ABCMdb

ABCC7 p.Ser341Ala

None has been submitted yet.

Publications

PMID: 16442101 [PubMed] Frelet A et al: "Insight in eukaryotic ABC transporter function by mutation analysis."

No. Sentence Comment

405 Alanine substitutions of these residues has been shown to strongly affect conductance, which is greatly reduced in F337A [190] and S341A [46] and significantly increased in T338A [187]. Login to comment

PMID: 10385235 [PubMed] Walsh KB et al: "Structural and ionic determinants of 5-nitro-2-(3-phenylprophyl-amino)-benzoic acid block of the CFTR chloride channel."

No. Sentence Comment

158 Based on the ®nding that the mutant S341A displays a 5 fold reduction in DPC anity, it was suggested that DPC interacts through the hydroxyl group at this residue (McDonough et al., 1994). 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

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
223 c P < 0.01 compared to block of S341A-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
256 Block by both drugs was reduced in S341A-CFTR (Fig. 9A and C). Login to comment
257 However, both S341A-CFTR and T1134F-CFTR responded differently to block by NPPB and DPC. Login to comment
258 Mutation S341A-CFTR altered the voltage-dependence for block by DPC but not for block by NPPB. 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
264 In contrast, the voltage dependence of block of S341A-CFTR was increased at pH 6.5 (P ‫ס‬ 0.038). 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
313 Block of single S341A-CFTR channels was not studied, due to the low single-channel conductance of this variant [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
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
442 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
476 In S341A-CFTR, the voltage dependence of block was increased 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

156 Alanine substitution for these three TM6 residues has been shown to strongly affect conductance, which is greatly reduced in F337A (21) and S341A (13), and significantly increased in T338A (16). 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
197 The shape of the I-V curve between -80 and ϩ60 mV was not affected by the K335A, T338A, T339A, or T1134A mutations, whereas S341A CFTR exhibited less outward rectification than WT CFTR. 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
227 The pattern was similar to that for T338A CFTR in that Px/PCl values for large anions were decreased in S341A CFTR, whereas Px/PCl values for small anions were increased. Login to comment
228 Except for PNO3/PCl, the magnitude of the effect in S341A CFTR was less than that seen in T338A CFTR. Login to comment
231 Interestingly, although GClO4/GCl was increased in T338A CFTR, GClO4/GCl was decreased in S341A CFTR. Login to comment
232 The pattern in S341A CFTR was similarly inverted for Gacetate/GCl compared with that in T338A CFTR. Login to comment
289 Comparing S341E with S341A CFTR and T338E with T338A CFTR, we can see that the introduction of a negative charge at S341 more strongly destabilized the binding of SCN- (which is pronounced in WT CFTR) than did the equivalent mutation at T338. Login to comment
291 In a previous work, McDonough et al. (33) identified S341 as a probable anion binding site based on the reduction in single-channel conductance observed in S341A CFTR. Login to comment
358 In contrast, among alanine substitution mutants, only S341A CFTR exhibited a significant effect on GS2O3/GCl. Login to comment
405 In the absence of this binding site, DPC appears to be able to permeate further toward the extracellular end of the pore as the voltage dependence of block is increased in S341A CFTR. Login to comment
406 S341 also appears to be a Cl-binding site because single-channel conductance was greatly reduced in S341A CFTR (33). Login to comment
446 Scenario 1 would predict that 1) selectivity between small anions would be more sensitive to mutations at T338 than at S341, 2) the effects on selectivity between Cl- and large anions would be missing for S341A CFTR, and 3) mutations at T338 would greatly affect block by DPC. Login to comment
454 1) Selectivity between Cl- and NO3 - as well as between Cl- and Br- was affected more in S341A CFTR than in T338A CFTR. Login to comment
455 2) Although relative permeabilities for the largest anions (acetate and larger) were affected greatly in T338A CFTR, they were also reduced significantly in S341A CFTR. Login to comment

PMID: 11927667 [PubMed] Gong X et al: "Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore."

No. Sentence Comment

12 Channel block by 100 mM Au(CN)2 _ , a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Login to comment
13 Relative Au(CN)2 _ permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. Login to comment
42 Some of these have previously been associated with altered anion selectivity (F337S, T338A; Linsdelletal.1998,2000),alteredanion:cationselectivity(R352Q; Guinamard & Akabas, 1999), or disrupted open channel blocker binding (S341A; McDonough et al. 1994). Login to comment
78 Currents carried by the CFTR mutants K329A, K335A, T338A, S341A and I344A were also stimulated an average of 2_3-fold by PPi (Fig. 2B). Login to comment
87 Comparison between different channel variants at _100 mV reveals the sensitivity to this concentration of Au(CN)2 _ is R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A. Login to comment
88 At depolarised voltages, where the blocking effects of 100 mM Au(CN)2 _ are weak, block of most mutants was not significantly different from wild-type; the only differences in Au(CN)2 _ sensitivity at +60 mV were R352Q > S341A > wild-type. Login to comment
101 Interestingly, S341A also significantly increased PAu(CN)2/PCl (Fig. 4), although to a lesser extent than T338A. Login to comment
123 At this voltage, block by 100 mM Au(CN)2 _ was significantly weakened in K335A, F337S, T338A and I334A, significantly strengthened in S341A and R352Q and unaffected in K329A (Fig. 3). Login to comment
124 The sequence of relative sensitivity to block by 100 mM Au(CN)2 _ at _100 mV (R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A) suggests that T338 normally makes the strongest contribution to Au(CN)2 _ binding within the pore, with nearby residues K335 and F337 also making large contributions. Login to comment
126 Interestingly, in spite of its previous association with disrupted anion binding within the pore (McDonough et al. 1994; Zhang et al. 2000), S341A showed significantly strengthened Au(CN)2 _ block at all potentials (Fig. 3), suggesting that the polar hydroxyl side chain of S341 does not contribute to lyotropic anion binding. Login to comment
140 However, the dramatic increase in PAu(CN)2/PCl observed in S341A (Fig. 4) suggests that the region of the pore which predominantly controls selectivity between different anions may extend further towards the intracellular end of TM6 than previously appreciated. Login to comment
147 Only mutations in the central portion of TM6 (F337S, T338A, S341A) affected both Au(CN)2 _ binding and Au(CN)2 _ permeability (Figs 3_5). Login to comment
157 Nevertheless, there does not seem to be a strong correlation between these two aspects of pore function, such that they may be controlled independently by the same structural featuresofthepore.Thus,F337Sisassociatedwithweakened Au(CN)2 _ binding and decreased Au(CN)2 _ permeability, T338A with weakened Au(CN)2 _ binding and increased Au(CN)2 _ permeability and S341A with strengthened Au(CN)2 _ bindingandincreasedpermeability(Figs3and4). Login to comment

PMID: 12172647 [PubMed] Chen JH et al: "CFTR is a monomer: biochemical and functional evidence."

No. Sentence Comment

54 Missense mutations S341A and R347D were generated by site-directed mutagenesis (Stratagene) and cloned into CFTR-M2 by replacing the A¯II-HpaI fragment, and into M2-CFTR by replacing the XbaI-HpaI fragment. Login to comment
79 To obtain approximately equal expression of dierent epitope-tagged CFTR-conduction variants, BHK cells were transiently cotransfected with cDNA in the following ratios: 6:1, S341A-M2:WT-HSV;7:5,R347D-M2:WT-HSV;1:1,S341A-M2:TT338, 339AA-HSV;3:11,R347D-M2:TT338,339AA-HSV;6:1,M2-S341A: HSV-WT; and 1:1,M2-R347D:HSV-WT. Login to comment
180 Mutations S341A and R347D (single-letter amino acid) dramatically lower chloride conductance (Tabcharani et al., 1993; McDonough et al., 1994) while the double mutation TT338, 339AA enhances chloride conduction (Linsdell et al., 1997). Login to comment
193 To the C-terminal ends of CFTR, we attached the M2 epitope to S341A and R347D and the HSV epitope to wild type and TT338, 339AA (S341A-M2, R347D-M2, WT-HSV, and TT338, 339AA-HSV, respectively). Login to comment
194 Single-channel chord conductances for S341A-M2, R347D-M2, WT-HSV, and TT338, 339AA-HSV at À100 mV were (in pS): 2.2, 5.1, 14.3, and 15.6, respectively (Figs. Login to comment
205 (C) Amplitude histograms and single-channel recordings of low-conduction mutants S341A and R347D C-terminally tagged with M2 (S341A-M2 and R347D-M2, respectively), and high-conduction variants WT and TT338, 339AA C-terminally tagged with HSV (WT-HSV and TT338, 339AA-HSV, respectively). Login to comment
207 (D) Single-channel current-voltage relationships of S341A-M2, R347D-M2, WT-HSV, and TT338,339A-HSV. Login to comment
210 (E) Tabulation of single-channel conductances from microsomes containing a low-conducting (S341A.M2 or R347D-M2) and a high-conducting species (WT-HSV or TT338,339AA-HSV). Login to comment
215 To assess CFTR stoichiometry, we co-expressed approximately equal amounts of a low-(S341A-M2 or R347D-M2) and a high-conduction species (WT-HSV or TT338,339AA-HSV) for single-channel analysis (Fig. 4E). Login to comment
223 (C) Amplitude histograms and single-channel recordings of S341A and R347D N-terminally tagged with M2 (M2-S341A and M2-R347D, respectively), and WT N-terminally tagged with HSV (HSV-WT). Login to comment
225 (D) Single-channel current-voltage relationships of M2-S341A, M2-R347D, and HSV-WT. Login to comment
227 Intermediate conducting channels were infrequently observed; membrane vesicles containing WT-HSV plus S341A-M2 or R347D-M2 produced 9.5 pS and 7.5 pS channels, and vesicles containing TT338,339AA-HSV plus S341A-M2 or R347D-M2 yielded 12 pS, 8 pS, and 2 pS channels. Login to comment
235 The chord conductances of M2-S341A, M2-R347D, and HSV-WT at À100 mV (in pS) were 2.2, 5.1, and 14.6, respectively (Fig. 5C and 5D). Login to comment
236 When co-expressed, microsomes containing HSV-WT and either M2-S341A or M2-R347D produced conductances that were predominantly channels of each constituent in their main conductance states and infrequently their subconductance states (Fig. 5E). Login to comment
237 The possibility that these intermediate conductances resulted from a hybrid assembly of multiple CFTR molecules was £ 3.3% and £ 2.1% for HSV-WT plus M2-S341A and M2-R347D, respectively. Login to comment
244 (E) Tabulation of single-channel conductances from microsomes containing HSV-WT and either M2-S341A or M2-R347D. Login to comment
247 Co-expressed CFTR proteins do not form a hybrid channel (95% CI £ 3.3% for M2-S341A and HSV-WT, and £ 2.1% for M2-R347D and HSV-WT). Login to comment

PMID: 12411425 [PubMed] Gong X et al: "Mechanism of lonidamine inhibition of the CFTR chloride channel."

No. Sentence Comment

7 5 Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) signi®cantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. Login to comment
116 As shown in Figure 7a, 55 mM lonidamine inhibited currents carried by R334C, K335A, F337S, T338A and S341A-CFTR. Login to comment
117 However, R334C, F337S and S341A were only weakly inhibited by this concentration relative to wild-type CFTR (see Figure 1). Login to comment
118 The eect of these mutations on block by lonidamine is more clearly seen in the dose-response curves shown in Figure 7b. Fits of these mean data by equation 1 suggests a Kd (at 7100 mV) of 58.5 mM for wild-type, 65.6 mM for K335A, 90.0 mM for T338A, 186 mM for F337S, 206 mM for S341A, and 338 mM for R334C. Login to comment
119 Similar analyses at other potentials showed a similar increase in Kd in R334C, F337S, S341A and (to a far lesser extent) T338A (Figure 7c). Login to comment
120 Fitting data from individual patches with equation 2 gave similar and, except in the case of K335A, signi®cant changes in Kd(-100): wild-type 60.6+5.2 mM (n=5), K335A 63.1+7.4 mM (n=5) (P40.05), T338A 93.4+4.1 mM (n=5) (P50.002), F337S 166+18 mM (n=5) (P50.0005), S341A 169+25 mM (n=5) (P50.005), R334C 260+19 mM (n=4) (P50.00001). Login to comment
121 These same ®ts also revealed changes in the voltage dependence of block, as judged by changes in d, although this was only statistically signi®cant in the case of R334C: wild-type 0.426+0.033 (n=5), K335A 0.484+0.024 (n=5) (P40.05), T338A 0.410+0.045 (n=5) (P40.05), F337S 0.365+0.015 (n=5) (P40.05), S341A 0.285+0.061 (n=5) (P40.05), R334C 0.233+0.066 (n=4) (P50.05). Login to comment
143 (a) Example I-V relationships for R334C, K335A, F337S, T338A and S341A-CFTR, before (solid lines) and following (dotted lines) addition of 55 mM lonidamine to the intracellular solution. Login to comment
145 (b) Concentration dependence of block at 7100 mV for wild-type, R334C, K335A, F337S, T338A and S341A. Login to comment
147 Each has been ®tted by equation 1, giving Kds of 58.5 mM (wild-type), 65.6 mM (K335A), 90.0 mM (T338A), 186 mM (F337S), 206 mM (S341A) and 338 mM (R334C). Login to comment
154 Lonidamine block was weakened in the TM6 mutants R334C, F337S and S341A (Figure 7), suggesting that these residues may normally contribute to lonidamine binding within the pore. Login to comment

PMID: 12745925 [PubMed] Gupta J et al: "Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore."

No. Sentence Comment

41 Example leak-subtracted I Á/V relationships obtained with different intracellular anions are shown for wild-type, R334C, F337A, T338A, T339V and S341A in Figure 2. Login to comment
45 The relative permeability of the lyotropic SCN( anion, which is high in the wild-type (PSCN/PCl 0/4.759/0.30, n0/6) (Table 1) was significantly altered in six out of eight mutants studied (Table 1 and Figure 3), with PSCN/PCl being greatly reduced in F337A and most strongly increased in T338A and S341A. Login to comment
49 Perhaps most significantly, the relative conductance of Br( , I( and SCN( were all increased in S341A, leading to a change in the conductance sequence to Br( !/I( Â/Cl( !/SCN( !/F( (Table 3). Login to comment
59 Wild type R334C K335A I336A F337A T338A T339V I340A S341A Cl 1.009/0.00 (6) 1.009/0.01 (6) 1.009/0.05 (5) 1.009/0.01 (5) 1.009/0.02 (6) 1.009/0.02 (8) 1.009/0.03 (6) 1.009/0.02 (5) 1.009/0.01 (6) Br 1.479/0.06 (6) 0.969/0.00 (5)** 1.529/0.03 (5) 1.359/0.05 (5) 0.669/0.03 (6)** 2.209/0.05 (5)** 1.829/0.24 (5) 1.409/0.09 (6) 2.459/0.20 (5)** I 0.819/0.04 (6) 0.729/0.05 (3) 1.579/0.06 (4)** 0.589/0.02 (4)* 0.389/0.15 (3)* 2.799/0.26 (7)** 0.769/0.02 (6) 1.249/0.07 (6)** 0.739/0.06 (6) F 0.119/0.01 (6) 0.099/0.01 (3) 0.139/0.02 (3) 0.079/0.01 (5) 0.409/0.02 (4)** 0.139/0.01 (6) 0.079/0.00 (5) 0.069/0.01 (5) 0.059/0.01 (6)* SCN 4.759/0.30 (6) 2.769/0.38 (6)** 3.989/0.16 (5) 3.709/0.11 (5)* 1.269/0.12 (5)** 7.509/0.29 (6)** 4.829/0.40 (5) 4.189/0.14 (7)* 10.09/1.8 (6)* Relative permeabilities for different anions present in the intracellular solution under bi-ionic conditions were calculated from macroscopic current reversal potentials according to Eq. (1) (see Experimental procedures). Login to comment
65 Wild-type R334C K335A I336A F337A T338A T339V I340A S341A Cl (G(50/G'50) 1.039/0.09 (6) 4.509/0.60 (6)** 1.399/0.09 (5)** 1.519/0.14 (5)* 1.189/0.22 (6) 1.779/0.25 (8)* 1.199/0.06 (7)* 1.419/0.11 (5)* 1.809/0.18 (5)** Cl (GCl/GCl) 1.009/0.08 (6) 1.009/0.13 (6) 1.009/0.07 (5) 1.009/0.09 (5) 1.009/0.22 (6) 1.009/0.14 (8) 1.009/0.06 (7) 1.009/0.09 (5) 1.009/0.10 (5) Br 0.649/0.05 (6) 0.329/0.02 (6)** 0.669/0.05 (5) 1.079/0.10 (5)* 0.359/0.06 (6)** 0.499/0.03 (5) 0.659/0.09 (5) 0.669/0.08 (6) 1.529/0.30 (4)* I 0.299/0.05 (6) 0.749/0.02 (3)* 0.279/0.01 (4) 0.109/0.02 (4)* 0.349/0.08 (3) 0.389/0.03 (5) 0.309/0.05 (7) 0.279/0.03 (6) 1.049/0.16 (7)** F 0.379/0.04 (6) 0.329/0.04 (3) 0.349/0.03 (3) 0.709/0.10 (4)* 0.129/0.02 (3)* 0.239/0.02 (6)* 0.509/0.10 (4) 0.309/0.02 (5) 0.519/0.07 (6) SCN 0.389/0.02 (6) 0.339/0.03 (6) 0.669/0.10 (5)* 0.279/0.02 (6)* 0.399/0.04 (5) 0.269/0.02 (5)* 0.269/0.02 (4)* 0.359/0.04 (6) 0.839/0.14 (6)* Relative conductances for different anions were calculated from the slope of the macroscopic I Á/V relationship for inward versus outward currents (see Experimental procedures). Login to comment
76 In the present study, large increases in the permeability of the lyotropic SCN( anion were observed in both T338A and S341A, and a dramatic decrease in SCN( permeability was observed in F337A (Figure 3), consistent with previous results with Au(CN)2 ( which suggest these residues are the main determinants of the permeability of strongly lyotropic anions [15]. Login to comment
78 Taken together, these anion permeability data suggest a relative loss of lyotropic anion selectivity in F337A and (to a lesser extent) R334C, strengthening of lyotropic selectivity in T338A and S341A, and only minor effects at other positions. Login to comment
86 Halide permeability sequence Eisenman sequence CFTR variants I( !/Br( !/Cl( !/F( I K335A, T338A Br( !/I( !/Cl( !/F( II I340A Br( !/Cl( !/I( !/F( III wild-type, I336A, T339V, S341A Cl( !/Br( !/I( !/F( IV R334C Cl( !/Br( !/F( !/I( V F337A Sequences were derived from the relative permeabilities given in table 1. Login to comment
105 As previously described by others [20], mutation of S341 was particularly associated with changes in anion relative conductance, consistent with weakened lyotropic anion binding in S341A. Login to comment
109 Lyotropic anion selectivity is disrupted in F337A and modified in R334C, T338A and S341A. Login to comment

PMID: 12820897 [PubMed] Ramjeesingh M et al: "Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane."

No. Sentence Comment

20 For example, co-expression of wild-type CFTR with a pore mutant, i.e. CFTR S341A (Ser341 → Ala), leads to the appearance of two distinct conductances, rather than a hybrid conductance path [12]. Login to comment

PMID: 14610019 [PubMed] Gong X et al: "Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore."

No. Sentence Comment

98 Block of R334C and S341A appeared somewhat weaker than for wild-type CFTR, whereas K335A and T338A showed a similar degree of block as wild-type (Fig. 5, A-C). Login to comment
145 (A) Example macroscopic currents carried by the CFTR mutants R334C, K335A, F337A, T338A, and S341A before (Control) and after addition of 300 ␮M Pt(NO2)4 2to the intracellular solution. Login to comment
147 Each plot has been fitted by Eq. 2; this provides a good fit of R334C (Kd(0) ϭ 2080 ␮M, z␦ ϭ -0.174), K335A (Kd(0) ϭ 418 ␮M, z␦ ϭ -0.317), T338A (Kd(0) ϭ 626 ␮M, z␦ ϭ -0.351) and S341A (Kd(0) ϭ 1362 ␮M, z␦ ϭ -0.249), but a poor fit of F337A. Login to comment

PMID: 18366345 [PubMed] Caci E et al: "Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis."

No. Sentence Comment

111 R334A and S341A showed reduced anion transport, although this was significantly greater than cells transfected with the fluorescent protein alone (Figure 1B). Login to comment
127 CFTR form CFTRinh-172 Ki (μM) Hill coefficient I- influx (mM/s) n Wild-type 1.32 + - 0.25 1.03 + - 0.07 0.1336 + - 0.0107 10 S341A 0.57 + - 0.17 1.21 + - 0.37 0.0297 + - 0.0064 4 T338A 3.20 + - 0.86 1.13 + - 0.20 0.1260 + - 0.0225 4 R347A 44.98 + - 4.71** 0.91 + - 0.04 0.1288 + - 0.0154 7 R334A 2.39 + - 0.74 0.93 + - 017 0.0313 + - 0.062 4 A349S 1.23 + - 0.41 1.11 + - 0.25 0.1500 + - 0.011 4 R347D >50 Not determined 0.1160 + - 0.0136 7 R347D/D924R >50 Not determined 0.1008 + - 0.0504 4 R347C >50 Not determined 0.1437 + - 0.0123 4 Mock 0.003 + - 0.001 10 introduced a mutation at position 349 (an alanine residue replaced by a serine residue). Login to comment

PMID: 20142516 [PubMed] Zhou JJ et al: "Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore."

No. Sentence Comment

268 This is consistent with previous work, for example with S341A (McDonough et al., 1994), showing that mutations at this position are associated with dramatic loss of Cl conductance. Login to comment

PMID: 9922376 [PubMed] Dawson DC et al: "CFTR: mechanism of anion conduction."

No. Sentence Comment

569 Substituting an alanine for serine-341 in TM6 virtually abolished theis the polarity and charge of each residue. Login to comment
575 The single-channel conductance of S341A was reduced to Ç1 pS, and the macro- that anion binding might be a generalized feature of bundles of a-helices containing arginines, although Dormanscopic i-V relation became slightly inwardly rectifying, suggesting to the authors that this polar amino acid may et al. (43), in their analysis of a model for ion permeation in the gramicidin channel, suggested that anion interac-be pore-lining and also constitute a binding site for DPC, via hydrogen bonding with the -OH group. Login to comment
577 One speculation is that TM5 and TM6 are both part of the ''lining`` of the pore such that the chargedaltered so as to match those surrounding S341 and then combined with the S341A mutation, binding was restored, residues in TM6 and the exact conformation of TM5 are both strong determinants of the types of interaction thatas if the binding site had been ''moved to TM12.`` The authors predicted that residues in TM6 and TM12 lying a permeating anion may experience with the pore wall. Login to comment

PMID: 9922377 [PubMed] Gadsby DC et al: "Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis."

No. Sentence Comment

50 The mutation S341A, for instance, altered the apparent affinity (and its voltage dependence) for block tions IV and V, the details of this complex interplay between phosphorylation of CFTR, ATP hydrolysis atof open CFTR channels by diphenylamine-2-carboxylate (133), and a cysteine-scanning method has demonstrated CFTR`s NBDs, and CFTR channel gating remain incompletely understood. Login to comment

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

No. Sentence Comment

242 The likely site of action of DPC in this mutation of serine-341 to an alanine caused a fivefold increase in the KD at 0100 mV (wild type, 276 mM vs.tissue is the inhibition of cyclooxygenase, the enzyme responsible for prostaglandin synthesis. Login to comment
243 Prostaglandins S341A, 1,251 mM). Login to comment
249 The interpretation of ar- residues immediately adjacent to S341, DPC bound with an affinity close to that of the wild-type channel (S341A-ylaminobenzoate inhibition of macroscopic Cl0 secretion is, at best, difficult because of their nonselectivity for Cl0 M1140I-T1142F). Login to comment

PMID: 22160394 [PubMed] Cui G et al: "Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers."

No. Sentence Comment

119 The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left). Login to comment
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
157 Out of 20 mutants in TM6 and 20 mutants in TM12, only two in TM6 (S341A and F337A) induced rectification in macropatch currents which were suggested to form the narrow part of the pore (see below, Fig. 7, Supplementary Fig. 3). Login to comment
158 Among the 20 single amino acid mutants of TM12 that we tested in this paper, none of them exhibited significant change in their single-channel conductance compared to WT-CFTR, while we know that mutations R334A, F337A, S341A, R347A, and R352A in TM6 all exhibited significant change in their single-channel conductance [11, 12, 29, and the present manuscript]; these data strongly suggest that TM6 and TM12 do not equally contribute to the pore of CFTR. Login to comment
166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
173 Mutation S341A caused the largest decrease in block by Glyb and Glip (aside from R347A and R352A, which have non-canonical effects as described above; Fig. 3). Login to comment
176 In contrast to the effects of mutation S341A, the reduction in block by Glyb in F337A reflected a substantial decrease in initial block without a change in the magnitude of time-dependent block (Figs. 5 and 6). Login to comment
184 Both mutations S341A and F337A significantly decreased single-channel conductance (Fig. 9 and Ref. [29]). Login to comment
185 Consistent with this designation, macroscopic chloride currents in S341A exhibited inward rectification while F337A/C/E exhibited outward rectification (Fig. 7; Supplementary Fig. 1) [28, 29]. Login to comment
193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
196 From the differences in the effects of mutations S341A and F337A on block by Glyb and Glip, and the similarity of effects of mutations M348A and V350A on block by the two drugs, we can infer that both drugs bind in the pore with the sulfonylurea-linked cyclohexamide end facing toward the cytoplasm. Login to comment
201 Similar to their effects on block by Glyb, both the S341A and F337A mutations decreased the efficacy of block by Meglitinide (fractional block was 0.35±0.04 and 0.45±0.04, p<0.01, respectively). Login to comment
206 Each of the functional parameters comprising the biophysical signature of CFTR (single-channel conduc- WT S341A F337A Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP -100 -50 50 100 -2000 -1000 1000 2000 ATP ATP +Glyb 50 μM ATP +Glyb 50 μMATP +Glyb 50 μM I (pA) Vm(mV) Vm(mV) -100 -50 50 100 -2000 -1000 1000 2000 ATP I (pA) Vm (mV) -100 -50 50 100 -2000 -1000 1000 2000 50 100 I (pA) ATP Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP Vm(mV) -100 -50 50 100 -800 -400 400 800 I (pA) ATP Vm(mV) -100 -50 50 100 -800 -400 400 800 I (pA) ATP ATP +Glip 200 μMATP +Glip 200 μMATP +Glip 200 μM I (pA) Vm(mV) -100 -50 50 100 -800 -400 400 800 ATP ATP +Tolb 300 μM ATP +Tolb 300 μMATP +Tolb 300 μM Fig. 7 I-V relationships for WT-CFTR and two important mutants, from inside-out macropatches in symmetrical 150 mM Cl- solution. Data were obtained by ramping the membrane potential from VM=-100 mV to +100 mV over 300 ms. Login to comment
208 Data for each CFTR variant is from a single patch expressing WT-, S341A-, or F337A-CFTR tance, rectification, selectivity, blocker pharmacology, etc.) can be compared between wildtype and site-directed mutants to infer channel structure. Login to comment
231 This conclusion is bolstered by the finding that the effects of mutations S341A and F337A on block by Glyb were the same as their effects on block by Meglitinide, which shares structure with the non-sulfonylurea end of Glyb. Login to comment
232 In conclusion with these results, for the following reasons, we believe that the narrow region in TM6 of the CFTR pore is located between F337 and S341: (1) mutations F337A/S/C/E/Y/L and S341A/E/T dramatically altered the relative permeability of different anions in the channel (Supplementary Tables 2, 3; Refs. Login to comment
235 (3) Both F337 and S341 mutations exhibited outward or inward rectification, respectively; and (4) both S341A and F337A affected block by all four sulfonylurea family blockers [8, 21, 40, 42, 50, 53]. Login to comment
238 Therefore, we cannot conclude that one part of the Glyb molecule binds exclusively to one section of the pore because: (a) mutations along the full length of the pore affected block by Tolb, and (b) mutations S341A and F337A affected block by both Tolb and Meglitinide, which represent the two disparate halves of the Glyb structure. Login to comment

PMID: 9379169 [PubMed] Linsdell P et al: "Multi-Ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

214 The mutation S341A also severely reduces channel conductance, suggesting that this amino acid also interacts with permeating Cl- ions (McDonough et al., 1994). Login to comment

PMID: 9379168 [PubMed] Linsdell P et al: "Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions."

No. Sentence Comment

171 In contrast, mutating serine 341 to alanine produced outward rectification of the i/V relationship, consistent with its proposed role as a binding site for permeating anions (McDonough et al., 1994). Login to comment
192 In contrast, mutating serine 341 to alanine produced outward rectification of the i/V relationship, consistent with its proposed role as a binding site for permeating anions (McDonough et al., 1994). Login to comment

PMID: 8744306 [PubMed] Cheung M et al: "Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment."

No. Sentence Comment

193 The mutation K335E, a water-accessible residue, altered the relative halide permeability and conductance sequences (Anderson et al., 199lb). Login to comment
195 Another mutation in M6, S341A, caused a fourfold reduction in the affinity for the voltage-dependent channel blocker diphenylamine-2-carboxylate (McDonough et al., 1994); we have shown that Ser341 is exposed in the channel lumen and thus could interact with a channel blocker. 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

51 Figure 3C shows data for CFTR in which S341 was mutated to alanine (S341A); the affinity of DPC for the mutant channel was reduced by approximately 5-fold compared with wild type (KD = 1251 PM). Login to comment
53 Figure4B shows thatthevoltagedependenceoftheweaker bindingfor S341A was changed only slightly from the wild-type. Login to comment
54 In the S341A mutation, Cl-permeation in the absence of blockers was also changed: the S341A whole-cell currents rectified inwardly (Figure 3C), opposite to wild-type currents. Login to comment
59 The S341T mutation gives a Kn for DPC binding intermediate to those of S341A and the wild type (Table 1) and a smaller degree of inward rectification than that of S341A (data not shown). Login to comment
68 (0 S341A mutant, showing strong inward rectification and decreased DPC block. Login to comment
69 (D) Triple mutation (S341A-M11401-Tll- 42F), showing strong inward rectification and restored DPC block compared with (C). Login to comment
73 S1141 could, however, participate in a binding site for DPC when the binding site including S341 was removed (S341A) and when the methionineand threonine residues immediatelyadja- cent to S1141 were changed to match the isoleucine and phenylalanine residues immediately adjacent to S341. Login to comment
74 That is, the triple mutant S341A-M11401-T1142F bound DPC more tightly than S341A alone, with an affinity close to that of the wild-type channel (Figure 3D; Figure 4). 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
84 E,,,, the reversal potential, determined empirically from the voltage steps; Triple, S341A-M11401-T1142F mutant. Login to comment
86 b p < .025 by unpaired t test compared with mutant S341A. 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
111 Open circles, wild-type; closed circles, T1134F; open boxes, S341A; closed boxes, triple mutation (S341A-Mll- 401-T1142F). 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
144 (B) S341A single-channel traces with expanded amplitudescale,filteredat F, = 100 Hz to resolve openings clearly. Login to comment
155 The Pore of CFTR Is lined by TM 6 and TM 12 We conclude that TM 6 lines the pore because mutation S341A lowers the single-channel conductance by a factor of 8, reverses the direction of rectification, and removes most binding of the open-channel blocker DPC. Login to comment
158 The sequence M11401-S1141-T1142F in TM 12 restores DPC binding to the S341A mutant. Login to comment
178 Mutations R334W, R347P, and S341A may reduce single-channel conductance by removing a Cl--binding site. Login to comment
219 The mutation that produces the largest effect on DPC block, S341A, also lowers the single-channel conductance from 8 to 1 pS and changes the rectification from outward to inward. Login to comment
223 The possibility that the effects of S341A are allosteric is also rendered less likely by the intermediate effect on both drug block and rectification of replacing S341 with another hydroxylated residue, threonine. Login to comment
227 This argues against the proposal that any alteration in sequencewithinTM6causes nonspecific effectsand isfurther evidenceforthespecificityof the S341A mutation. 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
309 For the S341A mutation, which results in singlechan- nels of very low conductance, currents were filtered at 100 Hz and 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

335 Mutation S341A in TM6 reduced affinity for DPC fivefold, induced inward rectification, and decreased the single-channel conductance to b03;1 pS (McDonough et al., 1994). Login to comment

PMID: 23463513 [PubMed] Cebotaru L et al: "Transcomplementation by a truncation mutant of cystic fibrosis transmembrane conductance regulator (CFTR) enhances DeltaF508 processing through a biomolecular interaction."

No. Sentence Comment

9 Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of èc;F508 CFTR. Login to comment
170 To explore more definitely which form of CFTR is generating the currents, we utilized the conduction mutant S341A. Login to comment
173 S341A has been shown to alter the ion selectivity of CFTR (30). Login to comment
174 Fig. 9 shows that the S341A mutation in wild type CFTR dramatically reduces the whole cell currents without affecting protein expression. Login to comment
175 With this result in hand, we then tested the double mutant èc;F508/ S341A CFTR (Fig 10). Login to comment
177 Interestingly, when èc;27-264 CFTR containing the wild-type CFTR conduction pore and the double mutant èc;F508/ S341A CFTR are cotransfected there are again hardly any currents detected despite the presence of ample amounts of protein. Login to comment
187 Functional study of S341A CFTR in CHO cells. Login to comment
188 S341A CFTR-expressing CHO cells have smaller whole cell currents than CHO cells expressing Waf9; CFTR (A and B). Login to comment
189 C, summary I/V data; afe;S.E. (S341A CFTR, red circles, n afd; 4; Waf9;CFTR, black squares, n afd; 3). Login to comment
201 We showed in a previous study of èc;264 CFTR our earlier truncation mutant transfected into IB3-1 bronchial epithelial cells that single channel currents occurred with open probability and conductance similar to wild-type CFTR (17) but the exact origin of the currents was FIGURE10.Dualexpressionofèc;508CFTRandèc;27-264CFTRresultsinèc;F508CFTR-mediatedwholecellcurrents.Expressionofèc;508/S341ACFTRalong with èc;27-264 CFTR results in robust expression, with slightly elevated B and C bands as compared with èc;508 CFTR/èc;27-264 CFTR expression (A), but with reduced whole cell currents (B) as compared with currents from cells expressing èc;508 CFTR/èc;27-264 CFTR (B), consistent with the previously described conductance defect in S341A CFTR (34). Login to comment
202 C, summary I/V data for CHO cells expressing èc;508/S341A CFTR and èc;27-264 CFTR (n afd; 7; red squares) and èc;508 CFTR and èc;27-264 CFTR (n afd; 5; black circles); afe; S.E. Login to comment
216 In this study, we address this question with the conduction mutant S341A. Login to comment
218 S341A has been shown to alter the ion selectivity of CFTR and the sensitivity to chloride channel blockers (30). Login to comment
220 Because we could rescue the èc;F508/S341A CFTR protein but not rescue channel currents because of the altered conduction pore, results showed conclusively that the current is indeed generated by rescued èc;F508-CFTR channel gating. Login to comment