ABCMdb

ABCC7 p.Thr1134Phe

None has been submitted yet.

Publications

PMID: 10744680 [PubMed] Yue H et al: "The two halves of CFTR form a dual-pore ion channel."

No. Sentence Comment

165 McDough observed (12) that wild-type CFTR and T1134F mutant CFTR occasionally showed a long-lived subconductance state with amplitude ϳ60% of the full conductance in Xenopus oocytes. Login to comment

PMID: 10811966 [PubMed] Zhang ZR et al: "Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes."

No. Sentence Comment

8 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 Construction of S341A-CFTR and T1134F-CFTR was described previously [35]. Login to comment
217 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
224 d P < 0.01 compared to block of T1134F-CFTR by DPC. Login to comment
253 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
257 However, both S341A-CFTR and T1134F-CFTR responded differently to block by NPPB and DPC. Login to comment
259 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 The order of sensitivity for block by NPPB at -100 mV was T1134F ‫ס‬ WT > S341A. Login to comment
261 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 For WT and T1134F-CFTR, the voltage dependence for block by DPC was decreased at pH 6.5 (P < 0.001). Login to comment
266 For WT, S341A-CFTR, and T1134F-CFTR, the voltage dependence for block by NPPB was decreased at pH 6.5. Login to comment
267 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 To determine the basis for this discrepancy, we turned to analysis of single T1134F-CFTR channels. Login to comment
269 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 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 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 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 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
350 (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 (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 Block of single T1134F-CFTR channels by NPPB. Login to comment
384 Mean values for kinetic parameters in T1134F-CFTR channels are given in Table 2. Login to comment
388 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 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 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 The order of sensitivity at -100 mV was: WT ‫ס‬ T1134F-CFTR > S341A-CFTR. Login to comment
424 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 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 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 Consistent with this hypothesis, the duration of ␶C2 in the presence of NPPB was greater for T1134F than for WT. Login to comment
465 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 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 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 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 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 In the WT channel and T1134F-CFTR, the voltage dependence of block by DPC was reduced at pH 6.5. Login to comment
478 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

PMID: 11124965 [PubMed] Kogan I et al: "Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity."

No. Sentence Comment

188 Such detailed molecular mapping studies have been initiated by McCarty and co-workers (52) in studies of the voltage-dependent block by DPC and NPPB in wild type and mutant (S341A and T1134F) CFTR. Login to comment

PMID: 11380256 [PubMed] Gupta J et al: "Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region."

No. Sentence Comment

158 Previously, a more dramatic mutation at one of these residues (T1134F) was shown to cause a slight reduction in unitary conductance (13). Login to comment

PMID: 11557589 [PubMed] McCarty NA et al: "Identification of a region of strong discrimination in the pore of CFTR."

No. Sentence Comment

60 Mutants K335E, K335F, T338A, T339A, S341A, S341T, T1134A, and T1134F were prepared as previously described (33). Login to comment
143 Relative permeabilities for WT and mutant CFTRs for monovalent anions CFTR n NO3 Br SCN I ClO4 Acetate Isethionate Glutamate Gluconate WT 16 1.35Ϯ0.01 1.19Ϯ0.02 2.42Ϯ0.06 0.36Ϯ0.01 0.10Ϯ0.01 0.15Ϯ0.00* 0.24Ϯ0.01 0.24Ϯ0.01 0.18Ϯ0.01 K335A 5 1.35Ϯ0.01 1.36Ϯ0.03 3.10Ϯ0.11† 0.75Ϯ0.02† 0.12Ϯ0.01 0.06Ϯ0.01† 0.07Ϯ0.01† 0.07Ϯ0.01† 0.08Ϯ0.01† K335F 7 1.51Ϯ0.03† 1.36Ϯ0.02† 2.73Ϯ0.14 0.99Ϯ0.03† 0.20Ϯ0.02† 0.13Ϯ0.01 0.18Ϯ0.03 0.30Ϯ0.02 0.20Ϯ0.02 K335E 5 1.24Ϯ0.04 1.17Ϯ0.02 2.60Ϯ0.06 1.10Ϯ0.03† 0.23Ϯ0.01† 0.10Ϯ0.01† 0.11Ϯ0.01† 0.10Ϯ0.01† 0.11Ϯ0.01† T338A 5 1.74Ϯ0.07† 1.59Ϯ0.02† 4.35Ϯ0.24† 2.56Ϯ0.13† 1.84Ϯ0.08† 0.07Ϯ0.01† 0.06Ϯ0.01† 0.08Ϯ0.01† 0.08Ϯ0.01† T338E 3 3.65Ϯ0.19† 1.94Ϯ0.04† 4.29Ϯ0.13† 2.41Ϯ0.24† 1.18Ϯ0.06† 0.16Ϯ0.03 0.37Ϯ0.05† 0.36Ϯ0.01† 0.22Ϯ0.03 T339A 5 1.47Ϯ0.01 1.29Ϯ0.03 2.65Ϯ0.06 0.57Ϯ0.02† 0.24Ϯ0.04 0.10Ϯ0.02 0.19Ϯ0.02 0.18Ϯ0.01 0.15Ϯ0.01 S341A 6 1.91Ϯ0.02† 1.42Ϯ0.01† 3.10Ϯ0.09† 0.59Ϯ0.00*† 0.09Ϯ0.00* 0.11Ϯ0.01† 0.12Ϯ0.00*† 0.11Ϯ0.00*† 0.12Ϯ0.00*† S341E 12 2.01Ϯ0.10† 1.46Ϯ0.05† 2.81Ϯ0.18 0.84Ϯ0.00*† 0.31Ϯ0.03† 0.20Ϯ0.01 0.23Ϯ0.02 0.19Ϯ0.01 0.19Ϯ0.02 S341T 5 1.81Ϯ0.05† 1.39Ϯ0.03 3.15Ϯ0.15† 0.41Ϯ0.01 0.07Ϯ0.00* 0.05Ϯ0.00*† 0.06Ϯ0.00*† 0.03Ϯ0.01† 0.06Ϯ0.01† T1134A 6 1.43Ϯ0.02 1.30Ϯ0.02 2.66Ϯ0.02 0.46Ϯ0.00*† 0.06Ϯ0.00*† 0.08Ϯ0.01† 0.10Ϯ0.01† 0.11Ϯ0.01† 0.10Ϯ0.00*† T1134F 5 1.31Ϯ0.07 1.17Ϯ0.05 2.50Ϯ0.10 0.63Ϯ0.01† 0.08Ϯ0.00* 0.13Ϯ0.01 0.09Ϯ0.01† 0.18Ϯ0.02 0.13Ϯ0.01 T1134E 4 1.68Ϯ0.02† 1.39Ϯ0.05† 2.37Ϯ0.18 0.19Ϯ0.03† 0.20Ϯ0.03 0.06Ϯ0.01† 0.09Ϯ0.01† 0.08Ϯ0.01† 0.10Ϯ0.01† Values are means Ϯ SE with only data from the hyperpolarizing ramp protocol; n, no. of oocytes. Relative permeability, permeability of anion x to that of Cl. Anions are listed in order of increasing ionic radius. Login to comment
167 Selectivity sequences for WT and mutant CFTRs CFTR Selectivity Sequence by Relative Permeability WT SCNϾϾNO3 ϾBrϾClϾϾIϾisethionateϭglutamateϾgluconateϭacetateϾClO4 K335A SCNϾϾBrϭNO3 ϾClϾIϾϾClO4 Ͼgluconateϭisethionateϭglutamateϭacetate K335F SCNϾϾNO3 ϾBrϾClϭIϾϾglutamateϾgluconateϭClO4 ϭisethionateϾacetate K335E SCNϾϾNO3 ϾBrϭIϾClϾϾClO4 Ͼgluconateϭisethionateϭglutamateϭacetate T338A SCNϾϾIϾϾClO4 ϭNO3 ϾBrϾClϾϾgluconateϭisethionateϭglutamateϭacetate T338E SCNϾNO3 ϾIϾBrϾClO4 ϾClϾϾisethionateϭglutamateϾgluconateϭacetate T339A SCNϾϾNO3 ϾBrϾClϾϾIϾϾClO4 ϭisethionateϭglutamateϭgluconateϾacetate S341A SCNϾNO3 ϾBrϾClϾϾIϾϾgluconateϭisethionateϭglutamateϭacetateϭClO4 S341E SCNϾNO3 ϾBrϾClϾIϾϾClO4 Ͼisethionateϭacetateϭglutamateϭgluconate S341T SCNϾϾNO3 ϾBrϾClϾϾIϾϾClO4 ϭisethionateϭgluconateϭacetateϭglutamate T1134A SCNϾϾNO3 ϾBrϾClϾϾIϾϾglutamateϭisethionateϭgluconateϭacetateϭClO4 T1134F SCNϾϾNO3 ϾBrϾClϾϾIϾϾglutamateϾacetateϭgluconateϾisethionateϭClO4 T1134E SCNϾNO3 ϾBrϾClϾϾClO4 ϭIϾgluconateϭisethionateϭglutamateϭacetate L856 A REGION OF STRONG DISCRIMINATION IN THE CFTR PORE AJP-Lung Cell Mol Physiol • VOL 281 • OCTOBER 2001 • www.ajplung.org out propagation to distant sites. Login to comment
191 Relative conductances for WT and mutant CFTRs for monovalent anions CFTR n NO3 Br SCN I ClO4 Acetate Isethionate Glutamate Gluconate WT 16 0.87Ϯ0.01 0.77Ϯ0.01 0.18Ϯ0.01 0.25Ϯ0.01 0.23Ϯ0.01 0.55Ϯ0.01 0.50Ϯ0.01 0.57Ϯ0.02 0.56Ϯ0.02 K335A 5 0.88Ϯ0.04 0.77Ϯ0.02 0.30Ϯ0.02† 0.35Ϯ0.02 0.24Ϯ0.02 0.33Ϯ0.01† 0.32Ϯ0.02† 0.37Ϯ0.02† 0.38Ϯ0.02† K335F 7 1.21Ϯ0.05† 0.87Ϯ0.02† 0.55Ϯ0.02† 0.36Ϯ0.01† 0.19Ϯ0.01 0.34Ϯ0.01† 0.34Ϯ0.01† 0.41Ϯ0.01† 0.37Ϯ0.01† K335E 5 1.16Ϯ0.05† 0.91Ϯ0.02† 0.59Ϯ0.02† 0.51Ϯ0.02† 0.28Ϯ0.01 0.22Ϯ0.01† 0.25Ϯ0.01† 0.22Ϯ0.01† 0.24Ϯ0.01† T338A 5 1.20Ϯ0.13† 1.03Ϯ0.06† 0.98Ϯ0.12† 0.82Ϯ0.02† 0.50Ϯ0.04† 0.18Ϯ0.05† 0.08Ϯ0.01† 0.31Ϯ0.05† 0.29Ϯ0.05† T338E 3 3.66Ϯ0.36† 1.53Ϯ0.09† 1.80Ϯ0.12† 1.39Ϯ0.11† 0.87Ϯ0.03† 0.36Ϯ0.04† 0.56Ϯ0.17 0.44Ϯ0.03† 0.48Ϯ0.03† T339A 5 1.01Ϯ0.02† 0.77Ϯ0.03 0.22Ϯ0.01 0.31Ϯ0.03 0.23Ϯ0.01 0.38Ϯ0.02† 0.48Ϯ0.01 0.48Ϯ0.01 0.52Ϯ0.01 S341A 6 1.67Ϯ0.01† 1.08Ϯ0.01† 0.63Ϯ0.03† 0.26Ϯ0.00* 0.15Ϯ0.01† 0.63Ϯ0.01† 0.54Ϯ0.02 0.63Ϯ0.01 0.63Ϯ0.01 S341E 12 1.74Ϯ0.11† 1.14Ϯ0.02† 1.81Ϯ0.06† 0.48Ϯ0.01† 0.35Ϯ0.02† 0.28Ϯ0.01† 0.69Ϯ0.02† 0.65Ϯ0.01† 0.68Ϯ0.01† S341T 5 0.85Ϯ0.02 0.82Ϯ0.01 0.29Ϯ0.01† 0.22Ϯ0.01 0.13Ϯ0.01† 0.48Ϯ0.01 0.45Ϯ0.02 0.43Ϯ0.02 0.55Ϯ0.01 T1134A 6 0.83Ϯ0.02 0.78Ϯ0.01 0.24Ϯ0.01† 0.21Ϯ0.01 0.09Ϯ0.01† 0.39Ϯ0.01† 0.38Ϯ0.01† 0.39Ϯ0.01† 0.40Ϯ0.01 T1134F 5 0.68Ϯ0.03† 0.69Ϯ0.03† 0.36Ϯ0.01† 0.07Ϯ0.01† 0.16Ϯ0.01 0.48Ϯ0.02 0.30Ϯ0.02† 0.22Ϯ0.01† 0.32Ϯ0.02† T1134E 4 0.99Ϯ0.02† 1.00Ϯ0.02† 0.50Ϯ0.02† 0.20Ϯ0.03 0.26Ϯ0.02 0.32Ϯ0.03† 0.34Ϯ0.01† 0.34Ϯ0.03† 0.34Ϯ0.03† Values are means Ϯ SE with only data from the hyperpolarizing ramp protocol; n, no. of oocytes. Relative conductance, conductance of anion x to that of Cl. Anions are listed in order of increasing ionic radius. Login to comment
213 Vrev Cl in ND96 bath solution for WT and mutant CFTRs CFTR n Vrev Cl WT 16 -21.24Ϯ0.59 K335A 5 -22.12Ϯ0.35 K335F 7 -21.92Ϯ0.90 K335E 5 -22.88Ϯ0.36 T338A 5 -26.97Ϯ0.79* T338E 3 -20.58Ϯ1.07 T339A 5 -22.21Ϯ0.98 S341A 6 -21.21Ϯ0.56 S341E 12 -28.77Ϯ1.36* S341T 5 -26.62Ϯ1.43* T1134A 6 -28.33Ϯ1.23* T1134F 5 -19.74Ϯ0.73 T1134E 4 -27.54Ϯ1.27* Values are means Ϯ SE; n, no. of oocytes. Login to comment
218 T1134F CFTR exhibited a similar pattern except that GNO3/GCl and GBr/GCl were decreased and GSCN/GCl was increased compared with T1134A CFTR. Login to comment
329 This behavior was retained in K335F and T1134F CFTR but lost in all other mutants. Login to comment
388 Selectivity between Cl- and the divalent anion S2O3 2CFTR n GS2O3/GCl WT 16 0.39Ϯ0.01 K335A 5 0.37Ϯ0.01 K335F 7 0.39Ϯ0.01 K335E 5 0.34Ϯ0.01* T338A 5 0.38Ϯ0.01 T338E 3 0.70Ϯ0.08* T339A 5 0.39Ϯ0.02 S341A 6 0.27Ϯ0.01* S341E 12 0.54Ϯ0.01* S341T 5 0.38Ϯ0.01 T1134A 6 0.34Ϯ0.02 T1134F 5 0.33Ϯ0.01* T1134E 4 0.44Ϯ0.05 Values are means Ϯ SE; n, no. of oocytes. Login to comment
430 Further evidence against a discrete selectivity filter is the greater than sixfold increase in relative affinity for I- exhibited by T1134F CFTR. Login to comment

PMID: 21796338 [PubMed] Qian F et al: "Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant."

No. Sentence Comment

212 In contrast, mutations in the analogous part of TM12 have been found to have little effect on conductance, which was reported as being unaltered in T1134A and M1137A [15] and slightly decreased in the less conservative T1134F [31]. Login to comment

PMID: 9922375 [PubMed] Sheppard DN et al: "Structure and function of the CFTR chloride channel."

No. Sentence Comment

183 site could be transferred from S341 to S1141 in M12 while another mutation in M12, T1134F, increased the affinityStudies of CFTR variants lacking the consensus glycosylation sequences in extracellular loop (ECL) 4 of DPC block (87). Login to comment

PMID: 9922378 [PubMed] Schultz BD et al: "Pharmacology of CFTR chloride channel activity."

No. Sentence Comment

252 nine residue 1134 to a phenylalanine caused a threefold improvement in the affinity for DPC (T1134F, 74 mM). Login to comment

PMID: 10542444 [PubMed] Hwang TC et al: "Molecular pharmacology of the CFTR Cl- channel."

No. Sentence Comment

102 Interestingly, the DPC-binding site could be transferred from Ser341 to Ser1141 in M12 by simultaneously mutating residues in M6 and M12, whereas another mutation in M12, Thr1134Phe, increased the affinity of DPC block, but decreased single-channel conductance37. Login to comment
104 They speculatedthatthehydroxylside-chainofSer341probably interacts with the carboxyl moiety of DPC via hydrogen bonding, and that this interaction is stabilized by the phenyl ring of the mutant Thr1134Phe interacting with the second phenyl ring of DPC (for a molecular model of MSD1 R MSD2 NBD1 NBD2 P P P ATP G O C O C O C ATP ATP ADP +Pi ADP +Pi ATP ADP +Pi Cl- Cl- Cl- PKA PP2A PKA PP2C trends in Pharmacological Sciences Fig. 3. Login to comment

PMID: 8810276 [PubMed] Price MP et al: "Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR."

No. Sentence Comment

263 First, the mutation T1134F decreases the single-channel conductance of CFTR (18). Login to comment
282 First, the mutation T1134F decreases the single-channel conductance of CFTR (18). Login to comment

PMID: 7522483 [PubMed] McDonough S et al: "Novel pore-lining residues in CFTR that govern permeation and open-channel block."

No. Sentence Comment

70 (E) T1134F mutation, showing increased block at all voltages. Login to comment
78 Affinity and Voltage Dependence for Block of CFTR Variants by DPC Construct TM Ko( - 100) (PM) 0 I-V Relation n Properties Wild type Wild type low [Cl-], (10 mM) K335E 6 K335F 6 T338A 6 T339A 6 S341A 6 S341T 6 S1118A 11 T1134A 12 T1134F 12 S1141A 12 Triple 6,12 276 f 14 181 f 13" 303 -t 14 351 * 15' 220 * 14 284 * 47 1251 f 116a 530 f 80" 243 * 37 230 * 20 74 * 3" 220 * 13 325 * 26b 0.41 f 0.01 0.32 f 0.02" 0.42 f 0.01 0.42 f 0.02 0.36 f 0.02" 0.44 * 0.12 0.49 * 0.03" 0.35 f 0.09 0.40 f 0.02 0.35 * 0.02" 0.41 f 0.01 0.42 f 0.03 0.21 * O.Ol",b Linear, E,,, = -8 f 1 mV Ere\ = +48+2mV Inward rectification Linear Linear Linear Strong inward rectification Inward rectification Linear Linear Linear Linear Strong inward rectification Affinity for DPC was determined empirically at -100 mV, from whole-cell currents measured in the presence of 200 uM DPC (see Experimental Procedures). Login to comment
93 In TM 12, the T1134F mutation increased DPC affinity to 74 f 3 PM but did not change 8 (Figure 3E; Figure 4), suggesting that this mutation stabilizes DPC binding at its main site, S341. Login to comment
95 The increased binding of DPC on the T1134F mutant was confirmed at the single-channel level, as described below. Login to comment
96 T1134F single channels recorded in excised, inside-out patches had a smaller amplitude than wild type, with conductance y = 5.8 f 0.2 pS (range 4.1-6.4 pS; n = 14; Figure 5) versus y = 8.0 + 0.4 pS for wild type (McCarty et al., 1993). Login to comment
98 As with S341A, however, the T1134F mutant channel kinetics were qualitatively similar to wild type, with seconds-long openings, uninterrupted at positive voltages and interrupted by brief closures at negative voltages (Figure 5; Figure 7). Login to comment
99 Also like the wild type, T1134F occasionally showed a long-lived subconductance state, with amplitude -60% of the full conductance. Login to comment
100 As noted previously for the wild-type channel (McCarty et al., 1993), the subconductance state in T1134F was not blocked by DPC (Figure 6B), as though DPC cannot reach its binding site at S341 when the channel is in this state. Login to comment
101 A Further Test for Open-Channel Block Application of DPC to the cytoplasmic surface of excised inside-out patches resulted in blocker-induced closures of 3-to Qfold longer duration for T1134F than for wild type, consistent with the increased DPC affinity of the T1134F mutation (Figure 6). Login to comment
102 The longer residence time of DPC on the T1134F channel enabled us to resolve the kinetics of blockade at several DPC concentrations with V, = -100 mV. Login to comment
104 The DPC-induced closed times of T1134F averaged 1.8 + 0.1 ms (averaged from 20, 50, and 100 PM) and were independent of DPC concentration (Table 2; Figure 7; Figure 8). Login to comment
111 Open circles, wild-type; closed circles, T1134F; open boxes, S341A; closed boxes, triple mutation (S341A-Mll- 401-T1142F). Login to comment
127 Exceptions were the blockade of outward currents for T1134F, owing to the greatly increased affinity for DPC, and the triple mutation, owing to the changed voltage dependence of block. Login to comment
137 For the T1134F mutation, which has kinetics favorable for detailed study, the rate constant for blocking (inverse of the open time constant) increases linearly with [DPC], but the rate constant for unblocking is independent of [DPC]. Login to comment
138 620 A Wild-type T1134F (TM-1 2) S341 A (TM-6) Figure 5. Login to comment
139 Mutations 5341A and T1134F Lower Single-Channel Conductance without Changing Kinetics Wild-type and T1134F traces are from inside-out patches excised in 1 mM ATP. Login to comment
143 (A) Comparison of wild-type, T1134F, and S341A conductances, all with V,, = -100 mV and F, flow-pass cutoff frequency) = 1 kHz. Login to comment
146 The same is true for wild-type (McCarty et al., 1993) and T1134F (data not shown) channels. Login to comment
151 Although many observations presented here are consistent with the simple view that DPC is an open-channel blocker at wild-type and mutant CFTR channels, a complication is added by the observations that unblocked T1134F displays an extra closed time constant compared with wild type, and that this time constant is similar to the residence time of DPC. Login to comment
153 We believe the unblocked T1134F channel only coincidentally has a time constant near that of the lifetime of DPC on the T1134F channel. Login to comment
157 In TM 12, mutation T1134F strongly increases DPC affinity, as confirmed by single-channel measurements, and significantly lowers the single-channel conductance. Login to comment
160 The identification of 629 B - * C T1134F T1134F Unblocked 50 pM DPC 810 / 0123456789 0 1 2 3 4 5 6 7 8 9 (msec) D Time Figure 6. Login to comment
161 The Residence Time of DPC Is Longer on the T1134F Channel than on the Wild-Type Channel Single-channel records are from inside-out patches excised in 1 mM ATP with V, = -100 mV, filtered at 1 kHz. Login to comment
168 (C) Representative closed time histogram for the unblocked T1134F channel. Login to comment
169 (D) Representative closed time histogram for T1134F with 50 uM DPC added to the cytoplasmic side. Login to comment
200 Time Constants for Block of T1134F Single Channels by Various DPC Concentrations IDPCI n Area, T,? Login to comment
208 Further Kinetic Analysis of the Data for DPC Blockade of T1134F Open circles, I/T, where T represents open time; closed circles, l/r, where T represents the second (DPC-induced) closed time constant within a burst. Login to comment
228 Similarly, in TM 12, mutation T1134F strengthens DPC binding and reduces single-channel conductance from 8 to <6 pS. Login to comment
229 DPC binds at the same electrical position, 40% of the distance through the membrane field, in the T1134F mutant as in the wild type, evidence that T1134F makes no gross disruption in the binding site. Login to comment
240 The relative orientation of the side chains on the phenylalanines of K335F and T1134F may explain the opposite results of phenylalanines placed at these nearly equivalent positions. Login to comment
241 In our model of TM 12, the phenyl ring of T1134F points directly into the pore and lies along and perpendicular to the second phenyl NeUrClfl 632 Figure 9. Login to comment
267 MDR has a rather broad substrate specificity, perhaps be- (C) Same as (B), with mutations K335F and T1134F highlighted. Login to comment
268 Note that K335F lies parallel to the second phenyl ring of DPC, and T1134F lies perpendicular. Login to comment
299 Single-channel currents were recorded from manually stripped oocytes injected with 70 ng of T1134F cRNA or with 100 ng of S341A plus 0.8 ng of &adrenergic receptor cRNA. Login to comment
308 T1134F single-channel traces were amplified at 20 dB per decade during acquisition. Login to comment

PMID: 10866956 [PubMed] Zhang ZR et al: "Interaction between permeation and gating in a putative pore domain mutant in the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

334 In particular, mutation T1134F in TM12 lowered single-channel conductance to b03;6 pS and increased DPC affinity without changing the voltage dependence of block (McDonough et al., 1994). Login to comment