ABCMdb

ABCC7 p.Thr338Ala

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: 11179391 [PubMed] Linsdell P et al: "Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

19 Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Login to comment
38 The present study seeks to shed new light on the relationship between anion binding and anion permeability in CFTR channels by comparing the anion binding properties of wild-type CFTR with two mutants with altered anion selectivity, F337S and T338A. Login to comment
42 The CFTR mutants F337A, L, S, W, Y and T338A were constructed and transfected into CHO and BHK cells by Alexandra Evagelidis and Shu-Xian Zheng in the laboratory of Dr John Hanrahan (McGill University, Montreal, Quebec, Canada), as described previously (Linsdell et al. 1998, 2000). Login to comment
43 In the present study, the permeation properties of two mutants, F337S and T338A, have been examined in detail. Login to comment
48 In contrast to mutations at F337, all mutations previously examined at T338 (T338A, N, S, V) significantly altered anion selectivity and Cl¦ conductance (Linsdell et al. 1998). Login to comment
49 For the present study, T338A was examined because: (1) it has a high single channel conductance, allowing single channel currents to be resolved, (2) its anion selectivity strongly follows the lyotropic sequence, in fact more strongly than that of wild-type CFTR, such that its effects on anion permeability might be considered 'opposite` to the effects of F337S, (3) replacing the threonine with a small, 'neutral` alanine is considered less likely to cause large changes in transmembrane helix structure, and (4) T338A is well expressed in both CHO and BHK cells (see Linsdell et al. 1998, for a full description of the permeation phenotype of T338A). Login to comment
83 Unitary properties of F337S and T338A CFTR A, single channel currents carried by wild-type, F337S and T338A, expressed in CHO cells, with symmetrical 150 mÒ NaCl, at a membrane potential of -50 mV. Login to comment
85 B, mean i-V relationships under these ionic conditions, wild-type 0, F337S 1 and T338A ±; mean of data from 3-9 patches. Login to comment
93 Estimation of unitary current amplitude from macroscopic current variance A, activation of macroscopic wild-type, F337S and T338A CFTR currents in BHK cell patches at +50 mV, in the symmetrical presence of 150 mÒ NaCl, by addition of PKA in the presence of ATP (see Methods). Login to comment
95 All three have been fitted by eqn (1) (see Methods), giving i = 0·118 pA and N = 374 for wild-type, i = 0·0387 pA and N = 1359 for F337S and i = 0·279 pA and N = 175 for T338A. Login to comment
105 Unitary Cl¦ currents carried by wild-type, F337S and T338A CFTR are compared in Fig. 3A. Mean slope conductance (Fig. 3B) was reduced in F337S (from 7·59 ± 0·10 pS (n = 12) to 1·76 ± 0·03 pS (n = 7)), and significantly increased in T338A (to 9·94 ± 0·14 pS, n = 6). Login to comment
112 Macroscopic current variance analysis of relative anion conductance in wild-type, F337S and T338A Macroscopic currents were activated in BHK cell patches at +50 mV, in the symmetrical presence of the anion named on the far left, by addition of PKA in the presence of ATP (see Fig. 4). Login to comment
117 Relative anion permeabilities and conductances for wild-type and mutant CFTR ------------------------------------------------------------ PXÏPCl gXÏgCl ------------------ --------------------------- WT a F337S a T338A b WT c WT d F337S d T338A d ------------------------------------------------------------ Cl 1·00 ± 0·01 1·00 ± 0·08 1·00 ± 0·02 1·00 ± 0·01 1·00 ± 0·10 1·00 ± 0·16 1·00 ± 0·09 (10) (3) (11) (12) (9) (7) (4) Br 1·37 ± 0·07 0·50 ± 0·04 1·74 ± 0·04 0·48 ± 0·01 0·48 ± 0·13 0·16 ± 0·03* 0·44 ± 0·02 (8) (4) (3) (6) (4) (4) (3) I 0·83 ± 0·03 0·23 ± 0·02 2·09 ± 0·16 - - - - (6) (4) (5) F 0·10 ± 0·01 0·43 ± 0·02 0·12 ± 0·02 - 0·094 ± 0·017 0·76 ± 0·19* 0·054 ± 0·011 (9) (4) (4) (3) (4) (3) SCN 3·55 ± 0·26 0·93 ± 0·10 5·85 ± 0·27 - 0·060 ± 0·012 0·17 ± 0·04* 0·085 ± 0·007 (7) (5) (4) (5) (3) (4) NO× 1·58 ± 0·04 1·08 ± 0·02 2·04 ± 0·08 0·60 ± 0·02 0·73 ± 0·07 0·29 ± 0·08* 0·96 ± 0·05 (10) (4) (3) (5) (5) (3) (3) ClOÚ 0·25 ± 0·01 0·19 ± 0·00 1·35 ± 0·08 - 0·059 ± 0·014 0·041 ± 0·008 0·082 ± 0·011 (8) (3) (3) (6) (2) (4) Formate 0·24 ± 0·01 0·27 ± 0·02 0·45 ± 0·04 0·35 ± 0·01 0·49 ± 0·01 0·17 ± 0·02** 0·46 ± 0·07 (9) (3) (3) (6) (5) (3) (3) ------------------------------------------------------------a From Linsdell et al. (2000); b from Linsdell et al. (1998); c by single channel recording, d by current variance analysis. Login to comment
124 Macroscopic current variance analysis was also used to compare the relative conductances of different anions in wild-type, F337S and T338A (Fig. 6; Table 1). Login to comment
127 Relative conductances in wild-type estimated by these two methods (Fig. 7), as well as those in F337S and T338A estimated from macroscopic current variance analysis, are summarised in Table 1. Login to comment
129 Although smaller differences are apparent for T338A, in no case were these significantly different from wild-type (Table 1). Login to comment
130 The conductance sequences for wild-type, F337S and T338A are summarised in Table 2. Login to comment
143 Permeability and conductance sequences for wild-type and mutant CFTR ------------------------------------------------------------ Wild-Type F337S T338A ------------------------------------------------------------ Permeability sequence SCN > NO× > Br > Cl > NO× > Cl ü SCN > Br > SCN > I ü NO× > Br > I > ClOÚ formate > F F > formate > I > ClOÚ ClOÚ > Cl > formate > F Conductance sequence Cl > NO× > Br ü formate > Cl > F > NO× > SCN Cl ü NO× > formate Br > F > SCN ClOÚ formate Br > ClOÚ SCN ClOÚ > F ------------------------------------------------------------ Permeability and conductance sequences are derived from data given in Table 1. Login to comment
159 The ability of permeant anions to block Cl¦ currents was also examined in selectivity altering mutants, using macroscopic current variance experiments (for the low-conductance F337S) or single channel recording (for the high-conductance T338A), at -50 mV (Fig. 11). Login to comment
162 T338A Cl¦ currents were only weakly blocked by SCN¦ and ClOÚ¦, and not significantly blocked by other permeant anions including I¦ (Fig. 11C). Login to comment
164 T338A Cl¦ currents were significantly increased in the presence of 25 mÒ intracellular NO×¦, suggesting that the high permeability and conductance of NO×¦ in this mutant (Table 1) allowed some degree of summation of Cl¦ and NO×¦ currents through the channel. Login to comment
165 The relationship between anion permeability and anion conductance The results summarised in Tables 1 and 2 indicate that the CFTR mutation F337S, which virtually abolishes the lyotropic pattern of anion permeability (Linsdell et al. 2000), also alters the relative conductance of different anions in the pore, whereas T338A, which strengthens the lyotropic nature of anion permeability (Linsdell et al. 1998), has no significant effect on relative conductance. Login to comment
171 Anion permeability in wild-type and T338A CFTR follows an approximate lyotropic sequence, with relative anion permeability being correlated with energy of hydration (Linsdell & Hanrahan, 1998; Linsdell et al. 1998; Fig. 12A). Login to comment
172 In contrast, there is no obvious relationship between relative anion conductance and energy of hydration in either wild-type or T338A (Fig. 12B). Login to comment
175 The relationship between anion permeability and energy of hydration observed in wild-type and T338A is lost in F337S (Fig. 12A), which we previously suggested reflected a reduction in the relative importance of anion dehydration in determining anion P. Linsdell J. Physiol. 531.160 Figure 11. Login to comment
176 Block of wild-type, F337S and T338A CFTR by intracellular permeant anions Relative current amplitudes (at -50 mV) in the presence of different intracellular permeant anions were estimated from single channel recording (for wild-type, see Fig. 10, and also for T338A), or from macroscopic current variance analysis for F337S. Login to comment
184 Interestingly, Cl¦ remains the anion with the highest conductance in this mutant, and the difference in conductance between Cl¦ and anions with similar energies of hydration (Br¦ and NO×¦) is greater than for wild-type or T338A (Fig. 12B). Login to comment
186 This applies both to lyotropic anions with increased permeability in T338A (Br¦, NO×¦, ClOÚ¦) and the kosmotropic anion F¦, which shows increased permeability in F337S. Login to comment
189 The conductance ratio, gIÏgCl, was 0·20 ± 0·03 (n = 4) for wild-type, 0·14 ± 0·05 (n = 4) for F337S, and 0·59 ± 0·09 (n = 4) for T338A. Login to comment
197 Dependence of relative anion permeability (A) and relative anion conductance (B) on anion-free energy of hydration in wild-type, F337S and T338A CFTR Values of PXÏPCl and gXÏgCl in each case are as given in Table 1. Login to comment
218 Macroscopic I-V relationships under bi-ionic conditions (intracellular I¦, extracellular Cl¦), for wild-type, F337S and T338A CFTR expressed in BHK cells The different degrees of outward rectification suggest different relative I¦ conductances in these three CFTR variants. Login to comment
242 In the present study, the anion binding properties of two mutants, F337S and T338A, were examined in detail. Login to comment
245 In contrast, T338A did not significantly affect the relative conductance of different anions (Table 1), although some small changes in the conductance sequence were apparent (Table 2). Login to comment
249 Block by SCN¦, I¦ and ClOÚ¦ was significantly weakened in both F337S and T338A, and F337S showed significant block by high concentrations of Br¦ not evident in wild-type (Fig. 11). Login to comment
250 These results suggest that lyotropic anion binding is weakened in both F337S and T338A, in spite of the 'opposite` effect of these two mutations on anion selectivity. Login to comment
256 Implications for the mechanism of anion selectivity The mutations F337S and T338A caused co-ordinated changes in the relative permeability and relative conductance of Br¦, F¦, NO×¦, ClOÚ¦ and I¦ ions, with high permeability being associated with high conductance (Table 1; Fig. 13). Login to comment
261 Thus, not only in wild-type CFTR but also both F337S and T338A, Cl¦ is the anion with the highest conductance (Table 1), even though other anions may have higher permeabilities. Login to comment
264 Optimization of Cl¦ conductance, rather than Cl¦ permeability, is likely to be of greater importance to the physiological function of CFTR and other Cl¦ channels, although in CFTR the mutations T338A and T338S increase Cl¦ conductance, apparently without adversely affecting other channel properties (Linsdell et al. 1998). 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

126 Although no information on the voltage dependence of SCN- block is obtained in this way, we have previously used this same protocol to show directly that intrapore anion binding is altered in the TM6 mutants F337S and T338A (21). Login to 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
164 However, as summarized in Table 1, TM12 mutations M1137A and N1138A did not alter the anion selectivity sequence, in stark contrast to the corresponding TM6 mutations F337A (20) and T338A (16). Login to comment
177 Similar weakening of SCN- block was previously observed in the TM6 mutants F337S and T338A (21). Login to comment

PMID: 11478590 [PubMed] Linsdell P et al: "Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

131 In spite of this caveat, it is interesting to note that the effects of mutations within the pore that drastically alter SCN- permeability but have only small effects on SCN- binding (F337S, T338A; Linsdell 2001) may result in such a model from alterations in barrier height, implying that the effects of these mutations may result primarily from changes in anion access to the pore. 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
221 Px/PCl values for small anions were increased in T338A CFTR, whereas Px/PCl values for large anions were decreased. Login to comment
224 Interestingly, Px/PCl for SCN- , the most permeant anion tested in WT CFTR, was nearly doubled in T338A CFTR, whereas PI/PCl was increased sevenfold. Login to comment
225 Gx/GCl values for large anions were also decreased in T338A CFTR, whereas Gx/GCl values for small anions were increased (Table 4). 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
256 For anions larger than isethionate (glutamate and gluconate), even the T338A mutation had only a very small effect on Gx/GCl. Login to comment
268 Px/PCl and Gx/GCl values for each anion x in each mutant were calculated for T1134A, K335A, T338A, T339A, and S341A CFTRs and normalized to Px/PCl and Gx/GCl values for WT CFTR. Login to comment
284 As in T338A CFTR, PClO4/PCl stands out as most sensitive to mutation at this position because PClO4/PCl was increased 18-fold in T338A CFTR and 11-fold in T338E 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
330 In contrast, mutation T338A induced significant hysteresis for all three of the large anions studied. Login to comment
359 Even mutation T338A, which had the most pronounced effects on selectivity between monovalent anions, did not affect selectivity between Cl- and S2O3 2- . 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
392 However, a subsequent study (24) found that the single mutation T338A led to an even larger single-channel conductance and that mutations here did not strongly affect the functional minimum pore diameter. Login to comment
398 McDonough et al. (33) previously showed that T339A CFTR was identical to WT CFTR with respect to blockade by DPC, whereas T338A CFTR exhibited identical affinity for DPC (at -100 mV) but slightly altered voltage dependence. 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
456 3) GClO4/GCl and Gacetate/GCl were oppositely affected by mutations T338A and S341A, as if these amino acids lie on opposite sides of a barrier that determines selectivity between these anions of very similar size. Login to comment

PMID: 11889571 [PubMed] Gupta J et al: "Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel."

No. Sentence Comment

4 Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Login to comment
63 While block of the TM12 mutants S1141A (Fig. 1) and T1134A and M1137A (data not shown) was indistinguishable from wild-type, block was significantly weakened in the TM6 mutants F337A and T338A, and significantly strengthened in the TM12 mutants N1138A and T1142A (Fig. 1). Login to comment
69 Mean fraction of control current remaining following addition of 60 µM glibenclamide (I/I0) is shown as a function of voltage for wild-type (q), T338A (s), N1138A (s), F337A (ss) and T1142A (xx). Login to comment
70 Mean of data from 5-10 patches, fitted by Eq. according to the mean parameters shown in Fig. 3 rent remaining following addition of glibenclamide (I/I0) was significantly reduced at all voltages in N1138A and T1142A (P<0.05), and significantly increased in F337A and T338A at negative membrane potentials (P<0.05). Login to comment
75 Consistent with the results shown in Fig. 2, Kd(0) was significantly increased in F337A and T338A, and significantly decreased in N1138A and T1142A (Fig. 3A). Login to comment
83 The extracellular Cl-concentration had a similar effect on Kd(0) in the TM6 mutants F337A (Fig. 5A) and T338A (Figs. 4, 5A). Login to comment
98 A Example I-V relationships for wild-type (left), T338A (center) and T1142A (right), recorded with 10 mM extracellular Clas described in Materials and methods, before (Ctrl) and immediately following addition of 60 µM glibenclamide (+Glib) to the intracellular solution. B Mean fraction of control current remaining following addition of glibenclamide, with 154 mM (q) or 10 mM (qq) extracellular Cl-, for wild-type (left), T338A (center) and T1142A (right). 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
100 Relative Au(CN)2 _ permeability was significantly decreased in F337S and significantly increased in T338A (Fig. 4), consistent with the previously described opposite effects of these mutants on CFTR lyotropic anion selectivity (Linsdell et al. 1998, 2000). 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
116 Au(CN)2 _ permeability of different CFTR variants A, example CFTR I-V relationships recorded with 150 m KAu(CN)2 in the extracellular solution and 150 m KCl in the intracellular solution, for wild-type, F337S and 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
139 The effects of F337S and T338A on PAu(CN)2/PCl are consistent with the disruption (F337S; Linsdell et al. 2000) and strengthening (T338A; Linsdell et al. 1998) of lyotropic anion selectivity previously described in these two mutants. Login to comment
142 Multiple TM6 residues contribute to anion binding, as determined by Au(CN)2 _ block of Cl_ permeation; the dramatic weakening of Au(CN)2 _ block in T338A suggests a particularly strong role of this residue in binding. 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: 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
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

PMID: 12679372 [PubMed] Gong X et al: "Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore."

No. Sentence Comment

40 conditions of high extracellular Cl_ concentration, Au(CN)2 _ block is weakened in the CFTR pore mutants K335A, F337S and T338A (Gong et al. 2002a), suggesting that these pore residues may contribute to lyotropic anion binding site(s) within the pore. Login to comment
60 In wild-type, K335A, F337S and T338A, high extracellular Cl_ significantly weakens Au(CN)2 _ block and (except in F337S) increases the fraction of the transmembrane electric field apparently experienced by the blocker. Login to comment
131 In contrast, mutation of other nearby TM6 residues associated with weakened Au(CN)2 _ binding (K335A, F337S, T338A) showed similar sensitivity to extracellular Cl_ concentration to that seen in wild-type (Figs 1 and 2). 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
44 Of eight mutants studied, only T339V was without any significant effect on anion permeability (Table 1), and five mutations (R334C, K335A, F337A, T338A, I340A) led to changes in the permeability sequence among halides (Figure 2 and Table 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
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
72 This halide selectivity sequence is changed to Eisenman sequence II in I340A, and Eisenman sequence I in both K335A and T338A (Table 2), consistent with a strengthening of lyotropic anion selectivity in these mutants. 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
109 Lyotropic anion selectivity is disrupted in F337A and modified in R334C, T338A and S341A. Login to comment

PMID: 14598388 [PubMed] Liu X et al: "CFTR: what's it like inside the pore?"

No. Sentence Comment

114 T338C CFTR undergoes pH-dependent changes in gCl and I-V shape that are not seen in wild type, T338A or T338S CFTR. 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
105 However, when we investigated the block at the most negative voltages that we were able to keep membrane patches (-150 mV) with a low extracellular Cl-concentration (4 mM), we noticed an anomalous voltage-dependent increase in Pt(NO2)4 2--blocked current in F337A but not in wild-type, F337Y or T338A (Fig. 6). Login to comment
106 Under these conditions, the strength of Pt(NO2)4 2- block in wild-type, F337Y, and T338A increases with increasingly negative voltages, eventually leading to a negative slope of the current-voltage relationship in the presence of blocker (Fig. 6 B). Login to comment
116 Thus, at very negative voltages, Pt(NO2)4 2- ions can escape from the F337A channel pore, but apparently not from the pore of wild-type, F337Y or T338A, by passing through the channel and into the extracellular solution-a process previously termed "punchthrough" (Nimigean and Miller, 2002). 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
190 In contrast, mutations of the adjacent TM6 residue (T338), including T338A, altered the selectivity between different lyotro- Figure 9. Login to comment
201 The slight Pt(NO2)4 2- permeability of F337A therefore suggests that this divalent anion might normally be prevented from passing through the pore for similar reasons that limit the permeability of kosmotropic anions like F-. In contrast, the T338A mutation appears to enhance unblock by permeation of the lyotropic Au(CN)2 - ion (Gong and Linsdell, 2003b). Login to comment

PMID: 15366420 [PubMed] Zhang ZR et al: "Steady-state interactions of glibenclamide with CFTR: evidence for multiple sites in the pore."

No. Sentence Comment

309 The most significant change was seen with the T338A mutant in transmembrane domain 6, although this only reflected a two-fold decrease in affinity. Login to comment

PMID: 15504721 [PubMed] Ge N et al: "Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

78 Only one mutation studied, T338A, led to a significant increase in channel conductance (Figs. 3 and 4) as described previously (35). Login to comment
93 Note that in the final panel, showing data from the high conductance mutant T338A, the current (i) scale is increased. Login to comment
102 The weakest block by Au(CN)2 - was observed in K95Q, T338A, R334K, and Q98A, consistent with these residues perhaps being associated with permeant anion binding sites inside the pore. Login to comment
109 Thiocyanate permeability was strongly increased in A96V and T338A, suggesting enhancement of lyotropic selectivity in these mutants, and dramatically reduced in F337A, which we previously suggested reflects the role of Phe-337 in contributing to an anion selectivity filter in the pore (11, 36). Login to comment

PMID: 15504728 [PubMed] Zhang ZR et al: "Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore."

No. Sentence Comment

93 Fig. 1 also contains records illustrating the subconductance behavior of two mutant CFTRs: R334C and T338A. Login to comment
94 The former exhibits a full conductance that is less than that of WT-CFTR under comparable conditions (14), and the latter exhibits an increased full conductance (9.8 pS in T338A-CFTR). Login to comment
97 This result suggests that neither the R334C nor T338A mutation, although they involve residues reputed to lie within the CFTR pore (14, 18), greatly altered the relative magnitude of the subconductance states. Login to comment
124 A-C, records for WT-, R334C-, and T338A-CFTR, respectively, were generated in excised, inside-out mode with asymmetrical [Cl- ], where the pipette was filled with 40 mM [Cl- ] and bath (cytoplasmic) solution contained 302 mM [Cl- ] in order to potentiate the single channel amplitude. Login to comment

PMID: 16436375 [PubMed] Liu X et al: "Variable reactivity of an engineered cysteine at position 338 in cystic fibrosis transmembrane conductance regulator reflects different chemical states of the thiol."

No. Sentence Comment

53 RESULTS T338C/WT CFTR Conductance Was Markedly Altered by 2-ME or DTT Prior to Exposure to Exogenous Thiol-directed Reagents5 - Exposing oocytes expressing T338C/WT CFTR to 2-ME or DTT during steady state activation led to increases in conductance (without any discernable change in reversal potential) that were rapid (t1/2 ϭ 20 s), and of variable amplitude and were not seen in oocytes expressing CFTR constructs lacking the cysteine at 338, such as WT, T338A, T338H, T338S CFTR, or Cys-less CFTR. Login to comment
102 This 6 Similarly, exposure to reducing agents was without effect on oocytes expressing either T338A or T338S CFTR, constructs that retain the 18 endogenous cysteines (see below). Login to comment
111 It should be noted that the cysteine at position 338 is essential for the effects of MTS reagents as well as 2-ME and DTT shown above, because neither the conductance due to T338A or T338S CFTR was sensitive to reducing agents or thiol-directed reagents.7 Trapping Thiols with an Alkylating Agent, IAM-The results presented so far are compatible with a scheme in which the total conductance of an oocyte expressing T338C/WT CFTR or T338C/Cys-less CFTR comprises at least three components that we will label as gSH, gSX1, and gSX2, where the total conductance, gCl, is given by Equation 1. gCl ϭ gSH ϩ gSX1 ϩ gSX2 (Eq. 1) 7 X. Liu and D. C. Dawson, unpublished observation. Login to comment
162 C, oocytes expressing T338A CFTR were first exposed to: 1 mM Au(CN)2 - (black circles), 1 mM IAM, 1 mM Au(CN)2 - . Login to comment
167 Fig. 4C contains the result of an experiment showing that IAM did not affect the function of T338A CFTR (n ϭ 3). Login to comment
169 Block of T338A CFTR was unaffected by exposure to IAM. Login to comment
192 Diamide-GSH had no discernable effect on conductance of oocytes expressing T338A CFTR.7 Oxidation by NO or H2O2 Did Not Reproduce the Signature Behavior of Spontaneously Oxidized T338C CFTR Channels-Fig. 7A depicts a typical experiment (n ϭ 4) in which an oocyte expressing T338C CFTR was first exposed to 1 mM DTT to increase the number of cysteines in the simple thiol form. Exposure to 1 mM SNAP, a commonly used NO donor (46, 65), produced a minimal effect on the conductance, but largely blocked the subsequent reaction with MTSES- , indicating oxida- tionofthecysteinetothenitrosothiol.Thisapparentoxidationwaswithout effect on the macroscopic conductance but was readily reversed by exposing oocytes to 1 mM DTT, as indicated by an 80% decrease in conductance followingthesecondexposuretoMTSES- .SNAPhadnodiscernableeffect on conductance of oocytes expressing T338A CFTR.7 Fig. 7B depicts a typical experiment (n ϭ 2) in which an oocyte expressing T338C CFTR was first exposed to 1 mM DTT to increase the number of cysteines in the simple thiol form. Exposure to 5 mM H2O2 FIGURE 5. Login to comment
214 At 1 ␮M, copper induced an 80% (Ϯ5%, n ϭ 5) decrease in T338C CFTR conductance, but was without effect on T338A or WT CFTR conductance.9 Washing often produced a slow recovery from inhibition that could vary from near zero to about 32% of the inhibited conductance. Login to comment

PMID: 16973827 [PubMed] Radpour R et al: "Two novel missense and one novel nonsense CFTR mutations in Iranian males with congenital bilateral absence of the vas deferens."

No. Sentence Comment

10 This approach allowed us to detect one novel nonsense mutation (K536X) in the nucleotide-binding domain 1 (NBD1) region and two novel missense mutations (Y122H and T338A) in the M2 and M6 regions of CFTR gene in our studied population, which were not reported previously. Login to comment
14 Because Y122H and T338A mutations were compound heterozygote with the IVS8-5T, it is difficult to judge the severity of these mutations and their role in the CBAVD phenotype. Login to comment
45 Mutation Location Mutation type Nucleotide alteration Allele frequency (%) K536X Exon 11 Nonsense 1738 A to T 1/224 (0.45) Y122H Exon 4 Missense 496 T to C 1/224 (0.45) T338A Exon 7 Missense 1144 A to G 1/224 (0.45) Figure 1. Login to comment
48 (C) Transabdominal ultrasonography in Patient no. 49 with T338A and IVS8-5T mutations. Login to comment
73 mutation of 1144A→G in exon 7 (Figure 2) which causes an amino acid change of threonine to alanine at position 338 of CFTR polypeptide. Login to comment
78 The numbering of the reported mutations is as follows: c.1738A>T or p.Lys536Stop (K536X), c.496T>C or p.Tyr122His (Y122H) and c.1144A>G or p.Thr338Ala (T338A). Login to comment
104 T338A An exon 7 missense mutation in TMD-M6 was found with 1144A→G in a CBAVD phenotype (Patient no. 49). Login to comment
105 T338A was Table II. Login to comment
110 Mutation type IVS8-(TG)mTn M470V n (%) K536X (TG)10 9T / (TG)10 9T M/V 1 (0.89) Y122H (TG)11 7T / (TG)13 5T V/V 1 (0.89) T338A (TG)11 7T / (TG)13 5T M/V 1 (0.89) Figure 3. Login to comment
121 Because Y122H and T338A mutations were compound heterozygote with the IVS8-5T, it is difficult to judge the severity of these mutations and their role in the CBAVD phenotype. Login to comment

PMID: 17314234 [PubMed] Radpour R et al: "Molecular study of (TG)m(T)n polymorphisms in Iranian males with congenital bilateral absence of the vas deferens."

No. Sentence Comment

77 CFTR gene mutations in 112 CBAVD patients and 7 CBAVD patients* Samples Mutation genotype3 (TG)m(T)n n (%) CBAVD Two mutations detected (5 /112 5 4.46%) F508del / R117H (TG)10 9T / (TG)10 9T 1 (0.89) F508del / 621+1G.T (TG)11 7T / (TG)11 7T 1 (0.89) 1540A/G / 1540A/G (TG)11 7T / (TG)11 7T 2 (1.79) R347H / R117H (TG)10 9T / (TG)11 7T 1 (0.89) One mutation detected with one 5T allele (32 / 112 5 28.57%) G551D / - (TG)10 7T/ (TG)13 5T 2 (1.79) F508del / - (TG)12 7T/ (TG)13 5T 8 (7.14) (TG)11 9T/ (TG)13 5T 6 (5.36) 1717-1G.A / - (TG)11 7T/ (TG)12 5T 4 (3.57) R117H / - (TG)12 7T/ (TG)13 5T 2 (1.79) 621+1G.T / - (TG)11 7T/ (TG)13 5T 3 (2.68) 2 (1.79) 1540A/G / - (TG)11 7T/ (TG)13 5T 2 (1.79) R553X / - (TG)12 7T/ (TG)13 5T 1 (0.89) Y122H / -4 (TG)11 7T / (TG)13 5T 1 (0.89) T338A / -4 (TG)10 7T / (TG)13 5T 1 (0.89) No mutation detected with two 5T alleles (11 / 112 5 9.82%) - / - (TG)12 5T / (TG)13 5T 3 (2.68) - / - (TG)13 5T / (TG)13 5T 8 (7.14) One mutation detected without 5T allele (35 / 112 5 31.25%) G85E / - (TG)11 7T / (TG)11 7T 2 (1.79) G551D / - (TG)10 9T / (TG)12 7T1 1 (0.89) 621+1G.T / - (TG)11 7T / (TG)11 7T 2 (1.79) (TG)10 9T / (TG)11 7T 1 (0.89) R334W / - (TG)12 7T / (TG)10 7T 1 (0.89) F508del / - (TG)11 7T / (TG)11 7T 7 (6.25) (TG)11 9T / (TG)12 7T 3 (2.68) (TG)10 9T / (TG)10 9T 2 (1.79) 1717-1G.A / - (TG)11 7T / (TG)12 7T 3 (2.68) (TG)10 9T / (TG)11 7T 2 (1.79) R117H/- (TG)12 7T / (TG)12 7T 2 (1.79) (TG)10 9T / (TG)11 7T 1 (0.89) 2789+5G.A / - (TG)10 7T / (TG)11 7T 1 (0.89) 3120+1G.A / - (TG)10 9T / (TG)11 7T 2 (1.79) R560T / - (TG)10 9T / (TG)11 7T 1 (0.89) N1303K / - (TG)10 9T / (TG)11 7T 1 (0.89) 1651A/G / - (TG)11 7T / (TG)12 7T 1 (0.89) R553X / - (TG)10 9T / (TG)10 7T 1 (0.89) K536X / -4 (TG)10 9T / (TG)10 9T 1 (0.89) No mutation detected with one 5T alleles (7 / 112 5 6.25%) - / - (TG)13 5T / (TG)12 7T 3 (2.68) - / - (TG)13 5T / (TG)10 9T 4 (3.57) No mutation detected (22 / 112 5 19.64%) - / - (TG)11 7T / (TG)11 7T 12 (10.71) - / - (TG)11 7T / (TG)12 7T 1 (1.79) - / - (TG)10 9T / (TG)10 9T 3 (2.68) - / - (TG)10 9T / (TG)11 7T 6 (5.36) CUAVD One mutation detected without 5T allele (2 / 7 5 28.57%) R334W / - (TG)10 9T / (TG)11 7T 1 (14.29) R117H / - (TG)11 7T / (TG)11 7T 1 (14.29) No mutation detected with one 5T alleles (3 / 7 5 42.86%) - / - (TG)11 9T / (TG)13 5T 2 (28.57) - / - (TG)10 7T / (TG)13 5T 1 (14.29) No mutation detected (2 / 7 5 28.57%) - / - (TG)10 9T / (TG)12 7T 2 (28.57) * CBAVD indicates congenital bilateral absence of the vas deferens; CUAVD, congenital unilateral absence of the vas deferens. Login to comment

PMID: 17849169 [PubMed] Liu X et al: "A possible role for intracellular GSH in spontaneous reaction of a cysteine (T338C) engineered into the Cystic Fibrosis Transmembrane Conductance Regulator."

No. Sentence Comment

4 Single-channel recordings indicated that T338C CFTR channels not exposed to 2-ME or DTT exhibited multiple conductance levels not seen in T338A CFTR channels. Login to comment
51 Summarized in Fig. 1 are results obtained from oocytes expressing T338C or T338A CFTRs that were either untreated, or exposed to 100 lM BCNU for 72 h prior to electrophysiological recording. Login to comment
59 (B) The initial steady state conductance of oocytes expressing T338A CFTR (black bars) and the conductance after exposure to 1 mM 2-ME (white bars) were summarized for the control oocytes and oocytes maintained in the storage solution (MBSH) containing 100 lM BCNU since injection of cRNA significantly from untreated controls, suggesting that the treatment may mimic the ''naturally modified`` state. Login to comment
60 Oocytes expressing T338A CFTR (Fig. 1B) exhibited no response to 2-ME with or without exposure to BCNU. Login to comment
61 The mean initial conductance of T338A CFTR was slightly lower in BCNU treated oocytes (67 ± 6 lS) than untreated ones (52 ± 9 lS), but the difference was not statistically significant. Login to comment
90 To verify that a cysteine was required for the multiple current amplitudes observed in T338C CFTR, I recorded single-channel currents of T338A CFTR. Login to comment
93 Thus exposure to BCNU did not result in any significant change in the fractional distribution of 0.9 pA events in T338A CFTR channels. Login to comment
94 No difference was detected among the apparent open probabilities (NPo /N) of T338A CFTR channels (pH 7.4) under control or BCNU treated conditions using records obtained at 500 lM intracellular ATP. Login to comment
116 Regardless, a cysteine at position 338 was essential for the GSH effect because at concentrations as high as 10 mM, GSH had no effect on conductance of oocytes expressing Cys-less CFTR (Fig. 5B, n = 2) or T338A CFTR (Fig. 5C, n = 2). Login to comment
120 These results indicate that although GSH is capable of breaking a mixed disulfide bond at 338, the reaction precedes at a much lower rate and required a much higher Fig. 4 BCNU had no effect on single T338A CFTR conductance. Login to comment
121 Fractional distribution of single-channel current amplitudes at pH 7.4 from patches obtained from T338A CFTR expressing oocytes that were: (A) incubated in MBSH, (B) incubated in MBSH containing 100 lM BCNU since injection of cRNA. Login to comment
122 Fractional distribution of single-channel current amplitudes at pH 6.0 from patches obtained from T338A CFTR expressing oocytes that were: (C) incubated in MBSH, (D) incubated in MBSH containing 100 lM BCNU since injection of cRNA. Login to comment
136 (C) Following activation (hatched bar and crosshair), an oocyte expressing T338A CFTR was exposed to 10 mM GSH (open triangles) et al. 1993; Ferreira et al. 1993). 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

110 T338A and R347A were similar to wild-type CFTR. 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: 18421494 [PubMed] Cui G et al: "Mutations at arginine 352 alter the pore architecture of CFTR."

No. Sentence Comment

105 Transitions to these subconductance levels occur rarely in WT-CFTR but more frequently in some pore-domain mutants, such as R334C and T338A, although the relative conductances between levels s1, s2, and f are maintained (Zhang et al. 2005a). Login to comment

PMID: 18597042 [PubMed] Mornon JP et al: "Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces."

No. Sentence Comment

194 Two mutations involving these residues (F337A and T338A) also significantly weakened the glibenclamide-mediated blocking of the channel [69], suggesting a direct interaction between the inhibitor and this region of the pore. Login to comment

PMID: 9729613 [PubMed] Linsdell P et al: "Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines."

No. Sentence Comment

91 Significant rectification resulting in a -I+50ÏI-0 ratio significantly less than one was observed in all mutants except T338A (Fig. 3). Login to comment
93 Single CFTR channel currents in inside-out patches excised from CHO cells stably expressing wild-type, T338A or T338S are shown in Fig. 4A. Login to comment
97 Mean slope conductance was increased from 7·9 ± 0·1 pS (n = 18) for wild-type to 10·4 ± 0·1 pS (n = 9) for T338A and 11·3 ± 0·2 pS (n = 5) for T338S. Login to comment
99 Although the gating of T338A and T338S channels was not studied in detail, no striking differences from wild-type were noted. Login to comment
107 Unitary properties of T338A and T338S CFTR A, examples of single channel activity for wild-type, T338A and T338S, recorded at -50 mV. Login to comment
109 B and C, mean single channel current-voltage relationships for wild-type, T338A (B) and T338S (C). Login to comment
142 Permeability of intracellular anions in wild-type and mutant CFTR Cl¦ channels ------------------------------------------------------------ Anion WT T338A T338S T338N T338V T339V ------------------------------------------------------------ Thiocyanate 2·63 ± 0·13 (6) 5·85 ± 0·27 (4)* 4·80 ± 0·19 (3)* 8·72 ± 1·03 (4)* 1·92 ± 0·35 (4)* 3·28 ± 0·08 (4)* Nitrate 1·53 ± 0·04 (7) 2·04 ± 0·08 (3)* 1·82 ± 0·03 (4)* 4·22 ± 0·22 (3)* 6·84 ± 1·18 (7)* 1·61 ± 0·02 (3) Bromide 1·23 ± 0·03 (5) 1·74 ± 0·04 (3)* 1·47 ± 0·07 (3)* 1·66 ± 0·15 (3)* 1·04 ± 0·09 (5) 1·39 ± 0·06 (4)* Chloride 1·00 ± 0·01 (10) 1·00 ± 0·02 (11) 1·00 ± 0·02 (6) 1·00 ± 0·03 (10) 1·00 ± 0·04 (11) 1·00 ± 0·06 (10) Iodide 0·84 ± 0·03 (5) 2·09 ± 0·16 (5)* 1·76 ± 0·09 (3)* 1·03 ± 0·05 (3)* 0·79 ± 0·11 (3) 0·84 ± 0·02 (3) Perchlorate 0·25 ± 0·02 (6) 1·35 ± 0·08 (3)* 0·66 ± 0·06 (3)* 0·41 ± 0·03 (3)* 0·54 ± 0·00 (3)* 0·24 ± 0·01 (4) Benzoate 0·069 ± 0·006 (6) 0·17 ± 0·03 (4)* 0·091 ± 0·019 (3) 0·089 ± 0·015 (4) 0·15 ± 0·02 (4)* 0·097 ± 0·014 (4) Hexafluorophosphate < 0·019 (4) 0·53 ± 0·01 (3)* 0·31 ± 0·02 (3)* 0·68 ± 0·02 (3)* 0·39 ± 0·05 (3)* 0·051 ± 0·010 (4)* Fluoride 0·11 ± 0·01 (7) 0·12 ± 0·02 (4) 0·095 ± 0·012 (4) 0·11 ± 0·01 (4) 0·093 ± 0·009 (3) 0·17 ± 0·02 (4)* Formate 0·25 ± 0·01 (8) 0·45 ± 0·04 (3)* 0·43 ± 0·03 (3)* 0·35 ± 0·04 (4)* 0·22 ± 0·01 (3) 0·28 ± 0·02 (3) Acetate 0·090 ± 0·004 (8) 0·19 ± 0·01 (3)* 0·18 ± 0·01 (3)* 0·10 ± 0·02 (5) 0·11 ± 0·02 (3) 0·16 ± 0·01 (3)* Propanoate 0·14 ± 0·01 (3) 0·18 ± 0·02 (4) 0·098 ± 0·010 (4)* 0·077 ± 0·013 (3)* 0·13 ± 0·02 (3) - Pyruvate 0·10 ± 0·01 (5) 0·20 ± 0·01 (3)* 0·13 ± 0·02 (3) 0·075 ± 0·015 (3) 0·17 ± 0·03 (3)* - Methane sulphonate 0·077 ± 0·005 (5) 0·14 ± 0·02 (4)* 0·079 ± 0·014 (3) 0·038 ± 0·004 (3)* 0·088 ± 0·007 (3) - Glutamate 0·096 ± 0·008 (4) 0·082 ± 0·009 (3) 0·080 ± 0·008 (3) 0·060 ± 0·012 (5)* 0·11 ± 0·01 (3) - Isethionate 0·13 ± 0·01 (4) 0·11 ± 0·01 (3) 0·086 ± 0·012 (5)* 0·043 ± 0·007 (3)* 0·067 ± 0·005 (3)* - Gluconate 0·068 ± 0·004 (36) 0·10 ± 0·01 (3)* 0·060 ± 0·004 (3) 0·044 ± 0·004 (3) 0·077 ± 0·009 (3) 0·088 ± 0·021 (5) ------------------------------------------------------------ Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.g. Figs 7 and 10) as described in Methods. Login to comment
150 Note that all lyotropic anions showed the permeability sequence T338A > T338S > wild-type, again suggesting that the effects of these mutations on pore properties are correlated with the size of the amino acid side chain substituted. Login to comment
153 Pore diameter of T338 mutants Previously, we suggested that the double mutant channel, TT338,339AA, had an increased minimum functional pore diameter, based on its increased permeability to extracellular formate, acetate, propanoate and pyruvate ions (Linsdell et P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.18 -------------------------------------------------------------------------------------------- Table 2 ---------------------------------------------- Wild-type SCN¦ > NO×¦ > Br¦ > Cl¦ > I¦ > ClOÚ¦ > formate > F¦ > PFÜ¦ T338A SCN¦ > I¦ ü NO×¦ > Br¦ > ClOÚ¦ > Cl¦ > PFÜ¦ > formate > F¦ T338S SCN¦ > NO×¦ ü I¦ > Br¦ > Cl¦ > ClOÚ¦ > formate > PFÜ¦ > F¦ T338N SCN¦ > NO×¦ > Br¦ > I¦ = Cl¦ > PFÜ¦ > ClOÚ¦ > formate > F¦ T338V NO×¦ > SCN¦ > Br¦ = Cl¦ > I¦ > ClOÚ¦ > PFÜ¦ > formate > F¦ -------------------------------------------------------------------------------------------- Figure 7. Login to comment
164 In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0·528 nm (wild-type), 0·576 nm (T338A), 0·549 nm (T338S), 0·510 nm (T338N) and 0·540 nm (T338V). Login to comment
168 In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0·528 nm for wild-type, 0·576 nm for T338A, 0·549 nm for T338S, 0·510 nm for T338N and 0·540 nm for T338V. Anions examined (in order of increasing diameter) were: SCN¦, Cl¦, NO×¦, Br¦, I¦, ClOÚ¦, benzoate and PFÜ¦. Login to comment
171 In this case, fits by eqn (2) suggested minimum pore diameters of 0·535 nm (wild-type), 0·615 nm (T338A), 0·505 nm (T338S), 0·503 nm (T338N) and 0·530 nm (T338V). Login to comment
179 As with T338A and T338S, T339V showed apparently normal channel gating, with open probability being time and P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.110 Figure 9. Login to comment
181 In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0·535 nm for wild type, 0·615 nm for T338A, 0·505 nm for T338S, 0·503 nm for T338N and 0·530 nm for T338V. Anions examined (in order of increasing diameter) were: formate, acetate, propanoate, pyruvate, methane sulphonate and gluconate.) Login to comment
196 Conversely, the elevated conductance of T338A and T338S might be advantageous in gene or protein replacement therapies for Alteration of CFTR anion selectivityJ. Physiol. 512.1 11 Figure 10. Login to comment
205 Interestingly, both T338A (10·4 pS; Fig. 4B) and T338S (11·3 pS; Fig. 4C) have higher conductances than that we reported previously for TT338,339AA (9·9 pS; Linsdell et al. 1997b). Login to comment
232 Thus in T338A, for example, the relationship between permeability and ionic hydration energy appeared much more linear than for wild-type (Fig. 12B). Login to comment
242 Relationship between relative permeability and free energy of hydration for different intracellular anions A, wild-type; B, T338A. 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

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
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
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
219 Gupta and Linsdell reported that mutation T338A reduced block by Glyb [21], but this was not the case in our experiments with either Glyb or Glip. Login to comment

PMID: 21940661 [PubMed] Stahl M et al: "Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101."

No. Sentence Comment

219 Gupta et al. (21) observed that two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block. Login to comment

PMID: 15361410 [PubMed] Liu X et al: "CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore."

No. Sentence Comment

64 The single channel current for T338A CFTR was measured using 150 mM symmetrical [Clÿ ]. Login to comment
121 (C) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338A CFTR. Login to comment
124 Changing the bath pH had essentially no effect on the conductances of oocytes expressing T338A CFTR (Fig. 2 C, n ¼ 3), nor did the same maneuver alter the conductances of oocytes expressing T338S (Fig. 3) or wt CFTR (Smith et al., 2001), consistent with the idea that the pH-dependent change in conductance of T338C CFTR was due to the titration of the cysteine substituted at 338. Login to comment
125 As an additional test of the hypothesis that the pH-induced response seen in T338C CFTR was due to the titration of the engineered cysteine, we exposed oocytes expressing T338C CFTR to NEM, a reagent that forms a thioether bond with the cysteine, and thereby blocks titration of the thiol group. Login to comment
155 Single-channel recording also indicated that the conductance of T338A CFTR channels was not sensitive to changes in bath pH (Fig. 5 C, inset). Login to comment
156 It is also of interest to note that the ratio of the single channel conductance at pH 6 and pH 7.4 for T338C CFTR was ;1.8, a value comparable to the ratio observed for the macroscopic conductances (1.7). Login to comment
164 No pH-induced change in Po* was observed in T338A CFTR, which averaged 0.85 6 1.2 at pH 6 and averaged 0.74 6 0.15 at pH 7.4 ([ATP] ¼ 1 mM). Login to comment
205 (Inset) T338A CFTR conductance was insensitive to changes in bath pH. Login to comment
206 Shown are examples of single-channel i-V plots obtained from inside-out patches detached from oocytes expressing T338A CFTR at pH 6 (solid circles) and pH 7.4 (shaded triangles). Login to comment
207 All the recordings for T338A CFTR were done in the presence of 150 mM symmetrical [Clÿ ]. Login to comment
208 NEM- or MTSESÿ -modified T338H/R334C CFTR were more titratable and the apparent pKa values were shifted toward more basic values. Login to comment
122 (C) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338A CFTR. Login to comment
165 No pH-induced change in Po* was observed in T338A CFTR, which averaged 0.85 6 1.2 at pH 6 and averaged 0.74 6 0.15 at pH 7.4 ([ATP] &#bc; 1 mM). Login to comment

PMID: 12054472 [PubMed] Tan AL et al: "Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

70 Hence it is conceivable that since the mutation T338A removes the hydrogen bond, the conformation of the hydrophilic Lys335 is perturbed, and as a result the permeability is altered. Login to comment

PMID: 9379166 [PubMed] Dawson DC et al: "Cystic fibrosis transmembrane conductance regulator. Permeant ions find the pore."

No. Sentence Comment

37 Of particular interest in this paper is the behavior of a mutant CFTR, T338A, T339A, in which two threonines in TM6 were substituted with alanines. 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

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
226 In addition, other mutations of hydroxylated residues in TM 6, namely T338A and T339A, affect neither DPC block nor rectification (Table 1). Login to comment

PMID: 10827976 [PubMed] Linsdell P et al: "Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

157 Under the macroscopic current recording conditions used here, the mutation T338A changes the halide selectivity from Eisenman sequence III to sequence I, consistent with the strengthening of lyotropic selectivity in this mutant (Linsdell et al., 1998). 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

338 Mutation T338A altered the voltage dependence of block by DPC without affecting affinity at afa;100 mV (McDonough et al., 1994). Login to comment
343 With respect to block by DPC, S1118F-CFTR had an effect much like that of T338A-CFTR, wherein affinity at afa;100 mV was not changed significantly but the voltage dependence was reduced. Login to comment

PMID: 16766608 [PubMed] Serrano JR et al: "CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore."

No. Sentence Comment

69 Similar responses to [Au(CN)2] were observed using oocytes expressing T338A CFTR (Fig. 1 C), and the apparent dissociation constants (in mM) were similar for the three constructs: Kwt &#bc; 0.754, KCys-less &#bc; 0.813, KT338A &#bc; 0.754. Login to comment
85 (C) A representative experiment showing the effect of [Au(CN)2] on T338A CFTR conductance. Login to comment
86 After activation by a stimulatory cocktail (Isop1IBMX, hatched bar), an oocyte expressing T338A CFTR channels was exposed to 1 mM DTT (open circles) and then to 10 mM, 100 mM, 1 mM, and 10 mM of [Au(CN)2] (solid circles). Login to comment
121 Block of the residual conductance by [Au(CN)2] was consistent with an apparent dissociation constant of ;0.8 mM, comparable to that seen with wt, T338A, and Cys-less CFTR. Login to comment
143 Exposure of an oocyte expressing a construct bearing a non-Cys substitution at position 338 (T338A CFTR) to IAM (2 mM) was without effect on reversible, lyotropic block by [Au(CN)2] (13). Login to comment

PMID: 23709221 [PubMed] Cui G et al: "Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function."

No. Sentence Comment

20 Infrequent subconductance behavior is seen in some CFTR mutants, such as T338A/Cand S1141A-CFTR (7, 12). Login to comment
213 We conclude that the subconductance states in CFTR probably also represent pore conformational change for the following reasons: 1) the CFTR channel pore forms from one polypeptide as a monomer and only bears one permeation pathway (12); 2) the s1 and s2 states occur as rare events in some point mutations, such as T338A/Cand K335A/C-CFTR, which do not appear to affect gross pore architecture, whereas they are frequent events in CFTR channels bearing salt bridge mutations, such as R352A- and R347A-CFTR, as discussed above; 3) mutations at sites involved in salt bridges (such as Arg347 , Arg352 , Asp924 , and Asp993 ) result in much more frequent occupancy of subconductance states; 4) mutations at sites involved in salt bridges (such as Arg347 and Arg352 ) lead to greatly altered sensitivity to pore blockers (7, 13); and 5) the subconductance behavior is not affected by different concentrations of Clafa; or by changes in membrane potential (12, 16). Login to comment

PMID: 23955087 [PubMed] Wang W et al: "Relative contribution of different transmembrane segments to the CFTR chloride channel pore."

No. Sentence Comment

31 Overall, the effects of the T338A mutation have been interpreted as reflecting an increase in functional diameter of the narrowest part of the pore [26] and a reduced barrier to the movement of permeant ions inside the pore [11]. Login to comment
70 Because the properties of T338A-CFTR channels have been investigated in detail previously, simplified protocols were used to facilitate comparison of the effects of the T338A mutation with those of other mutations (Figs. 5, 6, 7, and 8). Login to comment
87 T338A, as well as other substitutions at this position, cause complex changes in the relative permeability of different anions in CFTR [26]; however, the major changes observed in T338A can be summarized as (a) an increase in the relative permeability of lyotropic anions such as SCN- , NO3 - , Br- , I- , and ClO4 - , and (b) an increase in the relative permeability of extracellular organic (kosmotropic) monvalent anions including formate, acetate, propanoate, and pyruvate [26]. Login to comment
150 In addition, possible additive effects of reducing the side chain volumes of these two nearby residues was investigated using a T338A/S1118A double mutant. Login to comment
155 The depolarizing (rightward) shift in the current reversal potential indicates an increased PSCN/PCl in the T338A/S1118A double mutant. Login to comment
156 b Mean PSCN/PCl values calculated from reversal potential measurements under these conditions as described in the "Materials and methods" section. Mean of data from three to six patches. Asterisks indicate a significant difference from wild type (P<0.01), while hashtag indicates a significant difference from the T338A mutant (P<0.0002) Fig. 8 Acetate permeability of mutants. Login to comment
158 The hypolarizing (leftward) shift in the current reversal potential indicates an increased Pacetate/PCl in the T338A/S1118A double mutant. Login to comment
159 b Mean Pacetate/PCl values calculated from reversal potential measurements under these conditions as described in the "Materials and methods" section. Mean of data from three to five patches. Asterisks indicate a significant difference from wild type, while hashtag indicates a significant difference from the T338A mutant (P<0.01) very small (<5 %) reductions in conductance (Fig. 5). Login to comment
162 These very minor effects on conductance are in contrast with the large (>25 %) increase seen in T338A (Fig. 5), as reported previously [26]. Login to comment
163 The double mutant T338A/S1118A had a similarly elevated conductance that was not significantly different from that of T338A alone (P>0.75; Fig. 5). Login to comment
164 Altered movement of permeant ions in the pore of T338A-CFTR is also reflected by changes in the voltage-dependent block of Cl-currents by low concentrations of permeant Au(CN)2 - ions [11, 14, 15]. Login to comment
165 As shown in Fig. 6, channel block by intracellular Au(CN)2 - ions is altered in two distinct ways by the T338A mutation: (a) block is significantly weakened and (b) the I-V relationship in the presence of Au(CN)2 - shows an unusual "N"-shape (Fig. 6a), resulting in a "U"- shaped fractional current-voltage relationship (Fig. 6c) that indicates strongest block close to 0 mV membrane potential that is weakened at both more hyperpolarized and more depolarized voltages. Login to comment
166 This unusual shape is thought to reflect increased unblock by blocker permeation at hyperpolarized voltages, which may reflect a reduced barrier to Au(CN)2 - movement inside the pore in T338A [11]. Login to comment
168 Interestingly, block of the T338A/S1118A double mutant was slightly weaker than for T338A alone (Fig. 6c, d), suggesting that these two mutations might have additive effects on Au(CN)2 - binding in the pore. Login to comment
180 As proposed previously for TM6 residue T338 [42], the channel is shown as being in an "outward facing" configuration when closed (with T1115 and S1118 accessible from the outside), and switching to an "inward facing" configuration on opening (with T1115 and S1118 accessible from the inside) S1118A and T1115A, although this increase was much less than that observed in T338A (Fig. 7b). Login to comment
181 Interestingly, SCN- permeability was further increased in the T338A/S1118A double mutant (Fig. 7). Login to comment
184 Again, the increase in acetate permeability seen in T338A was significantly augmented in the T338A/S1118A mutation (Fig. 8), suggesting an additive effect of these two mutations on organic anion permeability. Login to comment
206 However, mutagenesis of S1118 to residues with smaller (alanine) or larger (glutamine, valine) residues had surprisingly small effects on channel functional properties, in particular compared to those of mutagenesis of T338A. Login to comment
207 The effects of the S1118A mutation on permeant anion (Au(CN)2 - ) binding (Fig. 6), permeability of the lyotropic SCN- anion (Fig. 7), and permeability of the organic acetate anion (Fig. 8) were qualitatively similar to, but generally smaller than, those of T338A, and in fact similar effects were seen in T1115A. Login to comment
211 Reduction of side chain volume in S1118A and T1115A, like T338A, led to an increase in the relative permeability of the small organic anion acetate, consistent with an increase in the apparent diameter of the narrowest region of the pore [25, 26]; however, introduction of side chains with larger volume (S1118Q, S1118V) did not lead to a decrease in acetate permeability (Fig. 8). Login to comment
213 Simultaneous mutagenesis of T338 and S1118 to small alanine residues also had only small additional effects compared to the T338A mutation alone (Figs. 5, 6, 7, and 8). Login to comment
214 Most striking here were a significantly increased permeability of the T338A/S1118A double mutant both to SCN- (Fig. 7) and to acetate (Fig. 8). Login to comment
215 Permeability of small lyotropic anions like SCN- might be influenced by interactions throughout the pore [18, 38] or might be determined predominantly at a localized "selectivity filter" [18, 24] and so the apparently additive effects of the T338A and S1118A mutations is difficult to interpret in terms of the relative roles or locations of these two residues. Login to comment
216 Permeability of large anions such as acetate is thought to be determined predominantly by steric factors at the narrowest part of the pore [25], and so the increase in acetate permeability in T338A/S1118A compared to either mutation alone might be considered evidence that these two mutations impact the dimensions of a common, narrow region of the pore. Login to comment
218 Nevertheless, T338A (which results in a similarly small reduction in side chain volume) is associated with much greater changes in pore properties. Login to comment

PMID: 26209275 [PubMed] Cui G et al: "Murine and human CFTR exhibit different sensitivities to CFTR potentiators."

No. Sentence Comment

131 WT 0.0 0.1 0.2 0.3 Fractional inhibition by 2.5 &#b5;M GlyH-101 # R334C R334A T338A R352A # # # 0.4 0.5 0.4 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO R334C- hCFTR A B C D ND96 ISO ISO+ GlyH ND96 ISO T338A-hCFTR 1 &#b5;A 50 s 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-hCFTR Fig. 5. Login to comment
132 Effects of GlyH-101 on wild-type (WT)- (A), R334C- (B), and T338A-hCFTR (C). Login to comment
163 Extracellular 25 òe;M GlyH-101 failed to block but only potentiated R334A-mCFTR. Summary data for fractional inhibition (E) and fractional potentiation (F) of WT-, V100L-, R147H-, I201V-, R334A-, and T338A-mCFTR with 25 òe;M GlyH-101. Login to comment
181 In contrast, 2.5 òe;M GlyH-101 exhibited strengthened block of both T338A- and R352A-hCFTR (Fig. 5, C and D). Login to comment