ABCMdb

PMID: 15767296

Bompadre SG, Cho JH, Wang X, Zou X, Sohma Y, Li M, Hwang TC
CFTR gating II: Effects of nucleotide binding on the stability of open states.
J Gen Physiol. 2005 Apr;125(4):377-94. Epub 2005 Mar 14., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Login to comment 4 ABCC7 p.Asp1370Asn Single-channel kinetic analysis of ⌬R/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. Login to comment 5 ABCC7 p.Glu1371Ser ⌬R/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of ~100 s, estimated from current relaxation upon nucleotide removal. Login to comment 7 ABCC7 p.Glu1371Ser To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Login to comment 9 ABCC7 p.Glu1371Ser Single-channel kinetic analysis for ⌬R/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. Login to comment 11 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. Login to comment 35 ABCC7 p.Lys464Ala Previous studies suggested that ATP hydrolysis at NBD1 was involved in the opening of the channel since mutations of the Walker A lysine at NBD1 (e.g., K464A) decrease the channel opening rate (Carson et al., 1995; Gunderson and Kopito, 1995; cf. Login to comment 37 ABCC7 p.Lys464Ala However, Powe et al. (2002) reexamined the gating kinetics of K464A mutant CFTR and found little difference in the opening rate between this mutant and WT-CFTR. Login to comment 45 ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser To study in more detail the effect of nucleotide binding on the open time of CFTR, we used both macroscopic current relaxation and single-channel kinetic analysis for CFTR mutants with impaired ATP hydrolysis: specifically, the D1370N and E1371S mutations in NBD2. Login to comment 47 ABCC7 p.Asp1370Asn Mutation of this aspartate to asparagine (D1370N) results in channels that exhibit longer open and closed times (Gunderson and Kopito, 1995; Vergani et al., 2003), resembling the elusive "slow gating" mode described in our previous paper (Bompadre et al., 2005). Login to comment 49 ABCC7 p.Glu1371Ser Mutation of this glutamate to serine produces a channel (E1371S) that presents long "locked-open" times (Aleksandrov et al., 2000; Vergani et al., 2003). Login to comment 51 ABCC7 p.Asp1370Asn Single-channel dwell-time analysis of ⌬R/D1370N-CFTR channels shows unequivocally that the open time of the channel is decreased in the presence of ADP. Login to comment 52 ABCC7 p.Glu1371Ser Relaxation analysis of macroscopic ⌬R/E1371S-CFTR currents upon nucleotide removal shows two different relaxation time constants in the presence of ADP and ATP, indicating that ADP induces a different locked-open state. Login to comment 53 ABCC7 p.Glu1371Ser Moreover, studies of ⌬R/E1371S-CFTR open time in patches containing a single channel at three different ATP concentrations show a change of the relative frequency of the different open times, suggesting the presence of an ATP-binding site, occupancy of which affects the stability of the open state. Login to comment 54 ABCC7 p.Glu1371Ser Similar results were obtained in E1371S mutants constructed in the WT background. Login to comment 56 ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser M A T E R I A L S A N D M E T H O D S Construction of CFTR Mutants CFTR mutations E1371S and D1370N were introduced into the plasmid pBQ4.7 WT-CFTR (Powe et al., 2002) by using the QuikChange kit (Stratagene). Login to comment 57 ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser The 0.9-kb PflMI-XhoI fragments containing the mutations from the pBQ4.7 were used to substitute the corresponding regions in pBudCE4.1 split ⌬R CFTR (Ai et al., 2004) to obtain pBudCE4.1 ⌬R/D1370N and pBudCE4.1 ⌬R/E1371S CFTR. Login to comment 58 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Similarly, the 0.9-kb PflMI-EcoRV fragments were used to replace the corresponding ones in pcDNA 3.1 WT-CFTR or pcDNA 3.1 K464A-CFTR (Powe et al., 2002) to generate pcDNA3.1 D1370N, pcDNA 3.1 E1371S, and pcDNA3.1 K464A/ E1371S CFTR constructs. Login to comment 66 ABCC7 p.Glu1371Ser For dwell-time analysis of ⌬R/E1371S-CFTR data, we pooled the current records obtained from several patches containing only one channel. Login to comment 81 ABCC7 p.Asp1370Asn Fig. S1 shows the ATP dose response for the D1370N mutant. Login to comment 82 ABCC7 p.Asp1370Asn R E S U L T S Effect of ADP on ⌬R/D1370N-CFTR In the accompanying paper (Bompadre et al., 2005), we demonstrated that ADP can shorten the open time of ⌬R-CFTR and this effect is more prominent when the channel is in the slow gating mode with longer Figure 1. Login to comment 83 ABCC7 p.Asp1370Asn ADP shortens the open time of ⌬R/D1370N-CFTR channels. Login to comment 89 ABCC7 p.Asp1370Asn Since the D1370N mutants exhibit open and closed times on the order of seconds (i.e., they mimic the slow gating mode described in Bompadre et al., 2005), we decided to use this mutant to further study the effect of ADP on the open time. Login to comment 90 ABCC7 p.Asp1370Asn Vergani et al. (2003) found that the ATP dose response for the D1370N mutant shifted to the right compared with that of WT channel. Login to comment 92 ABCC7 p.Asp1370Asn To study the effect of ADP on single-channel kinetics, we introduced the D1370N mutation into the ⌬R-CFTR background because ⌬R-CFTR is insensitive to dephosphorylation-induced rundown, and we can more easily obtain patches with fewer channels for kinetic analysis (Bompadre et al., 2005). Login to comment 93 ABCC7 p.Asp1370Asn In excised inside-out patches, ⌬R/D1370N-CFTR channels were opened with 1 mM ATP, and subsequent application of 1 mM ATP plus 1 mM ADP inhibited the currents by an average of 63 Ϯ 5% (n ϭ 5). Login to comment 102 ABCC7 p.Asp1370Asn Furthermore, the open time constant for D1370N-CFTR may still be too short for isolating the putative ADP-bound open state. Login to comment 103 ABCC7 p.Glu1371Ser We therefore constructed another mutant, E1371S, which has an open time on the order of tens or hundreds of seconds because the ATP hydrolysis is abolished at NBD2 (Aleksandrov et al., 2000; Vergani et al., 2003). Login to comment 105 ABCC7 p.Glu1371Ser Macroscopic current relaxation for ⌬R/E1371S-CFTR channels in the presence of ATP and ADP. Login to comment 106 ABCC7 p.Glu1371Ser (A) A sample trace of current relaxations for ⌬R/E1371S-CFTR channels opened with 1 mM ATP, and subsequently with 1 mM ATP ϩ 2 mM ADP. Login to comment 108 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser (C) The current decay upon removal of ATP plus ADP is fitted with a double exponential function with time constants of 12.9 Ϯ 0.1 and 105 Ϯ 3 s. ADP Effect on ⌬R/E1371S Mutant We first introduced the E1371S mutation into the ⌬R background in order to study the behavior of this channel in the presence or absence of ADP without having to worry about the possible effect of dephosphorylation on the channel open time. Login to comment 115 ABCC7 p.Glu1371Ser Compared with the relaxation time course with ATP alone, the current relaxation of ⌬R/E1371S-CFTR channels opened by ATP plus ADP is faster. Login to comment 124 ABCC7 p.Glu1371Ser Macroscopic current relaxation for ⌬R/E1371S-CFTR opened with 10 ␮M ATP. Login to comment 125 ABCC7 p.Glu1371Ser ⌬R/ E1371S-CFTR channels were activated with 10 ␮M ATP until the current reached a steady state. Login to comment 130 ABCC7 p.Glu1371Ser The dash line represents current relaxation of ⌬R/ E1371S-CFTR upon removal of 1 mM ATP (from Fig. 2 B). Login to comment 133 ABCC7 p.Glu1371Ser ATP Concentration Dependence of ⌬R/E1371S Current Relaxation Our experiments with ADP suggest the presence of a nucleotide binding site, occupancy of which by ATP or ADP can affect the stability of the locked-open state. Login to comment 137 ABCC7 p.Glu1371Ser Fig. 3 A shows a representative relaxation experiments for ⌬R/E1371S-CFTR channels opened with 10 ␮M ATP. Login to comment 142 ABCC7 p.Glu1371Ser Single-channel recording of ⌬R/E1371S-CFTR in the presence of 1 ␮M ATP. Login to comment 151 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Single-channel Analysis of ⌬R/ E1371S Mutant Fig. 4 shows a continuous recording of a single ⌬R/ E1371S mutant channel in the presence of 1 mM ATP that lasts for ~50 min. Login to comment 157 ABCC7 p.Glu1371Ser Single-channel recording of ⌬R/E1371S-CFTR in the presence of 10 ␮M ATP. Login to comment 172 ABCC7 p.Glu1371Ser We also analyzed one long recording (‫08ف‬ min) of ⌬R/E1371S-CFTR at 3 ␮M ATP (Fig. 6 B). Login to comment 183 ABCC7 p.Glu1371Ser Single-channel dwell time analysis of ⌬R/ E1371S-CFTR. Login to comment 223 ABCC7 p.Glu1371Ser ATP Concentration Dependence of E1371S-CFTR Current Relaxations Before we attempt to specify to which NBD ATP binds to affect the stability of the open state, we need to be sure that the [ATP] dependence of the open state stability is not solely due to deletion of the R domain. Login to comment 224 ABCC7 p.Glu1371Ser We therefore characterized the current relaxation for the E1371S mutation in the WT background. Login to comment 227 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Unlike ⌬R/E1371S-CFTR, the E1371S mutants in the WT background express very well. Login to comment 233 ABCC7 p.Glu1371Ser We would like to point out that the relaxation curve obtained for the ⌬R/E1371S mutant (Fig. 2 B) shows only one exponential decay (␶ ϭ 100 s) at 1 mM. Login to comment 235 ABCC7 p.Glu1371Ser Macroscopic current relaxation of E1371S-CFTR currents. Login to comment 236 ABCC7 p.Glu1371Ser (A) Sample trace of current relaxations for E1371S-CFTR channels activated with 10 ␮M ATP ϩ PKA, or with 1 mM ATP ϩ PKA. Login to comment 240 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser semble current for ⌬R/E1371S-CFTR, even with pooling of 47 patches, is still much smaller (‫58ف‬ pA) than the macroscopic currents obtained with the E1371S mutant in the WT background (‫085ف‬ pA). Login to comment 241 ABCC7 p.Glu1371Ser Therefore, the minor fast component of the exponential decay is difficult to resolve in the ⌬R/E1371S-CFTR. Login to comment 242 ABCC7 p.Glu1371Ser When performing the relaxation experiments in E1371S-CFTR channels, after several minutes of washout, we can still observe a single channel that remains open (Fig. 9). Login to comment 247 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser Current Relaxations of the K464A/E1371S Mutant According to the most recent model (Scheme 2 in the accompanying paper) for CFTR gating (Vergani et al., 2003), NBD1 has little role in the gating transitions since the off rate is extremely slow, and ATP binding at NDB2 precedes channel opening. Login to comment 249 ABCC7 p.Glu1371Ser Kinetic analysis of the last E1371S-CFTR channel that remains open after removal of ATP. Login to comment 250 ABCC7 p.Glu1371Ser (A) Sample trace of the current relaxation of E1371S-CFTR channels upon ATP washout. Login to comment 262 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser The K464A mutation shortens the locked-open time of E1371S-CFTR. Login to comment 263 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser (A) Sample trace of K464A/E1371S-CFTR channels in the presence of 1 mM ATP ϩ PKA. Login to comment 267 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser (C) Sample trace of K464A/E1371S-CFTR channels in the presence of 10 ␮M ATP (blue curve). Login to comment 273 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser Since the K464 mutation has a mild trafficking defect (Cheng et al., 1990; unpublished data), and ⌬R-CFTR already suffers from low expression, we decided to make the K464A/E1371S double mutant construct in the WT background. Login to comment 275 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser The current decay of the K464A/E1371S-CFTR channel currents is indeed faster than that of E1371S-CFTR, resulting in a shorter relaxation time constant (19.60 Ϯ 0.01 s) upon washout of 1 mM ATP (Fig. 10 B). Login to comment 276 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser This relaxation time constant is even shorter when the K464A/E1371S-CFTR channel is opened with 10 ␮M ATP (13.95 Ϯ 0.02 s). Login to comment 277 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser Since the number of K464A/E1371S-CFTR channels is relatively low due to a moderate trafficking defect, it is easier to observe microscopic channel behavior at 10 ␮M ATP (Fig. 10 C). Login to comment 278 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser As shown previously for ⌬R/E1371S-CFTR (Fig. 5), the current trace reveals that K464A/E1371S-CFTR channels also exhibit numerous brief openings that last for tens to hundreds of milliseconds in the presence of 10 ␮M ATP. Login to comment 281 ABCC7 p.Glu1371Ser (A) A representative ⌬R/E1371S-CFTR current trace from an excised inside-out patch exposed to ATP-free solution for several minutes before 1 mM ATP was applied. Login to comment 283 ABCC7 p.Glu1371Ser The open times of these spontaneous openings from several patches containing ⌬R/E1371S-CFTR (C) or ⌬R-CFTR (D) were pooled together to construct survivor plots. Login to comment 289 ABCC7 p.Glu1371Ser Excised inside-out patches from cells expressing ⌬R/E1371S-CFTR channels were exposed to ATP-free perfusion solution for Ͼ5 min to determine the opening rate of spontaneous opening events. Login to comment 302 ABCC7 p.Glu1371Ser Although the E1371S mutation increases the lifetime of ATP-opened channel by Ͼ100-fold, it only minimally affects the open time constant for these spontaneous opening events. Login to comment 303 ABCC7 p.Glu1371Ser Despite the similarity between these open time constants for the spontaneous openings and the brief open time constant for the ⌬R/E1371S-CFTR in the presence of 10 ␮M ATP (Fig. 6 D), it is doubtful that the observed events at 10 ␮M ATP solely come from spontaneous openings because they appear more frequently than the spontaneous openings. Login to comment 304 ABCC7 p.Glu1371Ser We then quantify the kinetic step to the brief opening events using the ⌬R/ E1371S-CFTR data in the presence of 10 ␮M ATP. Login to comment 313 ABCC7 p.Glu1371Ser We used macroscopic current relaxation upon nucleotide removal to quantify the locked-open times for E1371S mutant CFTR. Login to comment 321 ABCC7 p.Asp1370Asn From single-channel analysis, all we could observe was a shortening of the mean open time for ⌬R- (Bompadre et al., 2005) or ⌬R/ D1370N-CFTR (Fig. 1). Login to comment 322 ABCC7 p.Glu1371Ser In contrast, the macroscopic current relaxation of ⌬R/E1371S-CFTR clearly shows the presence of two components in the relaxation once the channels are opened by ATP plus ADP (Fig. 2), strongly suggesting the presence of two different locked-open states. Login to comment 323 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Furthermore, the relaxation of E1371S-CFTR channel currents in the presence of different ATP concentrations reveals the presence of two locked-open states, and an [ATP]-dependent shift in the distribution of each state (confirmed by ⌬R/ E1371S single-channel data). Login to comment 325 ABCC7 p.Glu1371Ser Perhaps because of a much smaller current amplitude, we did not resolve two relaxation time constants in the presence of 1 mM ATP for ⌬R/E1371S-CFTR channels. Login to comment 326 ABCC7 p.Glu1371Ser If we accept the fact that multiple locked-open states do exist for ⌬R/E1371S mutants at 1 mM ATP as shown by the single-channel analysis (Fig. 6), how can we ascertain that ADP induces another locked-open state rather than simply increases the relative occupancy of the short-lived locked-open state? Login to comment 342 ABCC7 p.Glu1371Ser Using similar dwell time analysis, we were able to detect multiple components in open time histograms (Fig. 6) for ⌬R/E1371S mutants. Login to comment 350 ABCC7 p.Glu1371Ser This same idea can also explain ADP`s effects on the current relaxation of ⌬R/E1371S mutants (Fig. 2). Login to comment 354 ABCC7 p.Lys1250Ala A similar short-lived open state was reported previously for K1250A-CFTR (␶o ϭ ‫052ف‬ ms), another hydrolysis-deficient mutant (Zeltwanger et al., 1999). Login to comment 356 ABCC7 p.Glu1371Ser Indeed, the mean lifetime of the spontaneous openings for ⌬R/E1371S is ‫004ف‬ ms. Login to comment 364 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser Furthermore, mutations that abolish ATP hydrolysis (e.g., K1250A and E1371S) dramatically prolong the open state (Gunderson and Kopito, 1995; Zeltwanger et al., 1999; Powe et al., 2002; Vergani et al., 2003). Login to comment 366 ABCC7 p.Lys464Ala Powe et al. (2002) showed that the K464A mutation shortens the open time by 40% at high [ATP]. Login to comment 367 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Interestingly, introducing the K464A mutations into the K1250A construct significantly decreases the locked-open time (Powe et al., 2002; Vergani et al., 2003). Login to comment 368 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Ser An equivalent observation is also made in the current report for K464A/E1371S mutants. Login to comment 371 ABCC7 p.Lys464Ala As mentioned above, Powe et al. (2002) showed that the K464A mutant exhibits a normal opening rate despite a lower ATP binding affinity at NBD1 for this mutant. Login to comment 380 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala This hypothesis is based on the observation that mutations that affect ATP binding at NBD1 (e.g., K464A) alter the stability of the open state of K1250A, suggesting an interaction between two ATP-binding sites. Login to comment 389 ABCC7 p.Glu1371Ser Indeed, the hydrolysis-deficient mutant, e.g., E1371S-CFTR, can assume a locked-open state for minutes, whereas WT channels only open for hundreds of milliseconds. Login to comment 394 ABCC7 p.Glu1371Ser In the current report, we show that the locked-open time of the E1371S mutant can be significantly shortened by all three maneuvers. Login to comment 397 ABCC7 p.Glu1371Ser Furthermore, from the energetic point of view, the absence of ligands at the dimer interface may also explain the short-lived openings observed for ⌬R/ E1371S-CFTR in the absence of ATP. Login to comment 398 ABCC7 p.Glu1371Ser Unsettled Issues One of the unsettled issues is the mechanism of short-lived openings of ⌬R/E1371S-CFTR that appear frequently in the presence of micromolar ATP. Login to comment 405 ABCC7 p.Lys464Ala Although we proposed that ATP binding at NBD2 plays a critical role in channel opening (see above), this idea is based more on default since we observed that the K464A mutation, which decreases ATP binding affinity at NBD1 (Basso et al., 2003), does not affect the opening rate (Powe et al., 2002). Login to comment 406 ABCC7 p.Lys1250Ala While we did observe a decrease of the opening rate by the K1250A mutation (Powe et al., 2002; cf. Login to comment 418 ABCC7 p.Glu1371Ser Single-channel kinetic analysis of the ⌬R/E1371S-CFTR mutant also revealed two ATP-dependent closed states (Fig. 6, A and B). Login to comment 429 ABCC7 p.Glu1371Ser A difference in closing transitions between WT and hydrolysis-deficient mutant CFTR may also explain the conundrum that ADP exerts a much larger effect on the locked-open time of ⌬R/E1371S channels than on the open time of the ⌬R channels. Login to comment 432 ABCC7 p.Glu1371Ser On the other hand, in E1371S-CFTR whose hydrolysis is abolished, thermoen- ergy is used for channel closing. Login to comment