ABCMdb

ABCC7 p.Thr1134Phe

Predicted by SNAP2:	A: N (53%), C: D (59%), D: D (80%), E: D (85%), F: D (80%), G: D (80%), H: D (85%), I: D (71%), K: D (91%), L: D (75%), M: D (75%), N: D (71%), P: D (85%), Q: D (80%), R: D (91%), S: N (82%), V: D (75%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: N, N: N, P: D, Q: N, R: N, S: N, V: N, W: D, Y: D,

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[hide] The two halves of CFTR form a dual-pore ion channe... J Biol Chem. 2000 Apr 7;275(14):10030-4. Yue H, Devidas S, Guggino WB
The two halves of CFTR form a dual-pore ion channel.
J Biol Chem. 2000 Apr 7;275(14):10030-4., 2000-04-07 [PMID:10744680]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8-4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br(-) > Cl(-) > I(-), whereas the ion selectivity of the small conductance channel is Br(-) = Cl(-) = I(-). The open probability (P(o)) of the large conductance is about 4-fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance.

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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

[hide] Direct comparison of NPPB and DPC as probes of CFT... J Membr Biol. 2000 May 1;175(1):35-52. 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 [PMID:10811966]

Abstract [show]
Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.

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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

[hide] Perturbation of the pore of the cystic fibrosis tr... J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21. Kogan I, Ramjeesingh M, Huan LJ, Wang Y, Bear CE
Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity.
J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21., 2001-04-13 [PMID:11124965]

Abstract [show]
Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl(-)) flux in affected epithelial tissues. CFTR is a Cl(-) channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl(-) permeates. Mutations of residues lining the channel pore (e.g. R347D) are typically thought to cause disease by altering the interaction of Cl(-) with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in the in vivo regulation of CFTR in health and disease.

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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

[hide] Asymmetric structure of the cystic fibrosis transm... Biochemistry. 2001 Jun 5;40(22):6620-7. Gupta J, Evagelidis A, Hanrahan JW, Linsdell P
Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.
Biochemistry. 2001 Jun 5;40(22):6620-7., 2001-06-05 [PMID:11380256]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore.

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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

[hide] Identification of a region of strong discriminatio... Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67. McCarty NA, Zhang ZR
Identification of a region of strong discrimination in the pore of CFTR.
Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67., [PMID:11557589]

Abstract [show]
The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

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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

[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.

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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

[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]

Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

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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

[hide] Pharmacology of CFTR chloride channel activity. Physiol Rev. 1999 Jan;79(1 Suppl):S109-44. Schultz BD, Singh AK, Devor DC, Bridges RJ
Pharmacology of CFTR chloride channel activity.
Physiol Rev. 1999 Jan;79(1 Suppl):S109-44., [PMID:9922378]

Abstract [show]
Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109-S144, 1999. - The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl- channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl-. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

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

[hide] Molecular pharmacology of the CFTR Cl- channel. Trends Pharmacol Sci. 1999 Nov;20(11):448-53. Hwang TC, Sheppard DN
Molecular pharmacology of the CFTR Cl- channel.
Trends Pharmacol Sci. 1999 Nov;20(11):448-53., [PMID:10542444]

Abstract [show]
Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is associated with a wide spectrum of disease. In the search for modulators of CFTR, pharmacological agents that interact directly with the CFTR Cl- channel have been identified. Some agents stimulate CFTR by interacting with the nucleotide-binding domains that control channel gating, whereas others inhibit CFTR by binding within the channel pore and preventing Cl- permeation. Knowledge of the molecular pharmacology of CFTR might lead to new treatments for diseases caused by the dysfunction of CFTR.

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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

[hide] Function of Xenopus cystic fibrosis transmembrane ... J Biol Chem. 1996 Oct 11;271(41):25184-91. Price MP, Ishihara H, Sheppard DN, Welsh MJ
Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR.
J Biol Chem. 1996 Oct 11;271(41):25184-91., [PMID:8810276]

Abstract [show]
To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, we studied Xenopus CFTR. We found that the anion permeability sequence of cAMP-activated Cl- currents in the apical membrane of Xenopus A6 epithelia differed from that of cAMP-activated Cl- currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length Xenopus CFTR cDNA from A6 cells. Expression of Xenopus CFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br- = I- > Cl- in Xenopus CFTR and Br- = Cl- > I- in human). In addition, the single-channel conductance of Xenopus CFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of Xenopus CFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br- = I- > Cl-) and single-channel conductance of hX1-6 resembled that of Xenopus CFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of Xenopus CFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and Xenopus CFTR and that the first extracellular loop influences channel gating.

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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

[hide] Novel pore-lining residues in CFTR that govern per... Neuron. 1994 Sep;13(3):623-34. McDonough S, Davidson N, Lester HA, McCarty NA
Novel pore-lining residues in CFTR that govern permeation and open-channel block.
Neuron. 1994 Sep;13(3):623-34., [PMID:7522483]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is both a member of the ATP-binding cassette superfamily and a Cl(-)-selective ion channel. We investigated the permeation pathway of human CFTR with measurements on conduction and open-channel blockade by diphenylamine-2-carboxylic acid (DPC). We used site-directed mutagenesis and oocyte expression to locate residues in transmembrane domain (TM) 6 and TM 12 that contact DPC and control rectification and single-channel conductances. Thus, TM 12 and the previously investigated TM 6 line the CFTR pore. In each TM, residues in contact with DPC are separated by two turns of an alpha helix. The contributions of TM 6 and TM 12 to DPC block and Cl- permeation, however, are not equivalent. The resulting structural model for the conduction pathway may guide future studies of permeation in other Cl- channels and ATP-binding cassette transporters.

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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

[hide] Interaction between permeation and gating in a put... Biophys J. 2000 Jul;79(1):298-313. Zhang ZR, McDonough SI, McCarty NA
Interaction between permeation and gating in a putative pore domain mutant in the cystic fibrosis transmembrane conductance regulator.
Biophys J. 2000 Jul;79(1):298-313., [PMID:10866956]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel with distinctive kinetics. At the whole-cell level, CFTR currents in response to voltage steps are time independent for wild type and for the many mutants reported so far. Single channels open for periods lasting up to tens of seconds; the openings are interrupted by brief closures at hyperpolarized, but not depolarized, potentials. Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. In addition, the open-channel burst duration is decreased 10-fold compared with wild-type channels. S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Selectivity of the channel to a range of anions is also affected by this mutation. Furthermore, the permeation properties change during the relaxations, which suggests that there is an interaction between gating and permeation in this mutant. The existence of a mutation that confers voltage dependence upon CFTR currents and that changes kinetics and permeation properties of the channel suggests a functional role for the 11th transmembrane domain in the pore in the wild-type channel.

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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