As mentioned above, Powe et al. (2002)
showed that the K464A mutant exhibits a normal opening rate despite a lower ATP binding affinity at NBD1 for this mutant. Furthermore, we interpret the [ATP]-dependent shift of the distribution of two locked-open states by proposing that the occupancy of NBD1 affects the stability of the locked-open state. Therefore, the shorter locked-open time represents an open state with ATP bound at NBD2 while NBD1 is vacant.
Structural Implications
Crystal structures of bacterial NBDs (e.g., Smith et al., 2002; Chen et al., 2003
) show a head-to-tail dimeric configuration. The two ATP-binding sites are buried at the dimer interface. The bound ligands as well as several amino acid residues participating in nucleotide interactions are intimately involved in forming a stable dimer. The dimer structure not only explains numerous biochemical data (e.g., Fetsch and Davidson, 2003
), but also places the signature motif (LSGGQ) that defines the ABC transporter family at the dimer interface actively involved in interactions with ATP. Importantly, this head-to-tail configuration of NBD dimers corroborates with the holoenzyme structure of Escherichia coli BtuCD protein (Locher et al., 2002
).
Although so far there is no structural evidence that equivalent dimerization of CFTR's NBDs occurs during gating transitions, we have proposed that the open state of the channel may be associated with a dimerized configuration of NBDs (Powe et al., 2002). This hypothesis is based on the observation that mutations that affect ATP binding at NBD1 (e.g., K464A) alter the stability of the open state of K1250A, suggesting an interaction between two ATP-binding sites. An NBD1NBD2 dimer with two ATP binding pockets sandwiched at the dimer interface seems a reasonable model to explain this result. Taking one step further, Vergani et al. (2003)
proposed a strict coupling of NBD dimerization/dissociation to channel opening and closing. More recently, using mutant cycle analysis, Vergani et al. (2005)
provided evidence for a direct interaction between R555 (in NBD1) and T1246 (in NBD2) in the open state. This new result strongly supports the idea that an NBD1NBD2 dimer is associated with the open state.
In light of these new findings, we feel compelled to make several structural implications of our results. First, in all the dimer structures published so far, there are extensive hydrogen bonds and van der Waals interactions between ATP and Walker A and B sequences in one subunit, and between ATP and the LSGGQ motif in the other subunit (Smith et al., 2002; Chen et al., 2003
). These extensive binding forces suggest that the dimer is an energetically stable state. Thus, it seems reasonable to propose that ATP binding elicits a large degree of molecular motion that involves closing of the dimer interface and that only hydrolysis of bound ATP could provide sufficient energy to destabilize the dimer (Hopfner et al., 2000
; Smith et al., 2002
). Indeed, the hydrolysis-deficient mutant, e.g., E1371S-CFTR, can assume a locked-open state for minutes, whereas WT channels only open for hundreds of milliseconds.
Second, once the dimer forms in the presence of high [ATP], two ATP molecules are sandwiched at the dimer interface (Smith et al., 2002; Chen et al., 2003
). Thus, the binding energy of the ligand should contribute significantly to the overall energy of the dimer. Weakening the binding energy is expected to shorten the lifetime of the dimer. This can be achieved by using a smaller ligand, such as ADP, by keeping the binding site empty (e.g., at low [ATP]), or by mutating the binding partner in the NBD. In the current report, we show that the locked-open time of the E1371S mutant can be significantly shortened by all three maneuvers.
Third, in the absence of ATP, the spontaneous opening rate for our R constructs is
0.006 s1, and the mean lifetime of these spontaneous opening events is
400 ms. If the spontaneous openings reflect an ATP-independent dimerization reaction, then these kinetic parameters may explain why the dimer is not detected biochemically in the absence of ATP (Moody et al., 2002
). Furthermore, from the energetic point of view, the absence of ligands at the dimer interface may also explain the short-lived openings observed for
R/E1371S-CFTR in the absence of ATP.
Unsettled Issues
One of the unsettled issues is the mechanism of short-lived openings of R/E1371S-CFTR that appear frequently in the presence of micromolar ATP. Admittedly, some of these events are due to spontaneous, ATP-independent openings. However, the opening rate to these brief openings in the presence of 10 µM ATP,
0.17 s1, is much larger than the spontaneous opening rate (
0.006 s1), suggesting that most of them are ATP dependent. We do not know to which NBDs ATP binds to increase the opening rate to this short-lived state. However, the extensive molecular interactions at the dimer interface suggest that the actual dimerization reaction takes multiple steps. Then, this short-lived open state could represent a transitional unstable dimer before an energetically stable dimer is formed. Perhaps ATP binding to either NBD or even both NBDs will increase the rate toward this state.
Although we proposed that ATP binding at NBD2 plays a critical role in channel opening (see above), this idea is based more on default since we observed that the K464A mutation, which decreases ATP binding affinity at NBD1 (Basso et al., 2003), does not affect the opening rate (Powe et al., 2002
). While we did observe a decrease of the opening rate by the K1250A mutation (Powe et al., 2002
; cf. Vergani et al., 2003
), this mutation at NBD2 likely affects both ATP binding and hydrolysis. To establish the role of ATP binding at NBD2 in controlling channel opening, future experiments need to identify amino acid residues, mutations of which only affect the ATP binding step.
Our demonstration of the presence of multiple open states already suggests that opening and closing of the CFTR channel is more complicated than a simple association/dissociation of NBD dimer. The presence of ATP-independent closed state further compounds the picture. By examining single-channel kinetics within a locked-open event, we observed an unexpected closed state with a mean lifetime of 100 ms (Fig. 9). This closed state is at least threefold more stable than the voltage-dependent flickery closings (Zhou et al., 2001
). The lifetime of this novel closed state is not voltage dependent (unpublished data) and these closings are present even after ATP is completely removed, suggesting that they are ATP independent. If a locked-open state represents a tight dimer configuration as proposed by Vergani et al. (2005)
, the presence of numerous transitions in and out of this ATP-independent closed state within a locked-open event indicates that the physical gate of CFTR can still open and close when a stable dimer is formed. This apparently violates the strict coupling hypothesis (Vergani et al. 2003
). On the other hand, if the strict coupling hypothesis is correct, this closed state may represent a transitional, partially separated dimer where ATP remains bound. In this latter scenario, the NBD dimer more likely assumes a dynamic and unexpectedly flexible structure.
Single-channel kinetic analysis of the R/E1371S-CFTR mutant also revealed two ATP-dependent closed states (Fig. 6, A and B). If our hypothesis that both NBDs are involved in CFTR gating is correct, as a minimum, two different configurations of the ATP-binding sites should exist: one with both sites vacant and the other with one site occupied. Since at least one of the ATP-binding sites is vacant in these two states, by definition, their lifetimes have to be ATP dependent. At millimolar [ATP], however, we only observe one long closed time constant probably because the channel has one of the binding sites (likely NBD1) occupied most of the time, and therefore, once the channel closes, the opening is initiated by ATP binding at NBD2. This latter, more restricted, scenario is actually very close to the model proposed by Vergani et al. (2003)
. Considering the difficulty of kinetically dissecting different ATP-dependent closed states, we are currently seeking independent methods to verify the existence of these states.
Our kinetic analyses and structural interpretations were all based on studies of hydrolysis-deficient mutant CFTR. Abolition of the ATPase activity by mutagenesis provides the advantage that CFTR can now be treated as a classical ligand-gated channel. If ATP hydrolysis dominates the closing transition for WT-CFTR gating, as described above, some of the closed states revealed in the current studies may not be discerned. Future studies of WT-CFTR gating at lower temperatures may confirm some of our findings. In this regard, the R-CFTR construct used in the current study could again be very valuable.
A difference in closing transitions between WT and hydrolysis-deficient mutant CFTR may also explain the conundrum that ADP exerts a much larger effect on the locked-open time of R/E1371S channels than on the open time of the
R channels. A much larger free energy is involved when ATP hydrolysis is used to close the channel. Thus the binding energy of nucleotide at NBD1 is relative small compared with the energy released from ATP hydrolysis. On the other hand, in E1371S-CFTR whose hydrolysis is abolished, thermoenergy is used for channel closing. Then the binding energy of nucleotide at NBD1 plays a more important role in deciding the rate of channel closing.
Conclusive Remarks
The most important conclusion from this study is that the gating of CFTR is more complicated than previously believed because we have demonstrated multiple open states and multiple closed states, both of which are affected by occupancy of nucleotides at each NBD. In view of this complexity, it is perhaps advisable to study CFTR gating from a reductionist point of view. It may be more fruitful in the near future to focus on quantifying limited kinetic steps rather than on elaborating a complete gating scheme.
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ACKNOWLEDGMENTS |
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This work is supported by the National Institutes of Health (T.C. Hwang, DK55835, HL53445; X. Zou, DK61529) and a beginning Grant-in-Aid from the American Heart Association (X. Zou). S.G. Bompadre is a recipient of NRSA (DK062565). Y. Sohma is supported by the Japan Society for the Promotion of Science (15590196).
Olaf S. Andersen served as editor.
Submitted: 3 December 2004
Accepted: 8 February 2005
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REFERENCES |
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