Correspondence to: David C. Gadsby, Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399. Fax:212-327-7589 E-mail:gadsby{at}rockvax.rockefeller.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cystic fibrosis transmembrane conductance regulator is a Cl- channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH2- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH2 terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH2 termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH2-terminal segment representing aa's 3622 (Flag3-622) or 3633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 6341480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.
Key Words: adenosine triphosphate-binding cassette transporter, domain structure, chloride channel, gating kinetics, coimmunoprecipitation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cystic fibrosis transmembrane conductance regulator is the ~170-kD protein product of the gene mutated in cystic fibrosis patients (
Unlike any other ABC protein, CFTR functions as a chloride channel (reviewed in
Further dissection of these roles would greatly benefit from structural information on each of these domains, which is presently lacking. Nevertheless, peptide models of the R domain (
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Biology
pGEMHE-WT was constructed by subcloning a CFTR cDNA fragment, excised from pBQ4.7 (a gift from Dr. Johanna Rommens, The Hospital for Sick Children, Toronto, Ontario, Canada), into the SmaI and XhoI (BioLabs, Inc.) sites of pGEMHE (
Isolation and Injection of Xenopus Oocytes
Stage VVI oocytes were isolated from adult female Xenopus laevis (Nasco) by partial ovariectomy under Tricaine (1% solution) anaesthesia and were defolliculated by treatment at room temperature (2123°C) for up to 2 h, with ~2 mg/ml collagenase (Type II; Worthington Biochemicals or Type I; GIBCO BRL) in nominally Ca2+-free oocyte Ringer's solution containing (mM): 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.5). Defolliculated oocytes were rinsed extensively with Ca2+-free Ringer's (three to five washes, 50-ml each), and then incubated at 18°C for several hours in Ringer's with 1.8 mM Ca2+ and 50 µg/ml gentamycin (GIBCO BRL) before they were pressure-injected (nanojet; Drummond Scientific) with cRNAs. Injection pipettes were pulled (PP83; Narishige) from glass capillaries (3-000-203-G/X; Drummond Scientific) and their tips were broken to an internal diameter of 1020 µm. Usually, and unless otherwise specified, 2.5 ng of each cRNA, premixed if for coexpression, in a constant total volume of 50 nl, were injected per oocyte. Injected oocytes were further incubated at 18°C for 23 d before they were used for recording or for preparation of membranes.
Electrophysiology
For two-microelectrode voltage-clamp measurements, oocytes in a plexiglass recording chamber (volume ~150 µl) were continuously superfused (~2 ml/min) at ~22°C with gravity-fed solutions selected by manual valves (Hamilton). The dead volume was ~100 µl, and solutions were completely exchanged in <8 s. The standard bath solution was Ca2+-free Ringer's solution. The chamber was connected to virtual ground circuitry through Ag/AgCl electrodes in 2.5% agar/3 M KCl bridges. Currents were measured by a voltage-clamp amplifier (OC-725A oocyte clamp; Warner Instrument Corp.), filtered at 50 Hz by an eight-pole Bessel filter (Frequency Devices, Inc.), digitized online at 100 Hz using a Digidata 1200 board (Axon Instruments, Inc.) with pCLAMP 6.0.2 software (Axon Instruments, Inc.), and stored on disk. Microelectrodes, pulled from glass capillaries (3-000-210-G; Drummond Scientific), were filled with 3 M KCl and had resistances of 0.52 M. Voltage steps were applied via the amplifier's toggle switch (duration 0.51 s) or using pCLAMP software. Steady state currents were averaged over 200 ms near the ends of 1-s voltage steps, and plotted against voltage. Conductance was calculated from linear fits to the steady currents between -60 and -20 mV. Average values were from at least five oocytes.
For patch-clamp recording, oocytes were shrunk for ~2 min in standard bath solution containing (mM): 138 NMG, 2 Mg-sulfamate, 5 HEPES, 0.5 EGTA, 134 sulfamic acid, pH 7.1 with sulfamic acid, supplemented with 100 mM NaCl. The vitelline membranes were removed manually and the oocytes were transferred to a recording chamber containing standard bath solution. Patch pipettes were pulled from borosilicate glass (N-51A; Drummond Scientific) using a vertical pipette puller, and fire-polished to a tip diameter of 12 µm (47 M). The pipette solution contained (mM): 138 NMG, 2 MgCl2, 5 HEPES, 136 HCl, pH 7.4 with HCl. 200300-G
seals were obtained by gentle suction. Patches were excised and transferred to a flow chamber, where the cytoplasmic surface was continuously superfused at 2123°C with standard bath solution containing various test substances. Switching between solutions was implemented by computer-driven electric valves (General Valve Corp.). With the dead volume of ~20 µl and flow rate of ~0.5 ml/min, complete solution exchange took 24 s. Solution-exchange rate was verified at the end of each recording by applying a brief pulse of 2 mM Ca-sulfamate, and observing the rate of decay of endogenous Ca2+-activated Cl--channel current upon Ca2+ removal. The bath electrode (Ag/AgCl pellet in 100 mM KCl) was connected to the flow chamber by an agar bridge (4% agar in 100 mM KCl). Outward unitary currents in CFTR channels were recorded at a pipette potential of -40 mV (Vm = +40 mV) via an Axopatch 200A amplifier (Axon Instruments, Inc.), filtered at 100 Hz with an eight-pole Bessel filter (Frequency Devices, Inc.), digitized online at 1 kHz using an ITC-16 board (Instrutech) and recorded on disk by PULSE software (Heka Elektronik). CFTR channels were activated by 300 nM catalytic subunit of protein kinase A purified from bovine heart (
|
|
|
|
|
|
|
|
For kinetic analysis, digitized (100 Hz bandwidth, 1 kHz sampling rate), baseline-corrected currents from records containing one to seven channels were idealized using conventional half-amplitude threshold crossing. The desired parameters were mean burst and interburst durations for the intact and severed CFTR constructs, and these were determined as follows. First, gating transitions were modeled as a simple closedopen scheme, while brief ("flickery") closures were attributed to pore blockage events (e.g.,
Then, for each record, the best set of rate constants, rCO, rOC, rOB, and rBO, was extracted from a simultaneous maximum likelihood fit to the dwell-time histograms obtained from all of the conductance levels, as described (ib = 1/rCO, while mean burst durations were given by
b = (1/rOC)(1 + rOB/rBO). As a control, because a similar burst-type gating to that of the C-O-B scheme arises from the alternative linear three-state model, closedclosedopen (C-C-O), the events list was sometimes also fitted assuming this C-C-O scheme; this yielded a new set of rate constants, but identical mean burst and interburst durations for the same record in every case. Open probabilities (Po) were calculated directly from the events lists as the time average of the idealized current divided by channel number (estimated for each patch as described above) and the measured single-channel current amplitude.
Macroscopic currents from patches containing 201,000 CFTR channels were digitally refiltered at 10 Hz using a Gaussian filter, and then sampled at 50 Hz. The average steady state current towards the end of 1030-s test applications of 50 µM MgATP was normalized to the mean of the steady currents at 2 mM MgATP before and after the test, and the ratios were used to estimate apparent affinities for ATP.
Single-channel conductances were estimated from amplitude histograms of excised-patch currents recorded at holding potentials of -80, -40, 0, +40, and +80 mV, in symmetrical 140 mM [Cl-]. The distances between adjacent peaks, from fits to sums of Gaussians, were plotted against voltage, and straight lines fitted to yield conductances. Results are presented as mean ± SEM of five or more experiments for kinetic data, and three or more experiments for apparent affinities and single-channel conductances. Statistical significances were evaluated by Student's t test.
Preparation of Oocyte Membranes
Oocytes injected with cRNA were incubated for 48 h, and then frozen in liquid nitrogen and stored at -80°C. Aliquots of ~150 frozen oocytes were homogenized at 4°C with 1 ml lysis buffer, containing (mM): 10 HEPES, pH 7.5 with NaOH, 6 EDTA, 50 NaCl, 1 mg/ml BSA, 1 PMSF, and protease inhibitor cocktail (Calbiochem; final concentrations were 1 mM AEBSF HCl, 300 nM aprotinin, 2 µM E-64, 2 µM leupeptin hemisulfate), and the suspensions were centrifuged at 3,000 g for 10 min. Supernatants were kept and centrifuged again at 3,000 g for 10 min. 2 ml of lysis buffer was added to the resulting supernatants before centrifugation at 173,600 g for 1 h. The pellets were washed with 3 ml modified lysis buffer (with 10% glycerol instead of BSA, and only 0.5 mM PMSF), the centrifugation was repeated and the pelleted membranes resuspended in 0.2 ml modified lysis buffer and stored at -80°C.
Western Blotting and Coimmunoprecipitation
Membrane proteins (75 µg total membrane protein per lane) were resolved by 7.5% SDS-PAGE and blotted onto nitrocellulose membranes using a semi-dry transfer cell (Bio-Rad Laboratories). Protein bands containing the R domain were detected with antiR-domain Ab (
Coimmunoprecipitation was by a procedure modified from that of
Deglycosylation and Protein Determination
Oocyte membrane protein samples (75 µg) were treated with N-glycosidase-F and endoglycosidase-H for 1 h at 37°C following the supplier's protocol (Boehringer). Protein concentrations were measured with bicinchonic acid (Pierce Chemical Co.).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Wild-Type CFTR Channels Display Basal Activity in Resting Xenopus Oocytes
Xenopus oocytes have proven to be a convenient expression system for functional and biochemical analysis of a variety of ion channels, including CFTR (e.g.,
|
Defining the COOH-terminal Boundary of NBD1 by Assessing Function of Coexpressed Severed CFTR Segments
The location of the boundary between NBD1 and the R domain in CFTR is presently unclear. The initial suggestion was that exon 13 of CFTR, comprising aa's 590830, encoded the R domain, and hence that the exon 12/13 junction marked the boundary between NBD1 and the R domain (
These results show that robust CFTR channel function is obtained when the COOH terminus of the segment Flag3-589 is extended to aa 633 (but not to aa 622) and when that construct is coexpressed with the appropriate complementary segment, suggesting that the COOH terminus of NBD1 lies between aa's 622 and 634. (In other words, it could lie at aa 623, or 624, or 625, etc., but could extend as far as aa 633.) An alternative possibility, that a crucial part of NBD1 extends beyond aa 633 but can nevertheless associate with the Flag3-633 segment to form a complete NBD1 domain, is rendered unlikely by our finding that strong CFTR channel activity was reconstituted by coexpression of Flag3-633 with 668-1480: despite this omission of the 34-residue segment aa's 634667, basal and activated conductances (16 ± 1 and 175 ± 7 µS, respectively; Fig 2 and Table 1) were similar to those of WT CFTR. On the other hand, oocytes coexpressing Flag3-622 plus 634-1480, in which any CFTR channels must lack aa's 623633, displayed negligible basal and activated conductances like those of uninjected oocytes.
Defining the NH2-terminal Boundary of NBD1
We used a similar strategy to incrementally extend the NH2 terminus of segment 590-1480 in an attempt to build a complete NBD1 there. Upon coexpression with Flag3-589, the extended segment 449-1480 yielded conductances indistinguishable from those of uninjected oocytes, but extended segment 433-1480 gave substantial activated (129 ± 12 µS) conductance, though still smaller than that of WT CFTR-injected oocytes (Fig 3). However, omission of the overlapping section (residues 433589) by coexpression of Flag3-432 plus 433-1480 resulted in somewhat larger basal (6 ± 1 µS) and activated (157 ± 5 µS) conductances that approached those of oocytes expressing WT CFTR channels. The fact that coexpression of Flag3-432 plus 449-1480 yielded no functional channels confirms that segment 433-448 is an essential part of NBD1. In contrast, coexpression of Flag3-414 plus 433-1480, which effectively deleted the 18-residue segment aa's 415432, nevertheless resulted in oocytes that displayed basal and activated conductances (7 ± 1 and 195 ± 5 µS, respectively) comparable with those of oocytes expressing intact CFTR (Fig 2 and Table 1). These data suggest that the NH2-terminal boundary of NBD1 lies between aa's 432 and 449.
As a control test, each individual segment of CFTR used to investigate the functional boundaries of NBD1 was expressed by itself in oocytes, and basal and activated conductances were measured in the usual manner. In all cases, the resulting conductances were similar to those of uninjected oocytes (Fig 4), confirming that none of these truncated CFTR fragments readily forms viable channels when expressed alone in Xenopus oocytes.
Expression and Maturation of Severed CFTR Segments
Western blots of proteins resolved from whole oocyte membrane preparations were used to evaluate expression of intact or truncated CFTR proteins. As expected, the R-domain antibody did not recognize the Flag3-622 or Flag3-633 segments (Fig 5 A, lanes 1 and 2), but did detect a single sharp band of ~95 kD (Fig 5 A, lane 3, thin arrow) when segment 634-1480 was expressed alone. However, an additional broad band of ~150 kD appeared when 634-1480 was coexpressed with Flag3-633 (Fig 5 A, lane 5, fat arrow), implying that segment 634-1480 was then glycosylated. Accordingly, the ~95-kD band could be digested to a perceptibly smaller band of ~90 kD (Fig 5 C, thin arrows) by either endoglycosidase-H or N-glycosidase-F, whereas the broad ~150-kD (Fig 5 C, fat arrow) band was insensitive to endoglycosidase-H, but was largely digested to a ~90-kD band after treatment with N-glycosidase-F (Fig 5 C). Because endoglycosidase-H may be expected to remove glycosyl moieties only from proteins not yet modified by the early Golgi enzyme mannosidase II, whereas N-glycosidase-F removes all asparagine-linked glycosyl groups, these results argue that the ~90-kD band corresponds to the deglycosylated form of segment 634-1480, while the ~95- and ~150-kD bands correspond to its core glycosylated and fully glycosylated forms, respectively. This, in turn, suggests that segment 634-1480 was merely core glycosylated when expressed alone (Fig 5 A, lane 3), but could be fully glycosylated when it was coexpressed with the contiguous segment Flag3-633 (Fig 5 A, lane 5). Together with the several-fold increase in density of the 634-1480 signal in the presence of Flag3-633 (compare lanes 3 and 5), these findings indicate that maturation and stabilization of segment 634-1480 are promoted by its coexpression with an appropriate complementary segment such as Flag3-633. Interestingly, however, the ~150-kD band was not detected when segment 634-1480 was coexpressed with Flag3-622 (Fig 5 A, lane 4). This, despite the fact that the Flag3-633 and Flag3-622 segments, detected by the antiFlag M2 antibody as single narrow bands (Fig 5 B), were both expressed at similar levels, and at almost the same level whether expressed alone or coexpressed with 634-1480. Thus, although removal of the eleven residues (aa's 623633) from Flag3-633 did not affect the stability of the resulting segment, Flag3-622 (Fig 5 B, lanes 2 and 5 vs. 1 and 4), this deletion abrogated the ability of that segment to promote glycosylation of the coexpressed fragment, 634-1480 (Fig 5 A, lane 5 vs. 4). These data indicate that completion of the COOH terminus of segment Flag3-622 by some or all of aa's 623633 is required for productive interaction with segment 634-1480, which, in turn, seems essential for maturation of the resulting complex. This is consistent with the conclusion from the functional measurements, using two-electrode voltage clamp, that the COOH terminus of NBD1 lies between residues 622 and 634.
Similarly, the R-domain antibody did not detect the Flag3-432 segment (which lacks an R domain; Fig 6 A, lane 1), but recognized segment 433-1480, which, when expressed alone, gave a sharp band at ~125 kD (Fig 6 A, lane 2, thin arrow). Coexpression of 433-1480 with Flag3-432 (Fig 6 A, lane 5) yielded that same band (thin arrow), together with an additional, broader band (fat arrow) of ~160 kD (together with weak degradation bands ~7075 kD), consistent with glycosylation of segment 433-1480. Indeed, after treatment with either endoglycosidase-H or N-glycosidase-F the lower (~125-kD) Mr band migrated as a slightly smaller fragment of ~120 kD (Fig 6 B, thin arrows). On the other hand, the higher (~160-kD; Fig 6 B, fat arrow) Mr band was insensitive to endoglycosidase-H, but was converted to a ~120-kD band by N-glycosidase-F. It seems, then, that the ~120-kD band represents the unglycosylated form of segment 433-1480, while the ~125- and the ~160-kD bands represent its core glycosylated and fully glycosylated forms. However, hardly any protein was detected (over the range ~45 to ~180 kD) by the R-domain antibody when segment 449-1480 was expressed alone (Fig 6 A, lane 3). Even when it was coexpressed with the Flag3-432 segment, only a relatively weak band of ~70 kD could be detected, presumably representing a degraded form of 449-1480 protein (Fig 6 A, lane 4, *). So, it appears that the stability of 449-1480 protein was slightly increased in the presence of Flag3-432, although no glycosylated form of 449-1480 was observed. Thus, deletion of aa's 433448 from 433-1480 yielded a segment that seemed highly unstable, even when coexpressed with a complementary segment of CFTR. These biochemical findings support our conclusion from the electrophysiological assays of function that the NH2 terminus of NBD1 lies between residues 432 and 449.
Physical Association between Complementary Fragments Starts in the Endoplasmic Reticulum
Coimmunoprecipitation tests were made using antiFlag M2 affinity beads to evaluate physical association between Flag-tagged NH2-terminal segments of CFTR and complementary COOH-terminal segments in membrane extracts from oocytes coinjected with cRNAs encoding severed CFTR molecules. Both segments Flag3-622 and Flag3-633 (detected with an antibody against an epitope near CFTR's NH2 terminus; Fig 7 A, bottom, lanes 2 and 3) were able to coimmunoprecipitate the core glycosylated (~95-kD; thin arrow) form of segment 634-1480 (top, lanes 2 and 3). However, the fully glycosylated (~150-kD; fat arrow) form of 634-1480 was coimmunoprecipitated only with Flag3-633 (Fig 7 A, top, lane 3) and not with Flag3-622 (lane 2). Because the immunoprecipitation was via the Flag tag, it appears that both Flag3-622 and Flag3-633 segments associate with the core glycosylated segment 634-1480 (Fig 7 A, top, lanes 2 and 3, thin arrow), presumably in the endoplasmic reticulum (ER), but only the Flag3-633 plus 634-1480 complex proceeds to the Golgi compartment, and eventually to the cell surface membrane as a mature, fully glycosylated channel. The Flag3-622 plus 634-1480 complex (Fig 7 A, lane 2), on the other hand, does not appear to leave the ER.
Correspondingly, immunoprecipitation of segment Flag3-432 (detected with the antiNH2-terminal antibody; Fig 7 B, bottom, lanes 1 and 2) resulted in coimmunoprecipitation of both core-glycosylated (thin arrow) and fully glycosylated (fat arrow) forms of segment 433-1480 (top, lane 2), but of little or no protein corresponding to segment 449-1480 (top, lane 1). The latter result corroborates the conclusion from the Western blot of Fig 6 A that segment 449-1480 appears to be rapidly degraded in the oocyte. Coimmunoprecipitation (using the antiFlag beads) of both immature and mature forms of 433-1480 (Fig 7 B, top, lane 2, thin and fat arrows), however, demonstrates that segments Flag3-432 and 433-1480 physically associate with each other, and that this association takes place in the ER (or, at the latest, in the early Golgi compartment). These data indicate that the complementary pairs of CFTR segments that constitute functional channels, whether severed just before or just after the intact NBD1, begin their association in the ER before proceeding through the Golgi to the cell membrane, whereas segment pairs that do not comprise an intact NBD1 fail to exit the ER.
The NH2-terminal Flag Epitope Lowers the Po of CFTR Channels by Slowing Opening
Forskolin activates CFTR channels indirectly via a pathway that involves adenylyl cyclase and PKA holoenzyme, and, in the oocyte, such activation appears to be subject to saturation (Csanády, L., K.W. Chan, D. Seto-Young, D.C. Kopsco, A.C. Nairn, and D.C. Gadsby, manuscript submitted for publication). Moreover, IBMX at millimolar concentrations likely directly stimulates CFTR channel activity (
|
|
Severing CFTR between NBD1 and the R Domain Lowers Po by Speeding Channel Closing
The more interesting question is whether severing CFTR, either just before or just after NBD1, affects channel gating. The results of the kinetic analysis (Fig 9 and Table 2) demonstrate that severed channels formed from segments 1-432 plus 433-1480 had a Po, a mean burst duration, and a mean interburst duration that were not significantly different from those of WT CFTR channels. Furthermore, this similarity was not influenced by addition of the Flag epitope, since severed channels formed from Flag3-432 plus 433-1480 segments showed Po, and burst and interburst durations, closely similar to those of Flag-WT channels (Fig 9 and Table 2). However, severed channels formed from CFTR segments 1-633 plus 634-1480 had a smaller Po than WT or 1-432 plus 433-1480 channels, attributable to a reduced burst duration (i.e., to an increased closing rate). This effect of severing CFTR channels just after NBD1 was independent of the presence or absence of the Flag epitope, because the mean burst duration of channels comprising segments Flag3-633 plus 634-1480 was significantly shorter than that of either Flag-WT or Flag3-432 plus 433-1480 channels (Fig 9 and Table 2). These findings indicate that severing CFTR just before NBD1 has no measurable effect on channel Po or gating kinetics, whereas severing CFTR between NBD1 and the R domain leads to a reduction in Po due to an increase in channel closing rate.
Neither Adding the Flag Epitope, nor Severing CFTR before or after NBD1, Affects Single-Channel Conductance or the Apparent Affinity for ATP
To verify that neither incorporating the Flag epitope nor severing CFTR before or after NBD1 causes a major structural alteration in the channel pore, we measured single-channel conductances of WT and Flag-WT CFTR channels, and of severed channels formed from segments 1-432 plus 433-1480, or Flag3-432 plus 433-1480, or 1-633 plus 634-1480, or Flag3-633 plus 634-1480, in excised patches exposed to symmetrical 140 mM Cl- solutions. Under these conditions, as illustrated for WT and for 1-432 plus 433-1480 channels, each channel type was characterized by an ohmic single-channel conductance: its average magnitude was ~7 pS and there was no significant difference between the conductance of WT CFTR channels and that of any of the other constructs (P = 0.1; Fig 10). This argues that severing CFTR, or adding the NH2-terminal Flag, did not grossly alter the pore architecture.
|
Assuming that a step after binding of ATP at one of the NBDs rate limits channel opening (see
|
Deletion of aa's 415432 in CFTR Does Not Alter the Apparent Affinity for ATP
The demonstration that effective deletion of aa's 415432, by coexpression of segments Flag3-414 and 433-1480, impairs CFTR channel activity very little, if at all (Fig 3), has ramifications for our understanding of the relationship between the structure of these channels and their function. This is because residues 415-432 in CFTR align (Fig 12, below) with those comprising the first ß strand in the recently solved high-resolution structures of HisP, the NBD of histidine permease (from Salmonella typhimurium;
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this work, we have demonstrated that physical and functional association of complementary segments of CFTR, severed just before or after NBD1, results in split channels with microscopic properties closely similar to those of intact WT CFTR. Further, by systematically shifting the sever point, we have been able to define the domain boundaries of NBD1 to within 1015 amino acids. The newly defined boundaries have important ramifications for structural and functional studies of CFTR's NBDs, whether as isolated polypeptides or in their native state, incorporated into full-length CFTR.
That a severed membrane transport protein may nevertheless function has been demonstrated previously. For example, E. coli cells expressing both putative hexahelical halves of lac permease, severed within its central cytoplasmic loop, transport lactose at about one third the rate of cells expressing intact permease, but no transport is seen in cells expressing either half alone (
Assaying the function of severed molecules to define domain boundaries is an approach well suited to multidomain, single-gene eukaryotic ABC transporters, because individual domains may each correspond to the product of a single ancestral gene and may therefore reasonably be expected to be capable of folding separately. Indeed, there are already several examples of putative links between domains of such eukaryotic ABC proteins being severed without destroying the molecule's overall function (as described above). In general, if severing the backbone of a protein does not detectably impair its function, it is reasonable to conclude that covalent linkage at that point is not required for the assayed function. It may further be concluded either that the cut occurs between functional domains, or that the sever point lies within such a domain but the native fold and function are nevertheless retained (
In the present work, we have made detailed single-channel measurements that show that individual severed CFTR channels, cut just before (1-432 plus 433-1480) or after (1-633 plus 634-1480) NBD1, have the same unitary conductance as WT CFTR channels (Fig 10) and almost identical microscopic gating characteristics in the presence of PKA catalytic subunit (Fig 9). Both severed constructs had the same opening rate as WT channels, and the same apparent affinity for activation of channel Po by MgATP, and both were locked open by PPi (Fig 8) or AMPPNP. Only the closing rate of channels cut after NBD1 differed from that of WT, and was ~40% faster, indicating that the bursting state is somewhat destabilized by that cut, due to a slight acceleration of the rate-limiting step for channel closing (likely reflecting speeding of the slowest step in the ATP hydrolysis cycle at NBD2; Csanády et al., 2000). Thus, we can conclude that the normal gating function of NBD1 (which we and others infer to be control of channel opening; for reviews, see, e.g.,
None of the half-molecule constructs gave measurable basal or activated Cl- conductance in two-microelectrode recordings of macroscopic currents, regardless of whether the oocytes were expressing the NH2-terminal half with (construct Flag3-835; not illustrated) or without (Flag3-633; Fig 4) the R domain, or the COOH-terminal half with (CFTR construct 634-1480; Fig 4) or without (construct 837-1480; not illustrated) the R domain. However, because we did not record from patches excised from those oocytes, we cannot rule out the possibility that an extremely low density of channels might be formed from certain half molecules. Our macroscopic current results contrast with a recent report that expression of only the COOH-terminal half of CFTR, either with or without the R domain, gave macroscopic, intracellular cAMP-activated, whole-cell currents in injected oocytes and in transfected IB3 cells, as well as unitary currents activated by PKA and ATP in patches excised from the IB3 cells (10 µS (
How do the boundaries of CFTR's NBD1 that we have functionally defined here compare with the crystal structures of the HisP and RbsA NBDs, whose close similarity to each other supports their being considered reasonable models for the structure of all ABC NBDs, including those of CFTR? Although the safe boundaries of CFTR's NBD1 (433-633) circumscribe only 200 residues, whereas the crystallized NBDs of HisP and RbsA included 259 and 241 residues, respectively, they still compare reasonably well because alignments (e.g., Fig 12; compare -phosphate of bound ATP (although nearby Leu435, if not Phe433 itself, is implicated in binding of the nucleotide base; see
The finding that severed CFTR channels lacking residues 415-432 (i.e., coexpressed Flag3-414 plus 433-1480) are opened by MgATP with the same apparent affinity as seen for WT CFTR channels (K1/2 ~ 50 µM; Fig 11) is intriguing. This is because a number of observations have suggested that it is binding of ATP at NBD1 (and possibly hydrolysis of that ATP) that is responsible for CFTR channel opening (reviewed in
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: aa, amino acid; ABC, ATP-binding cassette; ER, endoplasmic reticulum; MRP, multi-drug resistance-related protein; NBD, nucleotide binding domain; Pgp, P-glycoprotein; PPi, pyrophosphate; R domain, regulatory domain.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Roberto Sánchez and Andrej Sali for help with alignments and for structural modeling, David Kopsco and Atsuko Horiuchi for technical assistance, and Kate Hall, Peter Hoff, and Paola Vergani for help with the manuscript and figures.
L. Csanády is a William O'Baker Graduate Fellow of The Rockefeller University. This work was supported by National Institutes of Health grant DK-51767.
Luis Reuss served as guest editor.
Submitted: 25 February 2000
Revised: 5 May 2000
Accepted: 5 June 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Annereau, J.-P., Wulbrand, U., Vankeerberghen, A., Cuppens, H., Bontems, F., Tümmler, B., Cassiman, J.-J., Stoven, V. 1997. A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator. FEBS Lett 407:303-308[Medline].
Armstrong, S., Tabernero, L., Zhang, H., Hermodson, M., Stauffacher, C. 1998. The 2.5 Å structure of the N-terminal ATP-binding cassette of the ribose ABC transporter. Biophys. J. 74:A338.
Bear, C.E., Duguay, F., Naismith, A.L., Kartner, N., Hanrahan, H.W., Riordan, J.R. 1991. Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J. Biol. Chem. 266:19142-19145
Berkower, C., Michaelis, S. 1991. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO (Eur. Mol. Biol. Organ.) J 10:3777-3785[Abstract].
Berkower, C., Taglicht, D., Michaelis, S. 1996. Functional and physical interactions between partial molecules of STE6, a yeast ATP-binding cassette protein. J. Biol. Chem 271:22983-22989
Bibi, E., Kaback, H.R. 1990. In vivo expression of the lacY gene in two segments leads to functional lac permease. Proc. Natl. Acad. Sci. USA 87:4325-4329[Abstract].
Betton, J.-M., Hofnung, M. 1994. In vivo assembly of active maltose binding protein from independently exported protein fragments. EMBO (Eur. Mol. Biol. Organ.) J 13:1226-1234[Abstract].
Chan, K.W., Csanády, L., Nairn, A.C., Gadsby, D.C. 1999. Deletion analysis of CFTR channel R domain using severed molecules. Biophys. J 76:A405. (Abstr.).
Clancy, J.P., Hong, J.S., Bebök, Z., King, S.A., Demolombe, S., Bedwell, D.M., Sorscher, E.J. 1998. Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability. Biochemistry. 37:15222-15230[Medline].
Csanády, L. 2000. Rapid kinetic analysis of multi-channel records by a simultaneous fit to all dwell-time histograms. Biophys. J 78:785-799
Csanády, L., Chan, K.W., Angel, B.B., Nairn, A.C., Gadsby, D.C. 1998. R-domain serine 768, a negative regulator of CFTR chloride channel gating. J. Gen. Physiol. 112:26a-27a. (Abstr.).
Csanády, L., Gadsby, D.C. 1999. CFTR channel gating: incremental progress in irreversible steps. J. Gen. Physiol 114:49-53
Devidas, S., Yue, H., Guggino, W.B. 1998. The second half of the cystic fibrosis transmembrane conductance regulator forms a functional chloride channel. J. Biol. Chem 273:29373-29380
Dulhanty, A.M., Riordan, J.R. 1994. Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. Biochemistry 33:4072-4079[Medline].
Gadsby, D.C., Nairn, A.C. 1999. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol. Rev 79:S77-S107[Medline].
Gao, M., Loe, D.W., Grant, C.E., Cole, S.P.C., Deeley, R.G. 1996. Reconstitution of ATP-dependent leukotriene C4 transport by co-expression of both half-molecules of human multidrug resistance protein in insect cells. J. Biol. Chem 271:27782-27787
Groves, J.D., Wang, L., Tanner, M.J.A. 1998. Functional reassembly of the anion transport domain of human red cell band 3 (AE1) from multiple and non-complementary fragments. FEBS Lett 433:223-227[Medline].
Hartman, J., Huang, Z., Rado, T.A., Peng, S., Jilling, T., Muccio, D.D., Sorscher, E.J. 1992. Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product. J. Biol. Chem 267:6455-6458
Higgins, C.F. 1992. ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol 8:67-113.
Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F., Kim, S.-H. 1998. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature. 396:703-707[Medline].
Ishihara, H., Welsh, M.J. 1997. Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis. Am. J. Physiol. Cell Physiol 273:C1278-C1289
Jones, P.M., George, A.M. 1999. Subunit interactions in ABC transporters: towards a functional architecture. FEMS (Fed. Eur. Microbiol. Soc.) Microbiol. Lett 179:187-202[Medline].
Kaczmarek, L.Y., Jennings, U.R., Strumwasser, F., Nairn, A.C., Walter, U., Wilson, F.D., Greengard, P. 1980. Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture. Proc. Natl. Acad. Sci. USA. 77:7487-7491[Abstract].
Ko, Y.H., Delannoy, M., Pedersen, P.L. 1997. Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function. Biochemistry. 36:5053-5064[Medline].
Ko, Y.H., Pedersen, P.L. 1995. The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase. J. Biol. Chem 270:22093-22096
Liman, E.R., Tytgat, J., Hess, P. 1992. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9:861-871[Medline].
Loo, T.W., Clarke, D.M. 1994. Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. J. Biol. Chem 269:7750-7755
Mourez, M., Hofnung, M., Dassa, E. 1997. Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits. 1997. EMBO (Eur. Mol. Biol. Organ.) J 16:3066-3077
Moyer, B.D., Loffing, J., Schwiebert, E.M., Loffing-Cueni, D., Halpin, P.A., Karlson, K.H., Ismailov, I.I., Guggino, W.B., Langford, G.M., Stanton, B.A. 1998. Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in Madin-Darby canine kidney cells. J. Biol. Chem 273:21759-21768
Naren, A.P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J.E., Quick, M.W., Kirk, K.L. 1999. CFTR chloride channel regulation by an interdomain interaction. Science. 286:544-548
Ostedgaard, L.S., Rich, D.P., DeBerg, L.G., Welsh, M.J. 1997. Association of domains within the cystic fibrosis transmembrane conductance regulator. Biochemistry 36:1287-1294[Medline].
Picciotto, M., Cohn, J., Bertuzzi, G., Greengard, P., Nairn, A.C. 1992. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267:12742-12752
Ramjeesingh, M., Li, C., Garami, E., Huan, L.J., Galley, K., Wang, Y., Bear, C.E. 1999. Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator). Biochemistry 38:1463-1468[Medline].
Randak, C., Neth, P., Auerswald, E.A., Assfalg-Machleidt, I., Roscher, A.A., Hadorn, H.B., Machleidt, W. 1996. A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein. FEBS Lett. 398:97-100[Medline].
Randak, C., Neth, P., Auerswald, E.A., Eckerskorn, C., Assfalg-Machleidt, I., Machleidt, W. 1997. A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase. FEBS Lett 410:180-186[Medline].
Riordan, J.R., Rommens, J.M., Kerem, B.S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J. et al. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 245:1066-1073[Medline].
Sheppard, D.N., Ostedgaard, L.S., Rich, D.P., Welsh, M.J. 1994. The amino-terminal portion of CFTR forms a regulated Cl- channel. Cell. 76:1091-1098[Medline].
Sheppard, D.N., Welsh, M.J. 1999. Structure and function of the CFTR chloride channel. Physiol. Rev 79:S23-S45[Medline].
Shiba, K., Schimmel, P. 1992. Functional assembly of a randomly cleaved protein. Proc. Natl. Acad. Sci. USA. 89:1880-1884[Abstract].
Shyamala, V., Baichwal, V., Beall, E., Ames, G.F. 1991. Structurefunction analysis of the histidine permease and comparison with cystic fibrosis mutations. J. Biol. Chem 266:18714-18719
Smit, L.S., Wilkinson, D.J., Mansoura, M.K., Collins, F.S., Dawson, D.C. 1993. Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator. Proc. Natl. Acad. Sci. USA. 90:9963-9967[Abstract].
Stühmer, W., Conti, F., Suzuki, H., Wang, X., Noda, M., Yahagi, N., Kubo, H., Numa, S. 1989. Structural parts involved in activation and inactivation of the sodium channel. Nature. 339:597-603[Medline].
Vergani, P., Csanády, L., Basso, C., Sanchez, R., Nairn, A.C., Gadsby, D.C. 2000. Mutations near the predicted catalytic site of NBD1 affect CFTR Cl- channel function surprisingly little. Biophys. J 78:264A.
Walker, J.E., Saraste, M., Runswick, M.J., Gay, N.J. 1982. Distantly related sequences in the - and ß-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO (Eur. Mol. Biol. Organ.) J 8:945-951.
Wilkinson, D.J., Mansoura, M.K., Watson, P.Y., Smit, L.S., Collins, F.S., Dawson, D.C. 1996. CFTR: the nucleotide binding folds regulate the accessibility and stability of the active state. J. Gen. Physiol. 107:103-119[Abstract].
Yike, I., Ye, J., Zhang, Y., Manavalan, P., Gerken, T.A., Dearborn, D.G. 1996. A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms. Prot. Sci 5:89-97
Zeltwanger, S., Wang, F., Wang, G.T., Gillis, K.D., Hwang, T.-C. 1999. Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. J. Gen. Physiol 113:541-554