ABCMdb

PMID: 10811966

Zhang ZR, Zeltwanger S, McCarty NA
Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes.
J Membr Biol. 2000 May 1;175(1):35-52., 2000-05-01 [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Thr1134Phe In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. Login to comment 50 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe Construction of S341A-CFTR and T1134F-CFTR was described previously [35]. Login to comment 217 ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe Affinity and voltage dependence for block by NPPB and DPC Bath pH Construct NPPB DPC KD(-100) (␮M) ⍜ n KD(-100) (␮M) ⍜ n WT 87.2 ± 3.4a 0.35 ± 0.01 5 201.4 ± 11.3 0.37 ± 0.01 6 7.5 S341A 287.7 ± 19.3b,c 0.38 ± 0.01c 5 1553.9 ± 121.0a 0.47 ± 0.01a 4 T1134F 83.3 ± 3.9d 0.17 ± 0.01b,d 5 123.8 ± 9.2a 0.39 ± 0.01 4 WT 50.1 ± 2.9 0.24 ± 0.01f 4 124.6 ± 7.2 0.27 ± 0.01f 5 6.5e S341A 72.8 ± 4.5b 0.26 ± 0.01f 5 379.3 ± 21.1a 0.51 ± 0.01a,g 4 T1134F 41.8 ± 4.0 0.14 ± 0.01b,f 4 40.3 ± 3.8a 0.29 ± 0.01a 5 Affinity for NPPB and DPC were determined empirically at -100 mV from whole-cell currents measured in the presence of 100 ␮M drug; for pH 6.5 experiments, [NPPB] was reduced to 50 ␮M. Login to comment 223 ABCC7 p.Ser341Ala c P < 0.01 compared to block of S341A-CFTR by DPC. Login to comment 224 ABCC7 p.Thr1134Phe d P < 0.01 compared to block of T1134F-CFTR by DPC. Login to comment 253 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe PORE-DOMAIN MUTATIONS DIFFERENTIALLY AFFECT BLOCK BY DPC AND NPPB We have shown previously that mutations S341A and T1134F decrease and increase, respectively, affinity for DPC at -100 mV [35]. Login to comment 256 ABCC7 p.Ser341Ala Block by both drugs was reduced in S341A-CFTR (Fig. 9A and C). Login to comment 257 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe However, both S341A-CFTR and T1134F-CFTR responded differently to block by NPPB and DPC. Login to comment 258 ABCC7 p.Ser341Ala Mutation S341A-CFTR altered the voltage-dependence for block by DPC but not for block by NPPB. Login to comment 259 ABCC7 p.Thr1134Phe Similarly, instead of changing the affinity for NPPB, as was shown for DPC, T1134F-CFTR reduced the voltage dependence for NPPB (Fig. 9A, Table 1). Login to comment 260 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe The order of sensitivity for block by NPPB at -100 mV was T1134F ‫ס‬ WT > S341A. Login to comment 261 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe Low pH treatment (during drug loading and assay) did not shift the order of sensitivity between WT, S341A-CFTR, and T1134F-CFTR for block by DPC (Fig. 9D). Login to comment 263 ABCC7 p.Thr1134Phe For WT and T1134F-CFTR, the voltage dependence for block by DPC was decreased at pH 6.5 (P < 0.001). Login to comment 264 ABCC7 p.Ser341Ala In contrast, the voltage dependence of block of S341A-CFTR was increased at pH 6.5 (P ‫ס‬ 0.038). Login to comment 266 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe For WT, S341A-CFTR, and T1134F-CFTR, the voltage dependence for block by NPPB was decreased at pH 6.5. Login to comment 267 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe BLOCKADE OF SINGLE T1134F-CFTR CHANNELS BY NPPB Our recordings of macroscopic CFTR current indicated that mutation T1134F-CFTR affected block by NPPB and DPC in different ways: the mutation increased the affinity at -100 mV for DPC without changing voltage-dependence [35], but decreased the voltage-dependence of block by NPPB without changing the affinity at -100 mV (Table 1). Login to comment 268 ABCC7 p.Thr1134Phe To determine the basis for this discrepancy, we turned to analysis of single T1134F-CFTR channels. Login to comment 269 ABCC7 p.Thr1134Phe We have shown previously that T1134F-CFTR channels in the absence of blocker exhibit kinetics somewhat divergent from those of WT-CFTR channels. Login to comment 272 ABCC7 p.Thr1134Phe More importantly, unblocked T1134F-CFTR channels exhibit two closed time-constants compared to only one seen in WT-CFTR (Fig. 10). Login to comment 276 ABCC7 p.Thr1134Phe Kinetics of single-channel block in excised patches at Vm ‫ס‬ -100 mV CFTR Variant [NPPB] (␮M) ␶O (msec) ␶C1 (msec) ␶C2 (msec) Area ␶C2 (%) n T (sec) WT 0 8.93 ± 1.38 0.24 ± 0.02 7 415 5 4.12a ± 0.32 0.23 ± 0.01 2.07 ± 0.14 2.5 4 216 25 2.40a,b ± 0.28 0.29 ± 0.06 2.35 ± 0.47 10.8b 4 238 T1134F 0 17.63 ± 1.68 0.31 ± 0.05 1.33 ± 0.13 3.0 3 160 5 8.46a ± 0.59 0.49a ± 0.03 3.14a ± 0.24 3.3 3 156 25 5.72a,b ± 0.27 0.63a ± 0.10 2.82a ± 0.45 18.3a,b 3 174 a P < 0.05 Compared to unblocked channels. Login to comment 283 ABCC7 p.Thr1134Phe Using data combined from multiple patches, the affinity (KD (s-c) ) for NPPB of single T1134F-CFTR channels at -100 mV was calculated to be 75 ␮M, compared to 35 ␮M for WT-CFTR. Login to comment 284 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe In contrast, the affinity for DPC was increased in T1134F-CFTR channels: KD (s-c) for DPC was 175 ␮M for WT-CFTR [33] and 88 ␮M in T1134F-CFTR [35]. Login to comment 313 ABCC7 p.Ser341Ala Block of single S341A-CFTR channels was not studied, due to the low single-channel conductance of this variant [35]. Login to comment 350 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe (A and B) Voltage dependence of NPPB affinity for wild-type and two mutations. Apparent affinity for NPPB was measured at pH 7.5 (A) and pH 6.5 (B) for WT (circles) and the two indicator mutations S341A-CFTR (triangles) and T1134F-CFTR (squares) which had previously been shown to decrease and increase, respectively, affinity for DPC. Login to comment 353 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe (C and D) Voltage dependence of DPC affinity for wild-type and two mutations. Apparent affinity for DPC was measured at pH 7.5 (C) and pH 6.5 (D) for WT (circles) and the two indicator mutations S341A-CFTR (triangles) and T1134F-CFTR (squares). Login to comment 380 ABCC7 p.Thr1134Phe Block of single T1134F-CFTR channels by NPPB. Login to comment 384 ABCC7 p.Thr1134Phe Mean values for kinetic parameters in T1134F-CFTR channels are given in Table 2. Login to comment 388 ABCC7 p.Thr1134Phe This is, however, consistent with our previous kinetic measurements in excised patches [35], wherein the on-rate and off-rate at -100 mV were shown to be fast (6.4 × 106 M-1 sec-1 and 560 sec-1 , respectively, for DPC block of T1134F-CFTR). Login to comment 419 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe Consistent with our previous results, the order of sensitivity to DPC at -100 mV was as follows (Table 1): T1134F-CFTR > WT > S341A-CFTR. Login to comment 420 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe Block of T1134F-CFTR and WT-CFTR by DPC exhibited the same voltage dependence, while in S341A-CFTR the drug appeared to bind deeper in the pore (closer to the extracellular end). Login to comment 422 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe The order of sensitivity at -100 mV was: WT ‫ס‬ T1134F-CFTR > S341A-CFTR. Login to comment 424 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe WT-CFTR and S341A-CFTR exhibited voltage dependencies that were not significantly different, while in T1134F-CFTR the drug appeared to bind less deeply within the pore (closer to the cytoplasmic end). Login to comment 425 ABCC7 p.Thr1134Phe Finally, while mutation T1134F altered the kinetics of block of single-channels by both DPC [35] and NPPB (present study), the effects of this mutation were not the same for the two drugs. Login to comment 429 ABCC7 p.Thr1134Phe It is likely that the extended length of the NPPB molecule places the phenyl ring in closer apposition to the phenylalanine at T1134F, which may introduce electrostatic interactions that stabilize the drug at its binding site. Login to comment 430 ABCC7 p.Thr1134Phe Consistent with this hypothesis, the duration of ␶C2 in the presence of NPPB was greater for T1134F than for WT. Login to comment 436 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu Two mutants were studied by Walsh: K335E, predicted to be at the extracellular end of TM6, and R347E, predicted to be at the cytoplasmic end of TM6. Login to comment 437 ABCC7 p.Lys335Glu Blockade of K335E-CFTR was diminished (KD ‫ס‬ 371 ␮M) while the voltage-dependence was not affected. Login to comment 438 ABCC7 p.Lys335Phe This is similar to our results with K335F-CFTR [35]. Login to comment 439 ABCC7 p.Arg347Glu Block of R347E-CFTR was also reduced (KD ‫ס‬ 1573 ␮M) and the voltage-dependence was increased significantly. Login to comment 440 ABCC7 p.Lys335Glu The effect of the K335E mutation is probably representative of a through-space interaction, wherein the negative charge introduced impedes the approach of the negatively charged drug, rather than disruption of an intimate interaction between NPPB and this lysine. Login to comment 442 ABCC7 p.Ser341Ala In this regard, our whole-cell data suggested that S341 provides an important component to the binding site for NPPB and for DPC, as mutation S341A reduced the efficacy of both drugs. Login to comment 443 ABCC7 p.Arg347Glu The results of the R347E mutation studied by Walsh [56] are difficult to interpret because this mutation causes disruption of channel structure due to loss of a salt bridge with an aspartic acid in TM8 [5]. Login to comment 465 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe A second observation is that decreasing bath pH does not have the same fold-effect on the apparent KD for WTand T1134F-CFTR (Table 1). Login to comment 466 ABCC7 p.Thr1134Phe We can make use of the combined data from whole-cell experiments in this study to estimate the effective cytoplasmic DPC concentration, because we know the KD calculated from DPC block of single channels (KD s-c ) [33, 35], as follows: [DPC]cyto ‫ס‬ KD s-c /(I/(Io-I)) (5) Using Eq. (5) and I/Io data from a number of bath DPC concentrations for WT (10 ␮M to 1 mM) and one concentration for T1134F-CFTR (100 ␮M) allows us to estimate the effective [DPC]cyto as a function of [DPC]bath, which exhibits a linear relationship at pH 7.5 with r2 ‫ס‬ 0.98. Login to comment 467 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe With 100 ␮M drug in the bath and assuming that loading is allowed to run to completion, we calculate that the drug concentration in the cell reaches very similar values for WTand T1134F-CFTR (84.3 ± 4.3 ␮M for WT and 74.5 ± 4.9 ␮M for T1134F-CFTR (mean ± SD; P > 0.17)). Login to comment 468 ABCC7 p.Thr1134Phe If the effects of reduced bath pH only reflected alteration of the extent of drug loading, we would expect that this effect would impact equally the currents measured from oocytes expressing WT-CFTR and T1134F-CFTR. Login to comment 469 ABCC7 p.Thr1134Phe However, the calculated effective [DPC]cyto at pH 6.5 was significantly different between the two variants: 143.3 ± 7.4 ␮M and 216.8 ± 12.1 ␮M for WT and T1134F-CFTR, respectively (mean ± SD; P < 0.002). Login to comment 475 ABCC7 p.Thr1134Phe In the WT channel and T1134F-CFTR, the voltage dependence of block by DPC was reduced at pH 6.5. Login to comment 476 ABCC7 p.Ser341Ala In S341A-CFTR, the voltage dependence of block was increased at pH 6.5. Login to comment 478 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe In contrast to these results with DPC, the voltage dependence of block by NPPB was reduced by low pH in the WT channel and in both the S341A-CFTR and T1134F-CFTR channels. Login to comment