©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Two Nucleotide-binding Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Have Distinct Functions in Controlling Channel Activity (*)

(Received for publication, August 11, 1994; and in revised form, November 2, 1994)

Mark R. Carson Sue M. Travis Michael J. Welsh (§)

From the Howard Hughes Medical Institute, Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel contains two cytoplasmic nucleotide-binding domains (NBDs). After phosphorylation of the R domain, ATP interacts with the NBDs to regulate channel activity. To learn how the NBDs regulate channel function, we used the patch-clamp technique to study CFTR and variants which contained site-directed mutations in the conserved Walker A motif lysine residues in either NBD1 (K464A), NBD2 (K1250A and K1250M), or both NBDs simultaneously (K464A/K1250A). Studies in related proteins suggest that such mutations slow the rate of ATP hydrolysis. These mutations did not alter the conductive properties of the channel or the requirement for phosphorylation and ATP to open the channel. However, all mutations decreased open state probability. Mutations in NBD1 decreased the frequency of bursts of activity, whereas mutations in NBD2 and mutations in both NBDs simultaneously prolonged bursts of activity, as well as decreased the frequency of bursts. These results could not be attributed to altered binding of nucleotide because none of the mutants studied had reduced 8-N(3)ATP binding. These data suggest that the two NBDs have distinct functions in channel gating; ATP hydrolysis at NBD1 initiates a burst of activity, and hydrolysis at NBD2 terminates a burst.


INTRODUCTION

The cystic fibrosis transmembrane conductance regulator (CFTR) (^1)is a Cl channel with novel regulatory mechanisms that are attributed to the cytoplasmic R domain and two cytoplasmic nucleotide-binding domains (NBDs) (for reviews, see (1) and (2) ). Phosphorylation of the R domain by cAMP-dependent protein kinase (PKA) or by protein kinase C is required for the channel to open(3) . Once the R domain is phosphorylated, intracellular ATP interacts with the NBDs to regulate channel activity. The NBDs of CFTR share sequence similarity with the NBDs in a family of proteins called either the traffic ATPases or ATP Binding Cassette (ABC) transporters(4, 5) . Data from many members of the ABC transporter family have shown that the NBDs hydrolyze ATP to drive active transport of substrate across cell membranes. In CFTR, biochemical (6, 7, 8, 9) and functional (10) studies have shown that the NBDs interact directly with ATP and its analogs. Although CFTR forms a Cl channel in which substrate (ion) flow is passive rather than active, functional studies indicate that hydrolyzable nucleoside triphosphates and divalent cations are required for the channel to open(11, 12) . These considerations suggest that the NBDs of CFTR control channel gating by hydrolyzing ATP.

A notable feature of CFTR and many members of the ABC transporter family is the presence of two NBDs. In CFTR, the two NBDs share sequence similarity in certain conserved regions such as the Walker A (GXXGXGKT/S) and Walker B (R/KXh(4)D) motifs (where X refers to any amino acid and h refers to a hydrophobic residue) and in a short sequence (LSGGQ) that has been called a linker region by Ames (13) and which shares some similarity to sequences in G proteins(4) . However, the overall amino acid homology between the two NBDs of CFTR is only 29%. The difference in primary structure, as well as functional studies(10) , suggest that the two NBDs may have different functions. But how ATP controls channel opening and closing through the NBDs and what roles each of the two NBDs play in channel regulation is not known.

To better understand the function of each of the NBDs, we studied CFTR Cl channels containing mutations of the lysine in the Walker A motif. The importance of the Walker lysine is reflected by the fact that it is absolutely conserved in members of the ABC transporter family(4, 5) . Crystallographic and NMR studies of adenylate kinase have suggested that the Walker lysine comes into contact with either the alpha- or -phosphate of the bound ATP (14, 15, 16) and is important for hydrolytic function(16) . Functional studies of many ATP binding and hydrolyzing enzymes demonstrate that mutation of the invariant Walker lysine decreases the rate of ATP hydrolysis, while often maintaining ATP binding and the ability to undergo ATP binding-induced conformational changes. For example, mutation of the Walker lysine in adenylate kinase to methionine decreased k over a thousandfold with only a slight change in K, and a very small change in DeltaG, suggesting that these changes were not due to destabilization of overall structure (17) . Experiments with a member of the ABC transporter family, the mdr1 gene product, demonstrated that mutation of the Walker lysine in either of the two NBDs individually or simultaneously abolished drug resistance without altering binding to 8-N(3)ATP(18) .

These results suggested that mutation of the Walker lysines in CFTR may reduce the rate of ATP hydrolysis (if it occurs) without altering the ability of the protein to assume a normal tertiary structure, bind ATP, and undergo ATP binding-induced conformational changes. To investigate the role of each NBD in CFTR channel regulation, we studied CFTR variants in which the conserved Walker lysines in NBD1 (Lys) and NBD2 (Lys) were mutated either individually or simultaneously to either alanine or methionine. We studied these variants using the whole cell and excised inside-out configurations of the patch-clamp technique.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

CFTR mutants were constructed in the vaccinia virus expression plasmid pTM-CFTR4 (19) by the method of Kunkel(20) . A mutant containing two alterations was generated by simultaneously, including two mutagenic oligonucleotides in the reaction. Single letter amino acid abbreviations are used to refer to the residues mutated. For example, in the mutant K464A, the lysine residue at position 464 in wild-type CFTR is replaced with alanine. Mutants were verified by restriction enzyme analysis, DNA sequencing around the site of mutation, and in vivo expression. Some functional aspects of K464A, K1250M, K1250A, and DeltaR-S660A function have been described previously(10, 21, 22) .

Cells and Transfection Procedure

Wild-type CFTR was studied in either stably transfected NIH 3T3 fibroblasts, C127 mouse mammary epithelia cells, or transiently transfected HeLa cells as described previously(10) . Similar results were obtained with all three cell types, and the data are combined. Mutant CFTR was studied in transiently transfected HeLa cells with the vaccinia virus/bacteriophage T7 hybrid expression system as described previously (23, 24) . Briefly, cells were infected with a recombinant viruses (10-20 multiplicity of infection each), containing the bacteriophage T7 RNA polymerase driven by a viral promoter. A second recombinant virus containing CFTR cDNA driven by a T7 promoter was added simultaneously, or a plasmid containing the T7 promoter was transfected using Lipofectin (Life Technologies, Inc.) 1 h after infection. HeLa cells were plated at approximately 5 times 10^4 cells/cm^2 on collagen-coated glass coverslips 24 h before infection and were studied 18 h after infection. All mutants produced fully glycosylated mature protein (band C) as assessed by immunoprecipitation and phosphorylation.

Patch-clamp Technique

Methods for excised, inside-out, and whole cell patch-clamp recording are similar to those described previously(11, 24, 25) . An Axopatch 200 amplifier (Axon Instruments, Inc.) was used for voltage clamping and current amplification. A microcomputer (IBM AT compatible) and the pClamp software package (Axon Instruments, Inc.) were used for data acquisition and analysis. Data were recorded on videotape following pulse code modulation using a PCM-2 A/D VCR adapter (Medical Systems Corp., Greenvale, NY). Patch pipettes were fabricated as described(24) , with seal resistance routinely greater than 5 gigaohms. Voltages are referenced to the extracellular side of the membrane. Whole cell and excised macropatch experiments were performed at a holding potential of -40 mV; single channel data were recorded at a holding potential of -80 mV. Experiments were conducted at 34-36 °C using a temperature-controlled microscope stage (Brook Industries, Lake Villa, IL).

For whole cell and excised macropatch data, replayed records were filtered at 1 kHz using a variable 8-pole Bessel filter (Frequency Devices Inc., Haverhill, MA) and digitized at 2 kHz. For single channel analysis, replayed data were filtered at 1 kHz using a variable 8-pole Bessel filter, digitized at 5 kHz, and digitally filtered at 500 Hz. Idealized records were created using a half-height transition protocol; transitions less than 1 ms in duration were not included in the analysis.

Single channel open and closed time histograms were plotted with a logarithmic x axis with 10 bins/decade, and the maximum likelihood method was used to fit a one or two component exponential function, respectively. Burst duration data were plotted as histograms with a logarithmic x axis with 10 bins/decade and were fit with both one and two component exponential functions using the maximum likelihood method, with a lower fitting limit of 2.5 ms. To determine if the two component function fit statistically better than a one component fit, the log likelihood ratio test was used and considered significant at a value of 2.0 or greater(26) .

Burst analysis was performed with a t(c) (the time which delineates intraburst from interburst closures) of 20 ms using pClamp 6.0 software. This value was derived from analysis of single channel closed time histograms (see ``Results,'' Fig. 5A) and by the method of Sigurdson et al.(27) . Closures longer than 20 ms were considered to define gaps between bursts, whereas closures shorter than this time were considered gaps within bursts. In experiments where the membrane patch contained five or fewer active channels, bursts in which only one channel was open, which were separated from other bursts by greater than 20 ms, and which had no superimposed openings were included in the analysis. There was no statistical difference between burst durations derived from patches with only one active channel compared with patches with more than one active channel, nor was there a consistent trend toward an increase or decrease of burst values by inclusion of data from patches with more than one channel.


Figure 5: Burst analysis of wild-type CFTR and each Walker lysine mutant. A, closed time histogram of wild-type CFTR. Data are from an experiment in which the membrane patch had only one active channel, studied in the presence of 1 mM ATP and 75 nM catalytic subunit of PKA. Data are plotted with a logarithmic x axis and linear y axis. Burst duration delimiter, t(c), was determined as described under ``Results.'' B, mean channel burst duration for wild-type CFTR and each Walker lysine mutant. All measurements were made in the presence of 1 mM ATP and 75 nM PKA with the membrane voltage clamped at -80 mV. Burst durations were determined with a t(c) of 20 ms, as described under ``Experimental Procedures.'' For wild-type, K464A, K1250M, K1250A, and K464A/K1250A, n = 15, 12, 5, 16, and 6, respectively; p < 0.01 relative to wild type for all.



Results are means ± S.E. of n observations. Statistical significance was assessed using the log likelihood ratio test or a paired or unpaired Student's t test as appropriate.

[alpha-P]8-N(3)ATP Photolabeling

Photolabeling of membrane-associated CFTR was performed as described previously(9) , except CFTR was expressed in HeLa cells grown on 150-mm dishes. Membranes were prepared by differential centrifugation and resuspended in 20 mM HEPES, pH 7.5, 50 mM NaCl, 3 mM MgSO(4), with 2 µg/ml each of leupeptin, aprotinin, and pepstatin. Membrane-associated CFTR was phosphorylated prior to photolabeling by incubation with 100 nM PKA and 0.1 mM ATP in buffer containing 10 mM MgCl(2) and 50 mM PIPES, pH 6.8, for 20 min at 30 °C, then diluted in 12 volumes of buffer containing 20 mM Tris, pH 7.5, 0.2 mM EGTA, and 1 µM calyculin A. Membranes were collected by centrifugation and resuspended at a concentration of 2.5 mg of protein/ml in the same buffer.

Photolabeling was performed by preincubating membranes (50 µg of membrane protein/sample) on ice with the indicated amount of [alpha-P]8-N(3)ATP (6-12 Ci/mmol) diluted in 20 mM HEPES, pH 7.5, 50 mM NaCl, 3 mM MgSO(4), with 2 µg/ml each of leupeptin, aprotinin, and pepstatin in a total volume of 30 µl. After UV irradiation for 60 s, CFTR was solubilized and immunoprecipitated as described (9, 28) using antibodies raised against the R domain (M13-1, 0.5 µg/sample) and against the C terminus (M1-4, 5 µg/sample). Immunocomplexes were analyzed by SDS-polyacrylamide gel electrophoresis and incorporation of [alpha-P]8-N(3)ATP was quantitated with an AMBIS radioanalytic imaging system (AMBIS Systems, Inc., San Diego, CA).

Chemicals and Solutions

Catalytic subunit of PKA was obtained from Promega Corp. (Madison WI). [alpha-P]8-N(3)ATP was from ICN Radiochemicals (Irvine, CA). Adenosine 5`-triphosphate (ATP; disodium salt), 5`-adenylylimidodiphosphate (AMP-PNP, lithium salt), and all other reagents were from Sigma.

For experiments with excised inside-out membrane patches, the pipette (extracellular) solution contained (in mM): 140 N-methyl-D-glucamine, 100 aspartic acid, 35.5 HCl, 5 CaCl(2), 2 MgCl(2), 10 HEPES, pH 7.3, with 1 N NaOH. The bath (intracellular) solution contained (in mM): 140 N-methyl-D-glucamine, 135.5 HCl, 3 MgCl(2), 10 HEPES, 4 cesium, and 1 EGTA, pH 7.3, with 1 N HCl ([Ca] < 10M). For whole cell experiments, pipette (intracellular) solution contained (in mM): 120 N-methyl-D-glucamine, 115 aspartic acid, 3 MgCl(2), 4 cesium, 1 EGTA, and 1 mM Na(2)ATP, pH 7.3, with 1 N HCl ([Ca] < 10M). Bath (extracellular) solution contained (in mM): 140 NaCl, 10 HEPES, 1.2 MgSO(4), 1.2 CaCl(2), and 30 mM sucrose, pH 7.3, with 1 N NaOH.


RESULTS AND DISCUSSION

Effect of Walker A Motif Lysine Mutations on Channel Gating

Previous reports have shown that the CFTR variants K464A, K1250M, and K1250A produced functional channels(10, 22) . To learn whether a variant containing mutations in both Walker lysines could function, we studied K464A/K1250A. Cells expressing K464A/K1250A produced cAMP-activated Cl currents that retained many hallmarks of CFTR channel activity. Whole cell currents were time- and voltage-independent (Fig. 1A). Excised membrane patch experiments showed that K464A/K1250A required PKA phosphorylation and intracellular Mg and ATP for activity (Fig. 1B) and had ion conduction properties identical to that of wild-type CFTR (Fig. 1C). These findings suggested that mutation of both Walker lysines did not cause global alterations in CFTR channel structure or a loss of the normal requirements for function.


Figure 1: Properties of K464A/K1250A in transiently transfected HeLa cells. A, whole cell currents recorded under basal conditions, 2 min after addition of cAMP agonists (10 µM forskolin, 100 µM isobutylmethylxanthine, and 250 µM 8-(4-chlorophenylthio)adenosine-3`,5`cyclic monophosphate) to the bath solution, and 5 min after removing cAMP agonists. Voltage pulse protocol is shown. Each trace is the average of three voltage steps. B, time course of current from an excised membrane patch. Each point is average current during 1 s of data collection, with one point collected every 5 s. Bars indicate presence of ATP (1 mM), PKA (75 nM), and absence of Mg in bath (cytosolic) solution. The Mg-free solution contained 1 mM EDTA. C, single channel current-voltage relationship. Single channel conductance was unchanged relative to wild-type (9.7 ± 0.7 picosiemens, n = 4 versus 10.1 ± 0.4 picosiemens, n = 5, respectively). Data are mean ± S.E., n = 4, except +100 mV, where n = 2. Some error bars are hidden by data symbols.



However, mutation of the Walker A lysines did alter channel gating. Fig. 2shows examples of single channel tracings. The top tracing shows the characteristic pattern of gating in wild-type CFTR, with short bursts of activity (in which the channel flickers open and closed) separated by longer closings. In comparison, the duration of the closed intervals between bursts of activity was considerably increased in variants containing mutations of the Walker lysines. More strikingly, the duration ofbursts was much prolonged in variants containing mutation of Lys.


Figure 2: Single-channel traces of wild-type CFTR, and each Walker lysine mutant studied in excised, inside-out membrane patches from transiently transfected HeLa cells. All measurements were made in the presence of 1 mM ATP and 75 nM PKA with membrane potential clamped at -80 mV. Each tracing is 20 s long. Dashed lines represent channel closed state, and downward deflections correspond to channel openings. For purpose of illustration, traces were digitized at 2 kHz and filtered at 200 Hz and chosen to illustrate changes in closed times between bursts and burst duration and not changes in single channel open state probability (P(o)).



To quantitate these changes and to determine how these mutations altered CFTR Cl channel gating, we analyzed single channel open state probability (P(o)), single channel open and closed times, and the duration of bursts. Fig. 3shows that in the presence of 1 mM ATP and 75 nM PKA, all of the variants had a reduced P(o) compared with that of wild-type CFTR (0.44 ± 0.02, n = 15). Mutation of NBD1 decreased P(o) more than mutations in NBD2, although the difference did not achieve statistical significance between K464A and K1250A. Interestingly, when the K464A and K1250A mutations were combined in the same molecule (K464A/K1250A), P(o) (0.27 ± 0.05, n = 7) was greater than that observed with the K464A mutation alone (0.13 ± 0.02, p = 0.002, n = 12).


Figure 3: P for wild-type CFTR and each Walker lysine mutant. All measurements were made in the presence of 1 mM ATP and 75 nM PKA with membrane potential clamped at -80 mV. For wild-type CFTR, K464A, K1250M, K1250A, and K464A/K1250A, n = 15, 12, 6, 15, and 9. Asterisks indicate statistical significance (p < 0.001) relative to wild type.



To determine how these mutations decreased P(o), we analyzed open and closed time histograms from experiments in which the membrane patch contained only one channel. As we have described previously, open and closed time histograms were best fit with one and two component fits, respectively(29) . The open time constant ((o)), which describes the openings within a burst of activity, decreased to a roughly similar extent for all the mutants (Fig. 4A). In addition, the fast closed time constant (), which represents the brief closures within a burst of activity, approximately doubled in all the mutants (Fig. 4B).


Figure 4: Open- and closed-time constants from patches of membrane containing one channel. Histograms were plotted and fit as described in ``Experimental Procedures''. (o) is the open time constant; and are the fast and slow time constants from the closed time histogram, respectively. Data for mutants K1250A and K1250M were combined (K1250A/M; n = 1 and 2, respectively). For wild-type, K464A, and K464A/K1250A, n = 6, 6, and 4, respectively. Asterisks indicate statistical significance (p < 0.05) relative to wild type.



Although alteration of these fast events contributed to the decrease in P(o), a much more pronounced effect resulted from an increase in the slow closed time constant (), which describes the long closures between bursts of activity (Fig. 4C). The NBD1 mutation increased 5-fold, whereas similar mutations in NBD2 or both NBDs together produced a 30-50-fold increase. The finding that channels with a mutation in NBD2 have a similar or higher P(o) than K464A despite the fact that they remained closed longer is explained by the observation that the bursts of activity in channels with a mutated NBD2 are much longer than those of wild-type or K464A (see Fig. 2).

To quantitate how the Walker lysine mutations were altering the duration of channel opening bursts, burst analysis was performed using a t(c) (the time which separates interburst closures from intraburst closures) of 20 ms. This value was derived from analysis of wild-type CFTR closed time histograms derived from excised inside-out membrane patches containing a single channel studied in the presence of 1 mM ATP plus PKA. When plotted with a logarithmic x axis (Fig. 5A), the two closed states appear as distinct populations and the time constants differ by greater than 2 orders of magnitude (average fast closed time constant, , = 1.79 ± 0.22 ms, average slow closed time constant, , = 187 ± 13 ms, n = 6). The burst delimiter, t(c), was chosen as the nadir between the two populations of closures. The large difference between time constants suggests that misclassification errors made when defining bursts should be small(30) . A value of 20 ms was also derived using the method of Sigurdson et al.(27) .

Fig. 5B shows the effect of Walker lysine mutations on channel burst duration. K464A had a small but significant decrease in burst duration relative to that of wild-type channels (136 ± 7 ms for K464A versus 210 ± 22 ms for wild-type, p = 0.007). In contrast, both variants with mutation of the Walker lysine in NBD2 had a 4-5-fold increase in the burst duration relative to that of wild-type (881 ± 96 and 1118 ± 217 ms for K1250A and K1250M, respectively, p < 0.001 for wild-type versus either NBD2 mutant). Interestingly, when the Walker lysines in both NBDs were mutated (K464A/K1250A), the longer burst duration conferred by mutation of K1250 was dominant over the shorter burst conferred by K464A (1101 ± 310 ms versus 136 ± 7 ms for K464A, p < 0.001).

These results suggest that the NBDs are important not only in opening the channel, but also in closing the channel, specifically in ending bursts of activity. Because mutations of the Walker lysines are expected to slow the rate of ATP hydrolysis, and because such mutations in NBD2 markedly slowed the rate at which bursts of activity were terminated, we interpret these data to suggest that termination of a burst is an active event, mediated by NBD2, which involves ATP hydrolysis.

Effect of Mutating the Walker A Motif Lysines on 8-N(3)ATP Binding

Mutation of the Walker lysine slows the rate of hydrolysis in proteins that hydrolyze ATP. Although such mutations do not alter nucleotide affinity is some systems, in others they have produced either a decreased or increased affinity. We considered the possibility that mutation of the Walker lysines in CFTR might alter ATP binding affinity and thereby alter gating. Therefore, we measured incorporation of the photoreactive ATP analog 8-N(3)ATP into membrane-associated wild-type and mutant CFTRs. Our previous studies showed that membrane-associated wild-type CFTR was labeled in a specific and saturable way by 8-N(3)ATP, that 8-N(3)ATP supported channel function, and that ATP competed with 8-N(3)ATP(9) , suggesting that 8-N(3)ATP binding is an appropriate assay of the functional ATP binding sites of CFTR.

Mutation of both Walker lysines to alanine, or of Lys to methionine, did not significantly alter photolabeling by 8-N(3)ATP relative to that of wild-type (Fig. 6). Because we were not able to quantitate the amount of CFTR present, we could not determine the stoichiometry of binding. However, the absolute amounts of radiolabel incorporated were similar, suggesting that mutation of the Walker lysines did not change the number of ATP binding sites. These data suggest that the change in gating observed in the Walker lysine mutants was not due to an altered affinity of CFTR for ATP, but instead due to a change in some other intrinsic activity of the NBDs, perhaps ATP hydrolysis.


Figure 6: Binding of 8-N(3)ATP to wild-type and variant CFTR. Binding of [alpha-P]8-N(3)ATP to membrane-associated wild-type CFTR, K464A/K1250A, and K1250M, n = 9, 11, and 3, respectively. Data are expressed as amount of radiolabel incorporated relative to the amount bound at 20 µM to correct for variations in [alpha-P]8-N(3)ATP specific activity among experiments. A similar amount of photolabel incorporation was observed for all groups. Immunoprecipitation and phosphorylation suggest that similar amounts of wild-type or mutant protein were present in each reaction.



Effect of AMP-PNP on Channel Gating

If the speculation that termination of a burst of activity requires ATP hydrolysis at NBD2 is correct, then we would predict that a nonhydrolyzable analog of ATP, such as AMP-PNP, might also interact with NBD2, inhibit the hydrolysis step, and prolong the duration of bursts. Previous studies have shown that AMP-PNP cannot support channel activity on its own(12, 24) . However, Quinton and co-workers (31, 32) have shown that AMP-PNP in the presence of ATP and cAMP agonists can increase Cl current through the apical membrane in sweat gland duct and T84 intestinal epithelia. In addition, Hwang et al.(33) have reported that AMP-PNP could increase the activity of CFTR Cl channels in guinea pig cardiac myocytes when the channels were studied in the presence of ATP plus PKA at 25 °C. Therefore, we tested the hypothesis that AMP-PNP would prolong the burst duration when it was added in the presence of ATP and PKA.

When we added 1 mM AMP-PNP to excised membrane patches, most openings appeared to be of normal duration (Fig. 7A). However, there were occasional openings that had a substantially prolonged burst duration, similar to those observed in K1250A, K1250M, and K464A/K1250A. This observation was confirmed by an analysis of burst duration histograms. Prior to addition of AMP-PNP, there was only one population of bursts (Fig. 7B), since in five of six experiments a two component exponential function did not fit the data statistically better than a one component fit. After addition of AMP-PNP, a second longer, and statistically distinct population of bursts was evident in six of six experiments (Fig. 7C). Although this second population represented a small fraction of the total number of bursts, each individual burst within this population was substantially prolonged (note the log scale in Fig. 7, B and C). As a result, the mean burst duration (Fig. 7D) approximately doubled (p = 0.033) and P(o) increased 35% (p = 0.006) (Fig. 7E).


Figure 7: Effect of AMP-PNP on wild-type CFTR channel activity. A, current traces from a stably transfected C127 membrane patch with only a single active channel; data are plotted as in Fig. 3. The presence of both ATP (0.3 mM) and PKA were necessary to see the effect of AMP-PNP (1 mM). B and C, burst duration histograms of wild-type CFTR activity in the absence (B) and presence (C) of 1 mM AMP-PNP. Data for each histogram are derived from successive interventions in one experiment and plotted and fit as described under ``Experimental Procedures.'' Separate populations of bursts appear as different peaks. The solid line is the superimposed maximum likelihood fit. D and E, effect of 1 mM AMP-PNP on burst duration (D) and P(o) (E). Asterisks indicate statistical significance, p = 0.033 and p = 0.006, for burst and P(o), respectively; n = 6.



A Model of ATP Regulation of CFTR by the NBDs

Our data allow us to assign very different functions to the two NBDs, and we conclude that hydrolysis is involved in both channel opening as well as closing. To interpret our present data, as well as previous observations, we propose the model shown in Fig. 8, in which each of the NBDs have distinct functions in channel gating. Note that this model refers to the gating of CFTR by the NBDs after the R domain has been phosphorylated. The model shows the interaction of ATP with NBD1, with NBD2, and the resulting effect on channel gating (on the right). Four states in the gating cycle are shown.


Figure 8: Model of the interaction of the NBDs of CFTR with ATP and the effect on channel gating. Vertical columns from left to right represent events at NBD1, at NBD2, and the effect on channel opening and closing. ``C'' and ``O'' represent channel open and closed states, respectively. See text for a detailed description.



In state 1, no ATP is bound to the NBDs and the channel is closed. The requirement of ATP for the channel to open supports this conclusion.

In state 2, ATP binds to both NBDs, but the channel remains closed. We conclude that ATP binding alone is not sufficient to open the channel based on two types of observation. First, biochemical (9) and functional studies (31, 33, and our present data) have shown that nonhydrolyzable analogs can bind to and interact with the channel, but in the absence of ATP, they are not capable of opening it(11, 12, 24) . Second, biochemical studies have shown that nucleoside triphosphates can bind to the channel in the absence of Mg(6, 8, 9) , yet Mg is required for channel opening(11) . These results suggest that a step subsequent to ATP binding is required to open the channel.

In state 3, hydrolysis has occurred at NBD1, driving the channel into a bursting state. Within a burst, the channel flickers open and closed. We suggest that hydrolysis at NBD1 opens the channel, because mutation of K464 slows the rate of opening; in K464A the duration of the closed state between the bursts of activity was increased 5-fold. Our finding that mutation of Walker lysines did not alter binding of 8-N(3)ATP, and the fact that mutation of Walker lysines in the NBDs of many other ATPases slows the rate of hydrolysis, support the speculation that the transition from a closed to bursting state requires hydrolysis at NBD1.

In state 4, hydrolysis has occurred at NBD2, actively terminating the burst and closing the channel. Mutation of the Walker lysine in NBD2 will likely slow hydrolysis, delay the exit from state 3, and thereby increase the burst duration. Assigning hydrolysis at NBD2 to occur after that at NBD1 also explains the observation that mutations of Lys are dominant in determining burst duration. When both Lys and Lys are mutated in the same protein, the burst duration is the same as that observed in K1250A or K1250M. That is, once the channel is open in a bursting mode, an active step is required to close it.

Our observation that AMP-PNP prolongs the burst duration in a manner similar to mutations in NBD2 suggests that AMP-PNP is binding to NBD2 and supports the conclusion that hydrolysis is required to terminate a burst and close the channel. AMP-PNP appeared to be much less potent than ATP at interacting with CFTR as suggested by binding studies (AMP-PNP had approximately 5% the binding potency of ATP) (9) and by the data in Fig. 8C (which shows that the fraction of bursts with a prolonged duration was small). Nevertheless, once bound, the effect on burst duration was substantial.

In addition to the direct role of each NBD in opening and closing the channel, we speculate that there must also be some form of cross-talk between the NBDs to account for the finding that NBD2 mutations decrease the rate of channel opening. We indicate this in Fig. 8by the arrow from NBD2 to NBD1. Thus, it appears that normal function at NBD2 is necessary for normal function at NBD1, and it appears that events at NBD2 may regulate NBD1 function. In a similar way, NBD1 may also influence NBD2 function. Although we have not indicated it in Fig. 8because the effect is small, this is suggested by the observation that mutation of Lys slightly decreased burst duration. Overall, these results suggest that although each NBD may have a discrete function in channel gating, the function of one NBD may influence or modulate the function of the other.

In addition to the steps shown in the model, the channel might have additional states where there is either a phosphorylated intermediate, an ADP-bound state, or other conformational states which we cannot yet resolve and have therefore not included. Other steps involving release or removal of bound nucleotide may also occur in recycling of the channel from state 4 to state 1, but our studies do not address these steps. In addition, this model does not take into account the effect of different phosphorylation states on channel activity.

We previously suggested that hydrolysis is required for channel opening (11) . Our current findings support that conclusion. Gadsby and colleagues (33, 34) recently proposed that hydrolysis is not only involved in channel opening but that hydrolysis at one of the NBDs may be involved in closing. Our current findings support their conclusion to the extent that the data suggest that hydrolysis at one of the NBDs (NBD2) closes the channel by terminating a burst of activity. However, Baukrowitz et al.(34) concluded that hydrolysis at the NBD involved in closure only occurs at high phosphorylation states (i.e. in the presence of PKA). We found that the NBD2 mutants always prolonged CFTR bursts, even in the absence of PKA (n = 13, not shown), suggesting that NBD2 always participates in closing the channel. We previously reported that the N-terminal half of CFTR (which lacks NBD2) could produce functional channels(35) . Unfortunately that result does not address whether or not a single NBD (NBD1) is sufficient for normal gating, because the active channel was probably a dimer in which a second NBD1 could substitute for NBD2. The requirement for NBD2 function has also been suggested by the finding of Rich et al.(36) who found that a CFTR construct in which NBD2 was deleted was properly localized to the plasma membrane, but was not functional. Clearly, more studies are required to resolve this issue.

The model shown in Fig. 8does not explain some aspects of CFTR gating. One such aspect is the requirement for the presence of PKA to see a stimulatory effect of AMP-PNP. We and others (11, 12, 24) previously reported that when channels had been phosphorylated, but were studied in the absence of PKA, AMP-PNP had no effect. Using different preparations, Quinton (31, 32) and Hwang (33) found that the presence of either cAMP agonists or PKA was required to see an effect of AMP-PNP. These results suggest that PKA phosphorylates a labile site which in some way alters the ability of AMP-PNP to interact functionally with the NBDs. If AMP-PNP bound to, and prevented hydrolysis at NBD1, then we might have expected to observe an increase in the frequency of long closures between bursts. However that was not observed. Perhaps the affinity of AMP-PNP for NBD1 is less than that for NBD2; this difference might be assessed by biochemical binding experiments. Alternatively, the affinity of AMP-PNP for both NBDs may be equal, but an infrequent prolongation of the interval between bursts may well be within the range observed in the absence of AMP-PNP.

Another uncertainty of the model is that it assumes channel opening and closing require hydrolysis of ATP. Our current findings and previous data are consistent with the notion that these domains hydrolyze ATP. In fact, it would be difficult to explain the data if hydrolysis did not occur. Moreover, several other members of the ABC transporter family have been shown to hydrolyze ATP. However, at present there have been no biochemical studies that have directly demonstrated hydrolysis by CFTR, and therefore we cannot completely rule out mechanisms which do not involve hydrolysis.

The model shown in Fig. 8predicts that NBD2 bound to nucleoside triphosphate (state 3) is the active state and that hydrolysis to nucleotide diphosphate causes the transition to an inactive channel (state 4). This scheme is reminiscent of the way that G proteins function. In G proteins, the nucleoside triphosphate (GTP) bound state is the active state, and the protein remains active as long as bound nucleoside triphosphate remains unhydrolyzed (reviewed in (37) ). Once hydrolysis occurs, the G protein with bound nucleoside diphosphate (GDP) becomes inactive and remains inactive until GDP is replaced with GTP. In this way, the hydrolysis of GTP acts as a timing mechanism. When the rate of hydrolysis of GTP is reduced, as occurs in oncogenic ras mutations, the protein remains in the active state for a prolonged duration. Likewise, binding of nonhydrolyzable GTP analogs, such as GMP-PNP, produce a prolonged activation. These findings appear to parallel what we observed with Walker lysine mutations in NBD2 (functionally analogous to ras mutations) and with AMP-PNP (analogous to GMP-PNP). With both interventions, exit from the active state was delayed and the duration of bursts of activity was prolonged. This analogy and speculation suggests the intriguing possibility that other domains of CFTR, or other proteins, might act in a manner similar to the guanine nucleotide exchange factors or GTPase-activating factors (GAPs) to modulate CFTR Cl channel activity.

The suggestion that NBD2 might have a function similar to that of a G protein might also explain the effect of ADP. Our previous data indicated that ADP was a relatively potent inhibitor, prolonging the duration of the long closed state between bursts(29) . Mutation of Lys to methionine abolished inhibition by ADP, suggesting that ADP-dependent inhibition was mediated through NBD2. By analogy, in G proteins nucleoside diphosphate (GDP) potently inhibits activity by binding to the protein and thereby limiting the rate of nucleotide exchange(38, 39) . Thus, we speculate that in CFTR, ADP bound to NBD2 in state 4 might prevent the transition back to state 1 and then on to states 2 and 3.

In summary, the data indicate that the two NBDs in CFTR have different functions in controlling channel opening and channel closing and suggest that hydrolysis of ATP may be necessary for both processes. The model we have proposed explains many aspects of the data, but more importantly, it provides a framework from which future experiments can be designed to further elucidate the novel regulation of CFTR.


FOOTNOTES

*
This work was supported in part by the Howard Hughes Medical Institute, the NHLBI, and the Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242.

(^1)
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain; PKA, cAMP-dependent protein kinase; PIPES, 1,4-piperazinediethanesulfoninc acid; AMP-PNP, 5`-adenylylimidodiphosphate; GMP-PNP, 5`-guanylylimidodiphosphate


ACKNOWLEDGEMENTS

We thank Dr. David Gadsby for providing preprints prior to publication, Dr. John Marshall and Dr. Seng Cheng for the gift of M13-1 antibody, the University of Iowa DNA Core facility for oligonucleotide synthesis and DNA sequencing, L. G. DeBerg for the construction of mutants, S. P. Weber, P. H. Karp, S. R. Struble, J. A. Cieslak and E. C. Kasik for excellent technical assistance, T. A. Mayhew and D. R. Vavroch for secretarial assistance, and our laboratory colleagues for their advice and critical comments.


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