Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel (R-CFTR)
2 Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211
3 Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
Correspondence to Tzyh-Chang Hwang: hwangt{at}health.missouri.edu
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ABSTRACT |
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Key Words: chloride channel single-channel kinetics ABC transporter gating mode phosphorylation
Abbreviations used in this paper: ABC, ATP-binding cassette; BIM, bisindolylmaleimide; CHO, Chinese hamster ovary; CFTR, cystic fibrosis transmembrane conductance regulator; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; NBD, nucleotide binding domain; NMDG-Cl, N-methyl-D-glucamine chloride; PKI, PKA inhibitor; R, regulatory; WT, wild type.
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INTRODUCTION |
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Several different kinetic models have been proposed to explain ATP-dependent gating of CFTR (e.g., Zeltwanger et al., 1999; Ikuma and Welsh, 2000
; Vergani et al., 2003
), but it remains unclear how each NBD utilizes ATP to open and close the channel. Compared with voltage-gated cation channels, CFTR gating is slow, with each opening and closing events lasting for hundreds of milliseconds to seconds. Our understanding of CFTR gating relies heavily on single-channel kinetic analysis that demands recordings that last for tens of minutes in order to collect sufficient gating events. However, stationary recordings for this length of time are technically challenging because PKA-dependent phosphorylation is absolutely required for WT-CFTR function and CFTR is often dephosphorylated by membrane-associated protein phosphatases in inside-out patches. The channel rundown by dephosphorylation could affect not only single-channel kinetic parameters, but also "steady-state" doseresponse relationships (e.g., Szellas and Nagel, 2003
). The severity of this technical problem varies among different expression systems. In some cases, dephosphorylation-induced channel rundown in excised patches takes place within seconds upon removal of PKA (e.g., Weinreich et al., 1999
).
Another technical difficulty that hampers kinetic studies is to obtain membrane patches that contain only one channel. Although analytical methods have been developed to quantify gating kinetics from multichannel data (e.g., Fenwick et al., 1982; Csanády, 2000
), these methods still cannot replace classical single-channel dwell-time analysis because of the necessity of using a preconceived gating model. The discovery of phosphorylation-independent CFTR constructs provides a potential solution for the problems described above. Recently, Csanády et al. (2000)
constructed a CFTR whose R domain (amino acids 634836) is completely deleted. This construct, when expressed in Xenopus oocytes, exhibits robust basal activity. However, addition of PKA causes a further increase in the
R-CFTR currents (
30%), suggesting the presence of phosphorylation site(s) outside the R domain. This conclusion contradicts an early report by Rich et al. (1993)
that shows that all the functionally important phosphorylation sites are located in the R domain (see DISCUSSION for details).
One potential reason for this discrepancy is the difference in the expression system (mammalian cell line vs. Xenopus oocytes). We therefore examined if the R-CFTR construct made by Csanády et al. (2000)
is sensitive to modulation by PKA-dependent phosphorylation when it is expressed in a mammalian expression system. Our results show that the constitutive activity of
R-CFTR channels is not modified by application of cAMP agonists in cell-attached recordings or exogenous PKA in excised inside-out patches, indicating that these
R-CFTR mutant channels are completely phosphorylation independent, at least when expressed in Chinese hamster ovary (CHO) cells. Because the expression level is low, patches containing only a single channel can be frequently obtained. We found that gating parameters of
R-CFTR are very similar, if not identical, to those of WT-CFTR. These include single-channel Po, ATP doseresponse relationship, and opening and closing rates. It is therefore concluded that, contrary to Csanády's report, this
R-CFTR construct provides a valuable model system to study CFTR gating at a single-channel level when expressed in CHO cells. Indeed, our single-channel kinetic analysis in the presence of ADP reveals, for the first time, a new closed state, consistent with the idea that ADP inhibits CFTR opening by competing with ATP for a binding site. Surprisingly, ADP also reduces the open time of the channel, suggesting that binding of nucleotides affects the stability of the open state. This unexpected finding leads to further studies elaborated in the accompanying paper.
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MATERIALS AND METHODS |
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Construction of the CFTR Mutant
The DNA construct (pBudCE4.1 split R-CFTR) for expressing the
R-CFTR channel has been described in detail previously (Ai et al., 2004
). In brief, the cDNAs encoding CFTR residues 1633 and residues 8371480 were subcloned into the expression vector pBudCE4.1 (Invitrogen) under the control of the CMV and EF1-
promoters, respectively. The plasmids pGEMHE-1-633 and pGEMHE-837-1480 were a gift from D. Gadsby (Rockefeller University, New York, NY).
Transient Expression of CFTR
To transiently express CFTR, CHO cells were grown in 35-mm tissue culture dishes one day before transfection. The plasmid pBudCE4.1 split R-CFTR or pcDNA3.1 containing wild-type (WT)CFTR were cotransfected with pEGFP-C3 (CLONTECH Laboratories, Inc.) encoding green fluorescent protein using SuperFect transfection reagent (QIAGEN) according to manufacturer's protocols. The cells were incubated at 27°C for 12 d before use.
Whole-cell Experiments
Pipette electrodes were made from Corning 7056 glass capillaries (Warner Instrument). The pipette resistance was 3 M
in the bath solution. The membrane potential was held with an EPC9 patch-clamp amplifier (HEKA) at 0 mV, voltage ramps (±100 mV, 2 s in duration, every 6 s) or voltage steps were generated with Pulse software (HEKA) to create the IV relationships. Current traces were filtered at 1 kHz with a built-in four-pole Bessel filter and then digitized at 2 kHz into the computer. The currents were recorded at room temperature (
23°C). The pipette solution contained (in mM) 85 aspartic acid, 5 pyruvic acid, 10 EGTA, 20 tetraethylammonium-chloride, 5 tris creatine phosphate, 10 MgATP, 2 MgCl2, 5.5 glucose, and 10 HEPES (pH 7.4 with CsOH). Aspartate was replaced with Cl for some experiments under symmetrical [Cl] condition. The bath solution contained (in mM) 150 NaCl, 2 MgCl2, 1 CaCl2, 5 glucose, and 5 HEPES (pH 7.4 with NaOH). 20 mM sucrose was added to the bath solution to prevent activation of swelling-induced currents.
Single-channel Experiments
Patch-clamp experiments in excised inside-out mode were described in detail previously (e.g., Zeltwanger et al., 1999). In brief, single-channel CFTR currents were recorded at room temperature (
23°C) with an EPC9 or EPC10 amplifiers (HEKA). Data were filtered at 100 Hz with an eight-pole Bessel filter (Warner Instrument) and captured onto a hard disk at a sampling rate of 500 Hz. For cell-attached patches, the pipette potential is held at 50 mV. For excised inside-out patches, the membrane potential is held at 50 mV. The pipette solution contained (in mM) 140 N-methyl-D-glucamine chloride (NMDG-Cl), 2 MgCl2, 5 CaCl2, and HEPES (pH 7.4 with NMDG). The superfusion solution for inside-out patches contained (in mM) 150 N-methyl-D-glucamine chloride (NMDG-Cl), 10 EGTA, 10 HEPES, 8 TRIS, and 2 MgCl2 (pH 7.4 with NMDG).
Reagents
Forskolin, purchased from Alexis, was stored as 20 mM stock in DMSO at 4°C. Genistein were purchased from Sigma-Aldrich and stored as 100 mM stock in DMSO at 20°C. CPT-cAMP (8-(4-chlorophenylthio)-cAMP) and Mg-ATP, purchased from Sigma-Aldrich, were stored in water at 20°C. PKA was purchased from Promega. PKI was purchased from Alexis. Bisindolylmaleimide (BIM) and PMA were purchased from Sigma-Aldrich. ADP was purchased from Calbiochem.
Data Analysis
The steady-state mean current and single channel amplitudes were calculated with Igor software (Wavemetrics). Curve fitting of the doseresponse relationships were also performed with Igor. Single channel Po, mean burst duration, and mean interburst duration were obtained from patches with few channels (<4) with a program developed by Dr. Csanády (Csanády, 2000). To determine the true ATP-dependent parameters, the ATP-independent brief closures (flickers) observed within a burst had to be removed. To do that, we employed the three-state model shown below: scheme 1
where O and C are open and closed states, respectively. B is a blocked state induced by an intrinsic blocker (Zhou et al., 2001). Rate constants rCO, rOC, rOB, and rBO are extracted by a simultaneous fit to the dwell-time histograms of all conductance levels. Mean interburst, burst durations, and channel open probability (Po), were calculated as
ib = 1/rCO,
b = (1/rOC)(1 + rOB/rBO), Po = 1/(1 + rOC/rCO + rOB/rBO), respectively. Then, the burst time (
b) corresponds to the ATP-dependent open time of the channel (
o), and the interburst time (
ib) corresponds to the ATP-dependent closed time of the channel (
c). To avoid confusions, we will use throughout this manuscript the terms open and closed time to refer to these ATP-dependent events.
For single-channel dwell-time analysis (Fig. 8), we pooled the current recordings obtained from several patches containing only a single R-CFTR channel. The digitized current records were further filtered digitally at 50 Hz and analyzed with a program developed by Y. Sohma in our lab. The program automatically detects events using a 50% threshold-crossing method. The pooled open and closed interval durations were log-binned at a resolution of 10 bins per log unit and plotted as the square root of the number of intervals per bin with the constant bin width on a logarithmic time axis (Sigworth and Sine, 1987
). To exclude the short-lived, ATP-independent flickery events (Zhou et al., 2001
), we reconstructed dwell-time histograms by setting up a cutoff of 50 ms (e.g., Carson et al., 1995
; Li et al., 1996
; Zeltwanger et al., 1999
). Least square estimation with sums of exponential components was performed to obtain open and closed time constants. The intervals shorter than dead times were excluded from the fitting.
All values are presented as mean ± SEM. Student's t test was performed with Sigmaplot (SPSS Science), P < 0.05 was considered significant.
Online Supplemental Material
The supplemental material for this paper consists of two figures (available at http://www.jgp.org/cgi/content/full/jgp.200409227/DC1). Fig. S1 shows the ATP doseresponse for R-CFTR mutant channels. Fig. S2 shows the change from slow gating mode to fast gating mode as we switch from on-cell mode to excised inside-out mode in WT-CFTR channels.
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RESULTS |
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Although the cAMPPKA pathway constitutes the major regulatory mechanism for CFTR, PKC may also be involved in the phosphorylation of CFTR (Tabcharani et al., 1991; Button et al., 2001
; Chappe et al., 2003
; Chen et al., 2004
). We therefore tested whether
R-CFTR is sensitive to PKC modulation by using both a PKC activator, PMA, and an inhibitor, BIM. Fig. 2 A shows a continuous current trace of whole-cell
R-CFTR currents. A ramp voltage >100 mV was applied every 6 s to monitor any conductance change over the time course of the experiment. The basal
R-CFTR current is insensitive to 1 µM BIM (n = 7). In cell-attached patches (Fig. 2 B), the PKC activator PMA (500 nM) did not change
R-CFTR channel activity (n = 6). In both cases, however, genistein, serving as a positive control, increased
R-CFTR currents.
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The R-CFTR channel activity in the presence of different [ATP] was monitored to obtain a doseresponse relationship. The single channel open probability (Po) was plotted against [ATP], and a fit with the Michaelis-Menten equation yielded a K1/2 value of 89 ± 25 µM, which, within the error range, is quite similar to that of WT-CFTR (137 ± 28 µM; Zeltwanger et al., 1999
). Despite some scattered differences, the Po values for
R-CFTR are also very close to those of WT-CFTR (see Fig. S1, available at http://www.jgp.org/cgi/content/full/jgp.200409227/DC1).
Fig. 4 A shows representative single-channel traces of R-CFTR in the presence of different [ATP]. Similar to WT-CFTR,
R-CFTR exhibits two types of closings, an ATP-sensitive long closing and flickery closings that appear to be ATP-independent. For single-channel kinetic analysis, recordings from patches containing up to three channels were analyzed using the program developed by Csanády (2000)
. Fig. 4 B shows relationships between [ATP] and mean open time (
o), or mean closed time (
c). Both gating parameters are remarkably similar to those of WT-CFTR (Zeltwanger et al., 1999
). Thus, the function of NBDs, the gating machinery for CFTR, appears to be intact despite a complete removal of the R domain. We therefore conclude that
R-CFTR, being phosphorylation independent and behaving similarly to WT channels, is an ideal construct to explore more extensively the gating mechanisms of CFTR.
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Effect of ADP in Fast Gating Mode
It has been reported that ADP inhibits the ATP-dependent WT-CFTR activity (Anderson and Welsh, 1992; Gunderson and Kopito, 1994
; Winter et al., 1994
; Schultz et al., 1995
). Although it is proposed that ADP and ATP compete for a binding site that opens the channel (Anderson and Welsh, 1992
), a recent report by Randak and Welsh (2003)
, however, suggest a potential alternative mechanism for nucleotides' action on CFTR gating. Because of the structural similarity between ATP and ADP, the competitive mechanism seems appealing, but definite single-channel kinetic evidence is lacking. We first show that ADP inhibits ATP-induced
R-CFTR currents in a dose-dependent manner (Fig. 6). As predicted for a competitive mechanism, the magnitude of ADP-dependent inhibition is a function of [ATP]. As the [ATP] is increased, a higher [ADP] is needed to achieve a same magnitude of inhibition. The apparent Ki values for ADP inhibition are as follows: 26.3 ± 5.1 µM with 75 µM ATP, 51.2 ± 5.1 µM with 200 µM ATP, 180.1 ± 38.6 µM with 500 µM ATP, and 205.1 ± 43.7 µM with 1 mM ATP (Fig. 6 B). These results strongly suggest that ADP inhibits the CFTR channel by competitively binding to an ATP binding site.
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The patch membranes were exposed to 1 mM ATP for 12 min, then to 1 mM ADP plus 1 mM ATP for 13 min, and back to 1 mM ATP again. This bracketing was very important as a control because of the spontaneous switch of the gating mode. Fig. 9 A shows a continuous current trace obtained in a patch with a single channel. As expected, the closed time is dramatically prolonged in the presence of ADP. In addition, the current trace clearly shows a reversible shortening of the open time in the presence of ADP. Due to the short duration of the slow gating mode, it was very difficult to obtain a large number of patches that could be used for kinetic analysis since most of the time the channel switched to the fast gating mode before the appropriate bracketing was performed. Out of >30 patches, only 6 of them could be analyzed. The results showed that the open time was reduced by an average of 54 ± 6%. The mean open time was reduced from 828 ± 53 ms with 1 mM ATP alone, to 384 ± 41 ms with 1 mM ATP plus 1 mM ADP; the mean closed time increased from 614 ± 101 ms with 1 mM ATP to 1993 ± 373 ms for 1 mM ATP plus 1 mM ADP. Detailed studies of this novel effect of ADP are described in the accompanying paper.
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DISCUSSION |
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One known mechanism for this rundown is the dephosphorylation of the CFTR by membrane-associated protein phosphatases. Although including phosphatase inhibitors such as okadaic acid may be of some value to inhibit phosphatases 1 and 2A (e.g., Berger et al., 1993; Hwang et al., 1993
), no known specific inhibitors have been reported for phosphatase 2C, a major class of phosphatases that dephosphorylate CFTR (Travis et al., 1997
; Luo et al., 1998
). To complicate matters further, a recent study suggests that PP2C through its close association with CFTR is a strong candidate for the endogenous membrane-bound phosphatase responsible for dephosphorylating the channel inside an intact cell as well as in the membrane after patch excision (Zhu et al., 1999
). It is therefore questionable whether using phosphatase inhibitors can amend this problem.
In theory, one can also include PKA throughout the experiment to promote CFTR phosphorylation. The antagonism between added kinases and membrane-associated phosphatases allows for stable, steady-state conditions during recording. However, it is inevitable that the kinase/phosphatase-driven transitions between partially phosphorylated and dephosphorylated states become nested within transitions solely due to ATP-dependent gating, adding further variability to the extracted kinetic parameters.
Rich et al. (1991) first reported that CFTR channels become constitutively active after removal of part of the R domain (amino acids 708835). Since several of the PKA consensus sequences remain in this partial
R construct, the channel remains responsive to cAMP stimulation and therefore is not immune to dephosphorylation-dependent rundown in excised patches (compare Ma et al., 1997
). Later Rich et al. (1993)
reported a phosphorylation-independent channel, obtained after the mutation of serine 660 in their partial
R construct. More recently, Csanády et al. (2000)
obtained a functional CFTR channel with the R domain completely deleted (amino acids 634836). However, they reported that the basal activity of this
R-CFTR, expressed in Xenopus oocytes, could be increased
30% by the application of PKA. Although a suggestion of the presence of PKA phosphorylation site(s) outside of the R domain was made, so far there is no evidence that any serine residue outside of the R domain can be phosphorylated in intact CFTR in vivo or in vitro (Cheng et al., 1991
; Picciotto et al., 1992
; Neville et al., 1997
). In fact, using mass spectroscopic methods, the most sensitive biochemical technique for identification of phosphorylated peptides, Csanády et al. (2005)
failed to detect phosphorylation of extra R domain serine. Indeed, after removal of 15 PKA consensus sites (major as well as minor), 14 of which are located in the R domain, CFTR can no longer be activated by PKA (Seibert et al., 1999
).
We expressed the R-CFTR construct reported by Csanády et al. (2000)
in CHO cells and characterized in detail its regulation and gating mechanisms. Contrary to the results reported by Csanády et al. (2000)
, we found that the basal
R-CFTR currents cannot be enhanced by the application of cAMP agonists in cell-attached and whole-cell experiments, nor can the single-channel Po of
R-CFTR be increased by the presence of exogenous PKA in excised inside-out patches. The reason for this discrepancy is unknown. It should be noted, however, that two different expression systems were used: mammalian cells versus Xenopus oocytes. Nevertheless, the fact that
R-CFTR, expressed in CHO cells, is resistant to dephosphorylation makes this construct an ideal candidate for studying ATP-dependent gating, at least in CHO cells.
The current report shows that ATP-dependent gating of R-CFTR is very similar to WT-CFTR gating. The single-channel Po, the ATP dose response, and the open and closed times agree very well with our previous studies of WT-CFTR channels (Zeltwanger et al., 1999
). However the kinetic parameters in the current manuscript do not agree with those reported by Csanády et al. (2000)
. At maximal ATP concentration (2 mM), their Po is much lower than the one reported in this paper (0.19 ± 0.01 vs. 0.39 ± 0.01). The open time of the channel is about the same (297 ± 35 ms vs. 325 ± 21 ms), but their closed time is significantly longer (1297 ± 181 ms vs. 388 ± 34 ms). We do not know what accounts for these differences. Different expression systems may be partly responsible. Factors other than phosphorylation/dephosphorylation (e.g., PIP2, PDZ binding proteins) should also be considered (Wang et al., 2000
; Raghuram et al., 2001
; Himmel and Nagel, 2004
).
Perhaps because of its inefficient assembly, R-CFTR expression in the cell membrane is low. We were able to obtain a significant number of patches that contain only a single
R-CFTR channel. This distinct advantage, plus the fact that
R-CFTR is resistant to dephosphorylation-induced rundown, makes this
R-CFTR construct a useful tool to explore the mechanisms of CFTR gating. It may also provide a useful system to test the effect of pharmacological reagents since the phosphorylation step is bypassed (e.g., Ai et al., 2004
).
Different Gating Modes
Overcoming the dephosphorylation-induced rundown also allows us to obtain long-lasting steady recordings of R-CFTR channels, which reveal shifts of gating modes. Mode shifts are commonly observed in ion channel studies, reported for numerous other channels such as the Ca-activated maxi K channels (McManus and Mangleby, 1988
), fast Cl channels from rat skeletal muscle (Blatz and Mangleby, 1986
), and Na channels from mouse myocardial cells (Bohle and Benndorf, 1995
). Although in most cases the exact mechanism for mode shifts is yet to be identified, some are known to be phosphorylation dependent (e.g., Yue et al., 1990
). In the current study, we do not understand the mechanism for the mode shifts of CFTR, but it appears that CFTR's gating modes are not related to PKA-dependent phosphorylation.
Does WT-CFTR exhibit mode shifts? Although the current study does not provide direct evidence that these mode shifts occur in WT-CFTR, we speculate that they do exist for the following reasons. First, the reported Po values for WT-CFTR vary in the literature (see Table I). Some of these differences may be due to different levels of phosphorylation (e.g., a lower Po value reported by Mathews et al., 1998). However, there seems to be a pattern of observations regarding the Po of WT-CFTR. For example, most studies showed a Po of
0.4 for WT-CFTR, a number very similar to that of
R-CFTR in fast and slow gating modes, but much higher Po values have been reported (Gunderson and Kopito, 1994
; Ramjeesingh et al., 1999
). Interestingly, the Po value of
0.7 in these studies is similar to that of
R-CFTR in the high Po mode. Second, it is known for years that CFTR gating is much slower when studied in the cell-attached configuration than that in excised inside-out patches (compare Fig. 1 and Fig. 3; also Hwang et al., 1997
vs. Zeltwanger et al., 1999
). This change of gating behavior of WT-CFTR is consistent with our observation that
R-CFTR usually exhibits slow gating right after patch excision. Lastly, in a few patches with WT-CFTR activated with cAMP agonists in the cell-attached configuration before patch excision, we did observe a change from slow to fast gating mode after patch excision (Fig. S2).
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Effect of ADP on CFTR Gating
After establishing R-CFTR channels as useful tools for studying CFTR gating, we first investigated the mechanism of ADP-induced inhibition of CFTR channel activity. Previous studies showed that ADP can inhibit CFTR channel activity presumably by competing with ATP for a common binding site since the magnitude of inhibition is diminished upon increasing [ATP] (Anderson and Welsh, 1992
; Schultz et al., 1995
). Subsequently, Gunderson and Kopito (1994)
and Winter et al. (1994)
showed that ADP increased the closed time with little effect on the open time. Using noise analysis of macroscopic CFTR currents, Schultz et al. (1995)
confirmed that ADP decreases the Po of CFTR by decreasing the opening rate. However, since ATP is likely hydrolyzed during CFTR gating cycle, many potential kinetic steps can be affected to account for this inhibition. Without demonstrating an ADP-dependent closed state, a definite kinetic mechanism for this ADP inhibition of the opening rate remains to be determined.
In this paper we provide, for the first time, single-channel kinetic evidence that ADP competes with ATP by binding to the binding site that opens the channel. First, the ADP doseresponse curves shift to the right as the ATP concentration increases, so that the Ki is higher with higher ATP concentration, supporting a competitive mechanism (also see Anderson and Welsh, 1992). More importantly, using single-channel dwell time analysis, we were able to observe a new closed state in the presence of ADP. This new closed state was suggested by Schultz et al. (1995)
, but their only single-channel recording was not long enough to collect enough events to adequately separate it from the ATP-dependent closed state. Indeed, the fact that both ATP-dependent gating and ADP-induced inhibition are slow events demands extremely long recordings for single-channel analysis. The steady channel activity of
R-CFTR allows us to use a higher ADP/ATP ratio that effectively separates two closed time constants. With this result, we conclude then that ADP competitively inhibits CFTR channel opening. It should be noted that our results do not indicate that an ADP-bound channel cannot open. It simply means that ADP-bound channels have a much lower opening rate compared with ATP-bound ones.
Which NBD does ADP bind to inhibit channel opening? Recent biochemical studies (Szabo et al., 1999; Aleksandrov et al., 2002
; Basso et al., 2003
) suggest that ATP is occluded in NBD1 (i.e., the off rate is extremely slow). Therefore, association and dissociation of ATP at NBD1 cannot account for the electrophysiologically observed, millisecond-to-second gating transitions. Vergani et al. (2003)
found that mutations of either Walker A lysine, K464, or K1250 result in a rightward shift of the doseresponse of ATP in CFTR activation. The same paper shows that the D1370N mutation at the Walker B motif in NBD2 also decreases the apparent affinity of ATP (compare Bompadre et al., 2005
). Incorporating both electrophysiological and biochemical data, Vergani et al. (2003)
proposes (Scheme 2)
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Surprisingly, we found that ADP not only induces a new closed state but also shortens the open time. The open time of the channel was reduced in the presence of ADP by as much as 54%. An effect of ADP on the open time has been suggested by Weinreich et al. (1999). While performing macroscopic relaxation analysis, they observed that ADP increases the relaxation rate of macroscopic currents (reflecting channel closing) upon nucleotide removal. Like what has been discussed above, the mechanism of this effect of ADP is unknown without detailed single-channel kinetic analysis. Here we provide the first single-channel kinetic evidence that ADP affects the open state stability of the CFTR channel. This novel effect of ADP on the open time constant cannot be easily explained by the gating model described in Scheme 2 since both nucleotide binding sites have to be occupied by ATP for channel opening. We hypothesize that ADP binds at a site that is different from the binding site for channel opening, as proposed by Weinreich et al. (1999)
. In theory, one would predict the presence of a discrete open time constant corresponding to the new open state of the channel when ADP is bound. While our single-channel dwell time analysis shows a shortening of the open time in the presence of ADP, we were not able to resolve two open time constants in WT-CFTR. We reason that if the difference between the time constant of ADP-bound open state and that of ATP-bound one is not extremely large, dissecting different states is a technical difficulty that is hard to overcome. Nevertheless, the use of the
R-CFTR construct gives us a unique opportunity to be able to collect enough single-channel events that reveal this phenomenon. Observing the slow gating mode allows us to verify the ADP effect on the open time. In the accompanying paper, we used various CFTR mutants to further elaborate the kinetic mechanism for this interesting observation.
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ACKNOWLEDGMENTS |
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This work is supported by the National Institutes of Health (DK55835 and HL53445 to T.C. Hwang). T. Ai is a recipient of postdoctoral fellowship from the American Heart Association. S.G. Bompadre is a recipient of NRSA (DK062565). Y. Sohma is supported by the Japan Society for the Promotion of Science (15590196).
Olaf S. Andersen served as editor.
Submitted: 3 December 2004
Accepted: 8 February 2005
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REFERENCES |
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