Division of Medical Genetics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Submitted 17 November 2003 ; accepted in final form 4 May 2004
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ABSTRACT |
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site-directed mutagenesis; kinase-dependent activation; cell-attached patch clamp; open probability; mean open time
Studies aimed at determining key sites for PKA-dependent activation have used two approaches. In vivo phosphorylation has been used to identify sites that show increased phosphorylation during agonist-dependent activation. Results have differed between laboratories, but phosphorylation of serines at sites 660, 700, 737, 795, and 813 has been observed by several groups (11, 26, 27). While this does not demonstrate a role for these sites, these results decrease the likelihood that the other dibasic sites play a major role in the activation of wild-type CFTR. Studies in which serine has been mutated to alanine to prevent kinase-dependent phosphorylation have also been less than conclusive. No site appears to be essential for activation (11, 27). Removal of all dibasic sites reduces, but does not eliminate, PKA-dependent channel activity (8, 31). Studies in which single Ser-to-Ala mutations were made at dibasic sites have shown changes in the magnitude of channel activity, in the rate of channel activation, and in dose-response curves for agonist-dependent activation (4, 36, 37). The presence of serine at amino acid 737 or 768 appears to inhibit PKA-dependent channel activation. Serines at amino acid 660 or 813, and to a lesser extent 700 and 795, appear to stimulate channel activity. However, these studies had several flaws. Changes in apparent channel activity have not always been shown to be independent of CFTR expression. Changes in dose-response relationships do not permit a distinction to be made between changes in single-channel properties (Po or i) and changes in the amount of kinase activity needed to generate fully active channels. Last, how phosphorylation of CFTR solely at amino acid 737 or 768 affects channel activity is unknown. In general, there are three unresolved questions: 1) At what site or combination of sites is phosphorylation required to produce a maximally active channel? 2) Are there sites in CFTR where phosphorylation inhibits channel activation? 3) How does CFTR phosphorylation permit channel activation?
To address these issues, we measured CFTR channel kinetics in cell-attached and excised patches for a series of constructs with Ser-to-Ala mutations at PKA phosphorylation sites in the R domain. In addition to examining the impact of single Ser-to-Ala mutations at PKA phosphorylation sites, we studied a mutant in which the eight conserved phosphorylation sites in the R domain were mutated to alanine and a series of constructs in which one or two of these alanines were mutated back to a serine. Our studies were designed to allow us to measure not only the open probability (Po) but also mean open time (o) and mean closed time (
c) for each of these mutants. Our studies demonstrate that phosphorylation at S737 or S768 inhibits channel activity by decreasing Po. Last, our studies demonstrate that phosphorylation alters the rate of channel opening and not the rate of channel closing. In contrast to the conclusions of a recent labeling study (5), our data suggest that R-domain phosphorylation alters an equilibrium between conformations of CFTR with high and low affinities for ATP. Similarities between this mechanism for CFTR activation and the mechanism for ATP-dependent solute absorption by bacterial ATP-binding cassette (ABC) transporters (10) are described.
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MATERIALS AND METHODS |
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Electrophysiology.
Cells were used for patch clamping 13 days postinfection, with 2 days being optimal. Patch pipettes were pulled from PG52151-4 glass capillaries (World Precision Instruments, Sarasota, FL) using a vertical puller (PP-830; Narishige Instruments Laboratory, Tokyo, Japan). They had resistances of 46 M when filled with pipette solution of the following composition (in mM): 140 N-methyl-D-glucamine (NMDG)-Cl, 5 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.4 (adjusted with NMDG). The bath solution was composed of (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 5 glucose, and 5 HEPES, pH 7.4 (adjusted with NaOH). For inside-out patch experiments, the composition of the bath solution (IO buffer) was (in mM) 150 NaCl, 2 MgCl2, 5 EGTA, and 5 HEPES, pH 7.4 (adjusted with NaOH).
Data acquisition.
Cell-attached and excised inside-out patch currents were recorded at room temperature (22°C) under constant membrane potentials, using an EPC-9 patch-clamp amplifier controlled with the associated PULSE software (Heka Electronik, Lambrecht, Germany). The apparent membrane potential (bath minus the pipette potential) was 50 mV. Data were sampled at 200 Hz, filtered at 100 Hz with a built-in three-pole Bessel filter, and saved directly to a personal computer hard drive file for further offline analysis. Cells were patched in the absence of agonist, and, when patches with high-resistance seals (typically 50 G
) and no basal channel activity were obtained, 1 µM forskolin was added to the bath. Channel activity was typically recorded for at least 20 min. For inside-out patch experiments, the patch pipette tip was repositioned after patch excision in front of a three-barrel pipette outlet from a fast-switching, gravity-driven (0.2 ml/min) perfusion system (SF77A; Warner Instrument, Hamden, CT) to allow the application of IO buffer supplemented with 50 U/ml PKA plus 1 mM ATP, 1 mM ATP, or 10 mM ATP. Excised patches were exposed to PKA plus ATP for 10 min before activity with 1 and 10 mM ATP was recorded. For most patches, the number of active channels (N), determined by variance analysis, remained constant in our experiments; patches for which a decrease in channel number was observed were not analyzed.
Data analysis.
Data were digitally filtered at 10 Hz and analyzed using IGOR Pro software (WaveMetrics, Lake Oswego, OR). In records of at least 15-min duration for which the average current (I) was constant, Po was determined using the equation Po = (1 2/I·i), where
2 is the variance of I (13). Single-channel current i was determined from amplitude histograms, which were also used to establish that baseline current was constant. Mean open times were calculated with the formula
o = (T·I)/(i·n), where T is the length of the record (>180 s) and n is the total number of openings in the record (24). Mean closed times were subsequently derived from Po and
o. The number of channels in a patch (N) was calculated from
2, I, and i, N = (I/i)/[1
2/(I·i)], and then compared with the maximum number of observed channels.
Reagents. Forskolin (Calbiochem, La Jolla, CA) was kept as stock solution (10 mM) in DMSO at 20°C. Aliquots of PKA catalytic subunit (Promega, Madison, WI) were stored at 70°C and suspended in IO buffer just before use. All other chemicals were obtained from Sigma (St. Louis, MO).
Statistics. Calculated values are given as means ± SE. Data were compared using Student's t-test, with P < 0.05 considered significant.
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RESULTS |
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During these studies, we made five recordings in which only a single active channel per patch appeared. For these records, Po was calculated from all-points current histograms and from the variance in I, and the ratio of Po from variance analysis to Po from all-points histograms was determined. The mean ratio was 0.95 ± 0.06, suggesting that at least for single-channel patches, there were no significant differences between Po calculated by variance analysis and from all-points histograms. We also compared the maximum number of channels observed in our traces with the number calculated from our Po, i, and I values. While the maximum number of channels observed was often much less than the calculated number of channels, in none of the 67 patches analyzed was the maximum number of observed channels more than a fraction of a channel greater than the calculated number of channels. While these tests suggest to us that our values of Po are correct, we acknowledge that factors unknown to us could have caused our analysis to generate spurious Po values.
Our data show that single Ser-to-Ala mutations can have significant effects on Po, with 700A and 813A decreasing Po and 737A and 768A increasing Po relative to that of wild-type CFTR. However, in no case examined was phosphorylation at any one site essential for channel activity. This was also demonstrated by the observation that significant forskolin-dependent channel activity was observed with octa. The increased value of Po for 737A and 768A (relative to that of wild-type CFTR) suggested that phosphorylation at these sites might inhibit CFTR activation. However, an alternative explanation is that phosphorylation at these sites inhibits phosphorylation at activating sites. To distinguish between these explanations, single Ala-to-Ser mutations were generated in octa at amino acids 700 (700S), 737 (737S), 768 (768S), 795 (795S), and 813 (813S). Po values for 737S and 768S were not significantly different from the Po value for octa. In contrast, the Po values for 700S, 795S, and 813S were significantly greater than the Po value for octa. Double-mutant constructs were generated from octa with one serine at 813 and a second serine at 700 (700S/813S), 737 (737S/813S), 768 (768S/813S), or 795 (795S/813S). For 768S/813S, the Po was significantly greater than that for 768S and significantly less than that for 813S. A similar trend was seen with 737S/813S, where the Po was significantly less than that of 813S; however, while the Po of the double mutant was greater than that of 737S, the difference failed to reach significance. In contrast, a double mutation at two stimulatory sites, 795S/813S, had a Po that was greater than that for either 795S or 813S when present alone.
As shown in the expanded traces in Fig. 2B, it was possible to identify each channel opening in these records. By counting channel openings per unit time and measuring both i and I, mean open times were calculated. Mean closed times were then calculated from Po and the mean open time. Data are presented in Table 3. The method provides a measure of mean open time but provides no information about the distribution of mean open time. This is a limitation of the method, but it does not affect the validity of the calculated values. In addition, because mean open times are calculated from three readily obtainable parameters, they are likely to be more accurate than our Po values. For all but one form of CFTR tested (795S/813S), mean open times were not significantly different from those of wild-type CFTR. In 5 of 67 patches, we observed a single active channel. Because of the length of mean open and closed times, we were unable to obtain enough events for open- or closed-time histograms; however, for these patches, survivor plots were generated, and they are shown in Fig. 4. All forms of CFTR, with the exception of 795S/813S, had similar mean open times, but there was considerable variation in mean closed times. Thus changes in Po are largely a reflection of changes in mean closed time.
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DISCUSSION |
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Studies by Baldursson et al. (4) showed that in Fisher rat thyroid epithelial cells, cAMP-dependent chloride currents are greater for cells transfected with 737A than for cells transfected with wild-type CFTR. However, those authors failed to determine whether 737A expression was greater than that of wild-type CFTR, and only a small, apparently statistically insignificant difference in the k1/2 of dose-response curves for wild type and 737A was observed. With excised patches, Winter and Welsh (37) compared the Po values for wild-type CFTR with those for 737A, an inhibitory site mutation, and 660A, 795A, and 813A, activating site mutations. At low ATP concentrations, the Po of wild-type CFTR was greater than the Po for all other forms, including 737A; but at high ATP concentrations, there were no significant differences in Po for all forms of CFTR. On the basis of these studies, we concluded that the evidence for inhibitory effects of phosphorylation at S737 and S768 was not convincing.
Can better evidence for inhibitory effects of phosphorylation at S737 and S768 be obtained? The phosphorylation state of channels in a patch can never be determined, because in vivo labeling does not allow this issue to be resolved. As a consequence, the only measurable property is agonist-dependent CFTR activity. While differences between constructs that differ from wild-type CFTR by one Ser-to-Ala mutation are clearly due, at least in part, to differences in phosphorylation at the mutated site, additional effects due to alterations in phosphorylation at other sites cannot be ruled out. We attempted to circumvent this problem by limiting the number of PKA sites. We studied both Ser-to-Ala mutations on a wild-type background and Ala-to-Ser mutations on a background in which the eight conserved PKA sites in the R domain of CFTR were mutated to alanine, octa. As described by others (27, 31), a small level of forskolin-dependent channel activity was seen with octa. Moreover, the activity of 737S and 768S was not greater than that of octa, while that of 700S, 795S, and 813S was significantly greater than that of octa. When there is only a single phosphorylation site, forskolin-dependent activity can be due only to phosphorylation at that site. For constructs with two phosphorylation sites, when channel activity of the double mutant differs from that of both single-mutant constructs, both PKA sites must be phosphorylated in the double-mutant construct. A construct with an activating site and an inhibiting site should have an intermediate level of activity between the levels of the two single-site constructs. The Po of 813S/768S was less than that for 813S but greater than that for 768S, and similar effects were seen with 813S/737S. It is clear that, at least in the double mutations, phosphorylation occurs at S737 and S768 and that phosphorylation at these sites inhibits CFTR activity. Similarly, a construct with two activating sites should have channel activity that is greater than that of either single-site construct. Because the Po of 813S/795S is greater than that for either 813S or 795S, phosphorylation at both of these sites must be stimulatory. On the basis of this argument, we conclude that in our CFTR constructs, forskolin-dependent changes in channel activity were due to phosphorylation at the mutated sites. Our study also documents the presence of sites on CFTR where PKA-dependent phosphorylation is inhibitory. The presence of stimulatory and inhibitory phosphorylation sites may provide a mechanism for more precise regulation of CFTR channel activity.
While there is general agreement that R-domain phosphorylation is required before ATP can open CFTR channels, there is far less agreement with respect to how this is achieved. Two mechanisms have been proposed: 1) an R-domain blocking mechanism whereby an unphosphorylated R domain blocks ATP binding or hydrolysis and, conversely, a phosphorylated R domain could promote ATP binding (23, 37); and 2) a coupling mechanism whereby R-domain phosphorylation is required to couple ATP binding and hydrolysis to channel opening and closing (5, 20). The R-domain blocking mechanism is supported by observations made in several laboratories that deletion of the R domain produces a channel (R less CFTR) that has ATP-dependent but PKA-independent gating activity (23, 28). Moreover, the addition of PKA-treated R domain increased the channel activity of R-less-CFTR (23, 25, 37). The R-domain coupling mechanism, while difficult to reconcile with gating by R-less-CFTR, is supported by the observation that ATP analogs can photolabel CFTR in the absence of R-domain phosphorylation (5, 34). In addition, R-domain phosphorylation alters the Km but not the Vmax for ATP hydrolysis (20). Because cellular ATP concentrations are well in excess of the reported Km values, these data suggest that phosphorylation would have minimal effects on the rate of ATP hydrolysis under in vivo conditions. While these results can be viewed as supporting the second mechanism, they are not inconsistent with the first mechanism. If R-domain phosphorylation were to alter the affinity of CFTR for ATP, it would increase the Vmax/Km for ATP hydrolysis without changing Vmax. Phosphorylation would also increase the rate, but not the extent, of photolabeling. Because the rate of photolabeling has not been reported (5, 34), these studies do not rule out the blocking mechanism.
While there is considerable controversy with regard to the mechanisms that control CFTR gating, most models incorporate the following features: 1) kinase-dependent phosphorylation, most likely on the R domain; 2) channel gating coupled to ATP binding or hydrolysis; and 3) channel closing upon the loss of nucleotide. A kinetic schema incorporating these features is shown in Fig. 6. While the model cannot describe features of the channel that require multiple ATP binding sites (1, 5), it is sufficient for the purposes of this discussion. As shown in Fig. 6A, two conformations of the unliganded CFTR are envisioned: one, a closed form CFTRC, that can bind ATP and one, an inactive form CFTRI, that cannot bind ATP. Phosphorylation shifts the equilibrium between CFTRC and CFTRI, with phosphorylation at activating sites favoring CFTRC and phosphorylation at inhibiting sites favoring CFTRI. Dephosphorylation of activating and inhibiting sites by phosphatases has the opposite effect. The effects of individual phosphorylations are additive, so that phosphorylation on multiple activating sites shifts CFTRI/CFTRC equilibrium further toward CFTRC than does phosphorylation at a single activating site. However, at sufficiently high concentrations of ATP, all CFTR has bound ATP, regardless of the level of phosphorylation. Channel gating (Fig. 6B) is envisioned as involving four states: two closed states, one with bound ATP and one without bound ATP; and two open states, one with bound ATP and one with bound ADP-Pi. The open channel with bound ATP is necessitated by recent studies demonstrating channel gating in the presence of nonhydrolyzable ATP analogs, albeit with Po <5% of that for ATP (1, 35). While only one hydrolysis cycle is shown, a similar cycle exists for each of the closed forms of CFTR in Fig. 6A. The individual rate constants for each of these cycles may or may not differ from one another. In the presence of forskolin, or PKA for excised patches, our CFTR mutants are assumed to represent partially phosphorylated forms of CFTR, with intermediate values for the CFTRC/CFTRI equilibrium constant between those for unphosphorylated and fully phosphorylated CFTR. Both the R-domain blocking and R-domain coupling mechanisms are compatible with this model. If CFTRI is unable to bind ATP, the model describes the R-domain blocking mechanism; but if ATP can be bound and hydrolyzed by CFTRI without generating an open channel, this model describes the R-domain coupling mechanism.
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CFTR is a member of the ABC transporter superfamily. All other members of this family are thought to be ATP-dependent pumps that translocate substrates across, from, or within cell membranes. The mechanism for CFTR activity proposed in Fig. 6 is analogous to that for ABC transporters. One well-studied example is the maltose transporter in gram-negative bacteria (10, 12). A periplasmic binding protein, MalE, undergoes a conformational change upon binding maltose with high affinity. In the bound conformation, MalE binds to extracellular regions of a closed maltose transporter (MalFGK2). This induces a conformational change in the NBDs (MalK), allowing ATP to be bound and/or hydrolyzed. ATP hydrolysis causes a second conformational change in the transmembrane domains (MalFG) that 1) opens a transmembrane passageway for substrate entry into the cell and 2) distorts the conformation of MalE, thereby reducing its affinity for the substrate. Substrate is then able to enter the cell, and the release of nucleotide returns the transporter to the closed conformation, which is unable to bind unliganded MalE. Similar mechanisms are likely used by ABC transporters that bind substrate directly. For CFTR, phosphorylation of the R domain is analogous to the binding of liganded MalE. This allows ATP binding and/or hydrolysis to alter the conformation of the TMDs and open a channel. The channel remains open until nucleotide is released. For the maltose transporter, substrate transport is coupled to ATP hydrolysis by the fact that only when liganded MalE is present can ATP hydrolysis open the passageway, and the presence of the binding protein prevents the loss of ligand during transport. For CFTR, the R domain does not block the channel, and chloride can move into or out of the cell while the channel is open. Also, because the release of nucleotides does not dephosphorylate the R domain, multiple channel openings, each of which is coupled to ATP hydrolysis, are possible without rephosphorylating the R domain.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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