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J. Biol. Chem., Vol. 275, Issue 38, 29407-29412, September 22, 2000

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Differences between Cystic Fibrosis Transmembrane Conductance Regulator and HisP in the Interaction with the Adenine Ring of ATP^*

Allan L. Berger and 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

Received for publication, June 2, 2000, and in revised form, July 10, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel is a member of the ATP-binding cassette transporter family. The most conserved features of this family are the nucleotide-binding domains. As in other members of this family, these domains bind and hydrolyze ATP; in CFTR this opens and closes the channel pore. The recent crystal structures of related bacterial transporters show that an aromatic residue interacts with the adenine ring of ATP to stabilize nucleotide binding. CFTR contains six aromatic residues that are candidates to coordinate the nucleotide base. We mutated each to cysteine and examined the functional consequences. None of the mutations disrupted channel function or the ability to discriminate between ATP, GTP, and CTP. We also applied [2-(triethylammonium)ethyl] methanethiosulfonate to covalently modify the introduced cysteines. The mutant channels CFTR-F429C, F430C, F433C, and F1232C showed no difference from wild-type CFTR, indicating that either the residues were not accessible to modification, or cysteine modification did not affect function. Although modification inactivated CFTR-Y1219C more rapidly than wild-type CFTR, and inactivation of CFTR-F446C was nucleotide-dependent; failure of these mutations to alter gating suggested that Tyr¹²¹⁹ and Phe⁴⁴⁶ were not important for nucleotide binding. The results suggest that ATP binding may not involve the coordination of the adenine ring by an aromatic residue analogous to that in some bacterial transporters. Taken together with earlier work, this study points to a model in which most of the binding energy for ATP is contributed by the phosphate groups.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CFTR¹ is a regulated epithelial Cl channel (1) that belongs to the ATP-binding cassette (ABC) transporter family of membrane proteins (2, 3). The conserved features of this family are two membrane-spanning domains, usually with six transmembrane sequences each and two cytoplasmic nucleotide-binding domains (NBD). CFTR also contains a unique regulatory (R) domain. The membrane-spanning domains show topological similarity but little or no sequence similarity between different ABC transporters. In contrast, the NBDs show significant sequence conservation throughout the family.

In CFTR, the membrane-spanning domains form an anion-selective pore (4). The channel can open after the R domain is phosphorylated. Then ATP binding and hydrolysis by the two NBDs open and close the pore (5, 6). Previous studies have probed the structure and function of the CFTR NBDs by examining the functional consequences of applying nucleotide analogs and introducing site-directed mutations; those studies indicate that both NBD1 and NBD2 determine channel gating (for reviews see Refs. 5-7). Earlier work has also shown the importance of the highly conserved Walker A and B motifs in the NBDs. In addition, numerous mutations in the NBDs cause cystic fibrosis (8).

An important recent advance in understanding ABC transporters came when the three-dimensional crystal structures of three prokaryotic NBD proteins were solved (9, 10).² The structure of the ATP-binding subunit of the bacterial histidine transporter (HisP) provides a model for the binding of ATP to an NBD (9). ATP binds to the surface of the NBD near an angle formed between the two "arms" of the NBD. Most of the nucleoside is solvent-exposed, and the  phosphate is buried by extensive hydrogen bonding with the Walker A phosphate binding loop (Ser⁴¹ to Lys⁴⁵ and Ser⁴⁶). One residue (His¹⁹) contacts the ribose of ATP. In addition, the side chain of a tyrosine residue (Tyr¹⁶) binds to the adenine ring of ATP through a hydrophobic interaction to bury almost half of the surface area of the nucleotide base (172 Å²). No other residues from HisP contact the adenine ring. Mutational studies of HisP confirm that Tyr¹⁶ plays a critical role in nucleotide binding; mutation of Tyr¹⁶ to Ser abolished both ATP binding and histidine transport (10). The structures of LivF² and the amino-terminal NBD of RbsA (11) show a very similar binding interaction between the NBD and ATP. In both cases, an aromatic residue (phenylalanine) stacks in a hydrophobic interaction with the adenine ring of ATP, and the remainder of the nucleotide base and ribose are solvent-exposed. Moreover, sequence alignment of other ABC transporter NBDs reveals a conserved aromatic amino acid 20-30 amino acids upstream of the start of the Walker A motif, in a relative position similar to that of Tyr¹⁶ in HisP (12-14).

We hypothesized that each NBD of CFTR contains a phenylalanine or tyrosine residue upstream of the Walker A motif that is a contact for ATP binding. We predicted that changing the chemical structure of this side chain would decrease ATP binding and thereby reduce channel activity. To test this hypothesis, we mutated six candidate residues to cysteine (four in NBD1 and two in NBD2) and measured changes in channel activity. We also examined the effect of applying MTSET to the channel to learn whether modification of an accessible cysteine would alter function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- MTSET was purchased from Toronto Research Chemicals Inc. (Ontario, Canada). cAMP-dependent protein kinase was purchased from Promega (Madison, WI). Lipofectin was obtained from Life Technologies, Inc. All other reagents were obtained from Sigma.

Site-directed Mutagenesis and Transfection-- CFTR mutants were prepared in the pTM1-CFTR4 plasmid (15). Mutations were verified by both restriction enzyme analysis and by sequencing around the site of mutation. Wild-type and mutant CFTR proteins were transiently expressed in HeLa cells using the vaccinia virus/T7 expression system as described previously (16). Cells were studied 4-24 h after transfection, depending on the level of expression desired.

Electrophysiology-- Methods for excised, inside-out patch-clamp recordings were as described previously (17). Experiments were performed at 34-37 °C. The pipette (extracellular) solution contained: 140 mM N-methyl-D-glucamine, 100 mM aspartic acid, 35.5 mM HCl, 5 mM CaCl₂, 2 mM MgCl₂, 10 mM TES, pH 7.3, with 1 N HCl. The bath (intracellular) solution contained: 140 mM N-methyl-D-glucamine, 135.5 mM HCl, 3 mM MgCl₂, 10 mM TES, 4 mM Cs(OH)₂, 1 mM EGTA, 1 mM Na₂ATP, pH 7.3, with 1 N HCl. MTSET was added to the cytosolic surface of patches at 200 µM final concentration. MTSET stock solutions were made fresh in ddH₂O and stored on ice for less than 4 h.

Data from patches containing many channels and patches containing 1-3 channels were collected at 40 and 80 mV, respectively. Data were filtered at 1 kHz using a variable eight-pole Bessel filter (Frequency Devices Inc., Haverhill, MA) and digitized at 5 kHz. Open state probability was determined from patches containing 1-3 channels. Data analysis was done using the pClamp 6.0 software package (Axon Instruments Inc., Foster City, CA). Fitting of data from patches containing many channels was performed using IgorPro 2.0 (WaveMetrics, Inc., Lake Oswego, OR). Data are presented as mean ± S.E. for n observations. Statistical significance was determined as indicated in the figure legends. p values <0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Mutation of Residues That May Be Analogous to HisP Tyr¹⁶-- In HisP, Tyr¹⁶ binds the adenine base of ATP (9). To identify aromatic amino acids in CFTR that might have a function analogous to that of Tyr¹⁶, we compared the sequence of CFTR NBD1 and NBD2 to that of HisP (Fig. 1). From the alignment with CFTR NBD1, we identified three potential aromatic homologues for HisP Tyr¹⁶: Phe⁴²⁹, Phe⁴³⁰, and Phe⁴³³. In NBD2, only Tyr¹²¹⁹ was a good homology candidate. Phe⁴⁴⁶ (NBD1) and Phe¹²³² (NBD2) are also upstream of the Walker A domain in the predicted NBD domain. Although the HisP structure suggests that the side chains of Phe⁴⁴⁶ and Phe¹²³² face the protein interior where they stabilize domain structure, a phenylalanine is conserved at this position in CFTR from several species. Because of the sequence conservation, we chose to study these two residues in addition to the more obvious candidates.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of HisP with CFTR NBD1 and CFTR NBD2. The amino-terminal sequence of HisP from S. typhimurium and CFTR NBD1 and NBD2 is shown. Dots indicate gaps that were introduced to optimize alignment. Candidate aromatic residues for binding to the adenine ring of ATP are marked in bold. The Walker A motif is underlined.

To test potential interactions between these residues and ATP, we mutated each to cysteine. When expressed in HeLa cells, CFTR-F429C, F430C, F433C, F446C, Y1219C, and F1232C all generated Cl channel activity in excised, inside-out patches of membrane. Similar to wild-type CFTR, activity for each mutant required both cAMP-dependent protein kinase phosphorylation and ATP (data not shown). All the mutants had an open state probability (P_o) similar to that of wild-type CFTR (Table I). The apparent pattern of single-channel gating in patches with one to three channels was not remarkably different for any of the mutants (not shown). In contrast, previously studied mutations in the NBD Walker A and B motifs reduce P_o by inhibiting nucleotide hydrolysis and possibly binding (17-19), and most current models of CFTR gating predict that nucleotide binding to each NBD is necessary to support activity (6, 18, 20). These data suggest that mutation of the aromatic residues did not markedly disrupt the interaction of ATP with the NBDs. Because mutation of Try¹⁶ in HisP results in a large reduction in ATP binding and HisP function, our data suggest that the aromatic residues in CFTR have a role different from Tyr¹⁶ in HisP.

Effect of ATP, GTP, and CTP on Wild-type and Mutant CFTR-- One potential consequence of altering NBD interactions with the adenine ring of ATP would be a change in the rank order of activity supported by different nucleoside triphosphates. We found that ATP generated more Cl current than GTP or CTP in membrane patches containing multiple wild-type CFTR channel (Fig. 2A). These data are consistent with the results of earlier studies (21). HisP and the histidine transporter complex have a similar specificity (22). If the aromatic residues in CFTR interact with the adenine ring of ATP, mutating these residues could alter the nucleotide specificity of CFTR. However, we found that the three nucleoside triphosphates generated relative amounts of current in the mutants that were similar to that of wild-type CFTR (Fig. 2B). These data indicate that the aromatic residues do not play a key role in the ability of CFTR to discriminate between nucleoside triphosphate species.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Nucleoside triphosphate specificity of CFTR and cysteine mutants. A shows a representative current trace from an inside-out patch containing multiple wild-type CFTR Cl channels. ATP, GTP, and CTP (0.5 mM) were applied to the cytosolic surface during the times indicated by bars. B shows the percentage of current supported by ATP, GTP, and CTP. n = 3 for each.

MTSET Modification of Wild-type and Mutant CFTR-- The location and function of Tyr¹⁶ in HisP indicate that it is solvent accessible. To learn whether the residues we identified in the CFTR NBDs might play a role in CFTR function, we reacted each aromatic to Cys mutant with MTSET. If the candidate residues are accessible, covalent modification of the inserted cysteine would introduce a positive charge and create a local environment substantially different from the hydrophobic aromatic ring of the wild-type residues.

MTSET (200 µM) slowly decreased current of wild-type CFTR in the presence of ATP (Fig. 3), suggesting that the protein contains a cysteine that, when modified, inhibits channel function. Table II shows the apparent rate of inactivation based on an exponential fit to each curve. The NBD1 cysteine mutants were inactivated slowly at rates similar to wild-type (Fig. 3 and Table II). These data suggest that the cysteines introduced in NBD1 (at positions 429, 430, 433, and 446) either were not accessible for MTSET modification or their modification did not alter function (Fig. 3 and Table II). CFTR-Y1219C was more rapidly inactivated than wild-type, and CFTR-F1232C showed a tendency for more rapid inactivation (0.05 < p < 0.10). These data suggest that a cysteine inserted in NBD2 could be modified by MTSET to inhibit the channel, but the effect was small resulting in only a 2-3-fold increase in the reaction rate. If the residue was solvent-exposed, we expect a much more rapid inactivation (see below).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   MTSET inactivation of CFTR and the cysteine mutants. Data are from excised, inside-out membrane patches containing multiple channels. Arrow indicates application of MTSET (200 µM) to the cytosolic surface of the patch. ATP (1 mM) was present throughout. Each data point represents average current over 1 s. The line shown in each panel is a single exponential fit to the data. Rate constants are presented in Table II. WT, wild type.

View this table: [in this window] [in a new window]   Table II Apparent first-order rate constant of MTSET inhibition of CFTR current in the presence of 1 mM ATP Rate constants were determined from patches containing many channels by an exponential fit to the decrease in current following the addition of MTSET (200 µM). Asterisk indicates p < 0.05 compared to wild-type (n  3 for each construct).

Effect of MTSET Applied in the Absence of ATP-- If ATP normally binds an aromatic residue in NBD1 and NBD2, then it might interfere with the ability of MTSET to modify a cysteine at that position. That is, the presence of ATP might reduce access of MTSET to the introduced cysteine. At 1 mM ATP as we used above, the channel is almost maximally active; thus nucleotide occupancy of the NBDs may be high (23, 24), and ATP might have blocked accessibility of the introduced cysteine. Therefore, we tested the ability of MTSET to modify and inactivate the cysteine mutants in the absence of nucleotide. Fig. 4A shows an example of current from each of the cysteine mutants; MTSET was applied for 60 s in the absence of ATP. Upon readdition of ATP, current did not completely return to its starting values. This was due to a combination of current rundown (25, 26), which was also present in the negative control for each channel and MTSET inhibition. A summary of these data is shown in Fig. 4B. Wild-type, CFTR-F429C, F430C, F433C, and F1232C all exhibited similar levels of activity after treatment with 200 µM MTSET in the absence of ATP. We interpret these data to mean that ATP did not compete with the ability of MTSET inactivation of these channels.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   MTSET inactivation of CFTR and cysteine mutants in the absence of ATP. A shows representative current traces for the reaction of each CFTR construct with MTSET in the absence of ATP. Data from inside-out membrane patches were obtained as in Fig. 3. Solid bars indicate the presence of ATP (1 mM), and striped bars indicate the presence of MTSET (200 µM). B shows the percentage of current recovered after incubation in ATP-free solution either without (white bars) or with 200 µM MTSET (black bars) for wild-type and mutant channels. Current measurements were obtained upon readdition of 1 mM ATP as in A. Patches not treated with MTSET were also incubated in ATP-free solution to assess channel rundown. Statistical significance was determined using a one-way ANOVA (analysis of variance) to compare the MTSET treatment results (n  3 for each treatment). Asterisk indicates p < 0.05 compared with the group containing wild-type CFTR.

In these experiments of modification without ATP, MTSET inhibited CFTR-Y1219C to a greater extent than wild-type CFTR and the other NBD mutants (Fig. 4, A and B). However, because MTSET also inactivated CFTR-Y1219C faster than the other channels in the presence of 1 mM ATP (Table II), we expected a greater inhibition during the timed application of MTSET in the absence of ATP. In the presence of ATP (Fig. 3), a 60-s treatment with MTSET reduced CFTR-Y1219C current to 47 ± 7% (n = 8) of the initial value. In the absence of ATP (Fig. 4), a 60-s treatment with MTSET reduced current to 26 ± 12% (n = 6) of the initial value. When we subtract the measured channel rundown of 37 ± 16% for CFTR-Y1219C (because of the time for washing and the absence of ATP), we expected a current that was 30% of the basal value. The close agreement of the measured and predicted values indicates that ATP did not interfere with the ability of MTSET to inactivate CFTR-Y1219C.

To confirm more directly that the rate at which MTSET inactivated CFTR-Y1219C was independent of ATP, we examined the rate of inhibition in the presence of 25 µM ATP. This low ATP concentration is significantly below the EC₅₀ value of ATP for CFTR under the conditions used in this study (21). Thus ATP occupancy of the NBDs should be low. If ATP interferes with MTSET modification and inactivation of CFTR-Y1219C, we expect to see a faster inactivation rate constant. Fig. 5 shows an example. The rate of inactivation for CFTR-Y1219C in 25 µM ATP (140 ± 81 M¹ s¹, n = 4) was ~4 times the rate of inactivation for wild-type CFTR in 25 µM ATP (38 ± 3 M¹ s¹, n = 3), similar to the ratio of CFTR-Y1219C inactivation to wild-type CFTR inactivation in 1 mM ATP. The rates of inactivation for each channel in 25 µM ATP were not significantly different from the rates in 1 mM ATP. These results indicate that ATP did not alter MTSET modification of CFTR-Y1219C.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   MTSET inactivation of wild-type CFTR and CFTR-Y1219C-CFTR in 25 µM ATP. Data were obtained as in Fig. 3. The line shown in each example is a single exponential fit to data. WT, wild type.

In contrast to the other channels, CFTR-F446C showed a more striking inhibition by MTSET in the absence than in the presence of ATP (compare Fig. 4, A and B, with Fig. 3) that was not explained by the rate of inhibition observed in the presence of ATP (Table II). This difference suggested either that nucleotide protected Cys⁴⁴⁶ from modification by MTSET or that binding of nucleotide caused a conformational change in CFTR that protected Cys⁴⁴⁶. To distinguish between these possibilities, we measured MTSET inhibition in the presence of ADP. ADP binds to CFTR NBDs and competes with ATP but does not support channel activity (16, 23, 26, 27). Reaction of CFTR-F446C with MTSET for 60 s in the presence of 1 mM ADP preserved almost all CFTR activity (Fig. 6). These data suggest that ATP and ADP occupancy of the NBD protected the protein from MTSET modification. We interpreted this to mean that Phe⁴⁴⁶ was located near the nucleotide binding site.

View larger version (9K): [in this window] [in a new window]   Fig. 6.   MTSET inactivation of CFTR-F446C. Data were obtained as in Fig. 3, and graphs were generated as described in the legend to Fig. 4. The bar on the right shows reaction with 200 µM MTSET for 60 s in the presence of 1 mM ADP. The asterisk indicates p < 0.05 compared with absence of MTSET (unpaired t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The recent solutions of NBD crystal structures from prokaryotes provide a very important framework for understanding the structure and function of the NBDs of all ABC transporters, (9, 11).² Undoubtedly, the major features of these structures will apply throughout the family, but it is likely that there will be important structural and hence functional differences between individual members of the family. Here we tested one prediction of these structures in CFTR. An aromatic amino acid is conserved upstream of the Walker A motif of most ABC transporters (13, 14). The recent crystal structure of HisP implicates this residue, Tyr¹⁶, as a critical binding contact for the adenine ring of ATP. Mutation of this residue (Y16S) abolished the ability of HisP to bind ATP and consequently disrupted transport (10). In CFTR, we identified aromatic residues that might be analogous to Tyr¹⁶; we found four candidates in NBD1 and two in NBD2. However, mutating these residues to cysteine failed to reduce P_o. These results suggested that these residues do not have an important role in ATP binding.

The location and function of residue 16 in HisP indicates that it is solvent exposed. However, the rates at which MTSET inactivated the NBD1 mutants were not significantly different from wild-type CFTR. Even though CFTR-Y1219C showed a faster inactivation than wild-type CFTR, this difference was small. Importantly, the inactivation rates were very slow relative to the reaction rate of MTSET with free thiols (28). The inactivation rates were also much slower than those measured for cysteines introduced into the Walker A motif or the LSGGQ motif of CFTR (29). There are two interpretations of these data. The residues were not modified, suggesting that they are not solvent-exposed and not readily accessible. Alternatively the residues were modified but there were no major functional consequences. In either case, these results suggest that ATP binding may not involve the coordination of the adenine ring by an aromatic residue analogous to that in HisP, LivF, and RbsA. Although we think it less likely, an aromatic residue could stabilize ATP binding in CFTR; however, that residue would have to be located in a different region of the primary sequence.

CFTR-F446C was the only channel in which nucleotide altered the inactivation rate. In the presence of ATP or ADP, MTSET inactivated the channel at a rate indistinguishable from wild-type CFTR. But in the absence of nucleotide, inactivation was faster. These data suggest that either residue 446 is near the nucleotide binding site and there is competition between MTSET and nucleotide or perhaps there is not direct competition, but nucleotide binding makes residue 446 less accessible. Our data do not allow a definitive distinction between these possibilities. However, we favor the later alternative because the unchanged P_o relative to wild-type CFTR before covalent modification suggests that the phenylalanine side chain is not involved in stabilizing ATP binding. The data also suggest a relatively high degree of nucleotide occupancy at NBD1. That is, the time between ADP dissociation and ATP binding in the hydrolysis cycle, when Cys⁴⁴⁶ would be accessible to MTSET inhibition, must be relatively brief when the channel is studied in the presence of 1 mM ATP. This conclusion is consistent with nucleotide occlusion studies suggesting that NBD1 rapidly binds nucleotide, whereas ATP hydrolysis or ADP release are much slower (30).

The solvent accessibility of the nucleotide base and ribose in ABC transporter NBDs (9, 11)² contrasts with other nucleotide-binding proteins. Although there is considerable diversity in the structural elements that bind nucleotide, many other proteins bind nucleotide in a cleft, with protein contacts on either side of the aromatic ring. Examples include: Ras (31), transducin (32), cAMP-dependent protein kinase regulatory and catalytic domains (33, 34), motor proteins (35), and CMP kinase (36). These interactions provide varying degrees of nucleotide specificity because of hydrogen bonding between the protein and the atoms associated with positions 1, 2, and 6 of the purine ring. These contacts allow discrimination between ATP, GTP, and pyrimidines. However, in CFTR and other ABC transporters, most of the energy for nucleotide binding may be contributed by the phosphate binding loop (Walker A). Alternatively, other regions of the protein outside a monomeric NBD might contribute to a pocket that binds the nucleotide base and ribose. The former possibility is supported by the low degree of nucleotide specificity in ABC transporters (22, 37-40). Although polyphosphate does not support CFTR channel activity (40),³ nucleoside triphosphates other than adenosine do.

Taken together, our findings of poor nucleotide discrimination for CFTR and the absence of a conserved critical aromatic contact in CFTR NBDs suggest that ATP is even more solvent-exposed when bound to CFTR NBDs than when bound to the bacterial NBDs. This structural model differs substantially from the nucleotide binding clefts of other superfamilies of nucleotide-binding proteins. This model may explain the finding that peptides that contain the CFTR Walker A motif but not much sequence closer to the amino terminus still bind ATP with high affinity (41, 42). The superficial nature of the ATP binding model we propose for CFTR could also explain the observation that cysteine residues in the Walker A motifs of P-glycoprotein are accessible for disulfide-mediated cross-linking to large protein domains that would not be expected to enter a binding cleft (43).

Our data suggest that the aromatic residue, which binds the adenine ring of ATP in the structure of several prokaryotic NBDs, is not conserved in CFTR. Our data also highlight the considerable flexibility in the nucleotide-binding site of CFTR and to a model in which most of the binding energy for ATP is contributed by the phosphate groups.

	ACKNOWLEDGEMENTS

We thank Pary Weber, Phil Karp, Tamara Nesselhauf, and Theresa Mayhew for excellent assistance and our laboratory colleagues for helpful discussions. We thank Dr. Philip J. Thomas for helpful discussions and for sharing information about the structure of LivF prior to publication.

	FOOTNOTES

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

Associate of the Howard Hughes Medical Institute.

^§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail: mjwelsh@blue.weeg.uiowa.edu.

Published, JBC Papers in Press, July 11, 2000, DOI 10.1074/jbc.M004790200

² P. J. Thomas, personal communication.

³ A. L. Berger and M. J. Welsh, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ABC transporter, ATP-binding cassette transporter; NBD, nucleotide-binding domain; HisP, ATP-binding subunit of the histidine transporter; MTSET, [2-(triethylammonium)ethyl] methanethiosulfonate; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/38/29407    most recent M004790200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berger, A. L. Articles by Welsh, M. J. Search for Related Content PubMed PubMed Citation Articles by Berger, A. L. Articles by Welsh, M. J. Social Bookmarking                      What's this?