ABCMdb

PMID: 11557589

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

Sentences

No. Mutations Sentence Comment

60 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser341Thr ABCC7 p.Thr339Ala Mutants K335E, K335F, T338A, T339A, S341A, S341T, T1134A, and T1134F were prepared as previously described (33). Login to comment 61 ABCC7 p.Thr1134Glu ABCC7 p.Thr338Glu ABCC7 p.Lys335Ala Mutants K335A, T338E, and T1134E were prepared with the QuickChange protocol (Stratagene, La Jolla, CA). Login to comment 62 ABCC7 p.Ser341Glu S341E CFTR was a generous gift from D. Dawson (Oregon Health Sciences University, Portland, OR). Login to comment 65 ABCC7 p.Thr1134Glu ABCC7 p.Thr1134Glu Capped transcripts (mMessage mMachine, Ambion, Austin, TX) for WT CFTR and for each of the variants (2-35 ng for most and 340 ng for T1134E CFTR) along with 0.6 ng of beta2-adrenergic receptor cRNA were injected into each oocyte. Login to comment 94 ABCC7 p.Ser341Glu All data are background subtracted, with the currents measured in the absence of Iso in the same oocyte as background, with the exception of experiments with S341E CFTR. Login to comment 97 ABCC7 p.Ser341Glu However, currents in S341E CFTR-expressing oocytes were increased by ϳ30% on further stimulation with Iso and exhibited time-independent behavior in response to steps in membrane potential, indicating that these currents reflect activity of CFTR. Login to comment 98 ABCC7 p.Ser341Glu S341E CFTR expressed well in oocytes so that the signal-to-noise ratio (compared to a background of an uninjected oocyte) was at least 40 for each experiment. Login to comment 99 ABCC7 p.Ser341Glu Furthermore, the selectivity pattern described in Characterization of the WT CFTR pore: lyotropic selectivity and anion binding for S341E CFTR differed considerably from the recently described pattern for the endogenous Ca2ϩ -activated Cl- chan- nel of oocytes (34). Login to comment 100 ABCC7 p.Ser341Glu Hence, we felt confident in ascribing anion currents in oocytes expressing this cRNA to the S341E CFTR mutant channels rather than to the endogenous Ca2ϩ -activated Cl-channel. Login to comment 143 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser341Thr ABCC7 p.Thr339Ala ABCC7 p.Thr1134Glu ABCC7 p.Thr338Glu ABCC7 p.Lys335Ala ABCC7 p.Ser341Glu 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 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser341Thr ABCC7 p.Thr339Ala ABCC7 p.Thr1134Glu ABCC7 p.Thr338Glu ABCC7 p.Lys335Ala ABCC7 p.Ser341Glu 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 169 ABCC7 p.Arg347Asp For instance, the R347D mutation has been shown to have nonspecific effects due to destruction of a salt bridge (8). Login to comment 191 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser341Thr ABCC7 p.Thr339Ala ABCC7 p.Thr1134Glu ABCC7 p.Thr338Glu ABCC7 p.Lys335Ala ABCC7 p.Ser341Glu 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Thr339Ala ABCC7 p.Lys335Ala 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 200 ABCC7 p.Thr339Ala T339A CFTR exhibited altered Gx/GCl values only for acetate and NO3 - (Table 4). Login to comment 201 ABCC7 p.Thr339Ala The limited effects of the T339A mutation are consistent with the hypothesis that this position is non-pore lining. Login to comment 203 ABCC7 p.Thr1134Ala ABCC7 p.Lys335Ala K335A and T1134A CFTR exhibited decreased Gx/GCl values for most anions, with radii as large as or larger than acetate. Login to comment 204 ABCC7 p.Thr1134Ala ABCC7 p.Lys335Ala However, neither Px/PCl nor Gx/GCl values for the smallest anions (NO3 - and Br- ) were altered in K335A CFTR or T1134A CFTR compared with WT CFTR. Login to comment 213 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser341Thr ABCC7 p.Thr339Ala ABCC7 p.Thr1134Glu ABCC7 p.Thr338Glu ABCC7 p.Lys335Ala ABCC7 p.Ser341Glu 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 217 ABCC7 p.Lys335Phe ABCC7 p.Lys335Ala At K335, Gx/GCl values for all small anions were increased in K335F CFTR compared with K335A CFTR, whereas Px/PCl values for these anions were not affected by the bulky side chain. Login to comment 218 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Ala T1134F CFTR exhibited a similar pattern except that GNO3/GCl and GBr/GCl were decreased and GSCN/GCl was increased compared with T1134A CFTR. Login to comment 221 ABCC7 p.Thr338Ala Px/PCl values for small anions were increased in T338A CFTR, whereas Px/PCl values for large anions were decreased. Login to comment 224 ABCC7 p.Thr338Ala 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 ABCC7 p.Thr338Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala Except for PNO3/PCl, the magnitude of the effect in S341A CFTR was less than that seen in T338A CFTR. Login to comment 231 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala Interestingly, although GClO4/GCl was increased in T338A CFTR, GClO4/GCl was decreased in S341A CFTR. Login to comment 232 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala The pattern in S341A CFTR was similarly inverted for Gacetate/GCl compared with that in T338A CFTR. Login to comment 234 ABCC7 p.Ser341Thr Previous results by McDonough et al. (33) indicated that the nature and orientation of the aliphatic hydroxyl side chain at S341 was critical to pore characteristics in that S341T was not identical to WT CFTR with respect to blockade by DPC. Login to comment 236 ABCC7 p.Ser341Thr We asked whether side-chain orientation at S341 was important to anion selectivity by performing selectivity experiments in the S341T mutant. Login to comment 237 ABCC7 p.Ser341Thr Relative permeabilities to large anions were greatly reduced in S341T CFTR. Login to comment 255 ABCC7 p.Thr1134Ala The one exception to this pattern is the decreased GClO4/GCl in T1134A CFTR. Login to comment 256 ABCC7 p.Thr338Ala For anions larger than isethionate (glutamate and gluconate), even the T338A mutation had only a very small effect on Gx/GCl. Login to comment 261 ABCC7 p.Leu859Ala Discrimination between anions could result from differences in energy barrier heights (reflected in Px/PCl) or from differences in well depths (reflected in L859A Gx/GCl). Login to comment 268 ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Thr339Ala ABCC7 p.Lys335Ala 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 275 ABCC7 p.Thr1134Glu In contrast, T1134E CFTR exhibited a leftward shift in Vrev Cl . Login to comment 276 ABCC7 p.Thr338Glu ABCC7 p.Ser341Glu S341E CFTR exhibited a larger shift in Vrev Cl and pronounced outward rectification, whereas T338E CFTR exhibited pronounced inward rectification. Login to comment 277 ABCC7 p.Thr338Glu ABCC7 p.Ser341Glu The opposite patterns of macroscopic rectification in T338E CFTR and S341E CFTR suggest that these two positions may lie on opposite sides of a barrier in the pore. Login to comment 278 ABCC7 p.Lys335Glu K335E CFTR exhibited decreased Px/PCl for large anions and increased Px/PCl for the "sticky" anions I- and ClO4 - (Table 2). Login to comment 279 ABCC7 p.Thr1134Glu Px/PCl values for all anions except SCN- and ClO4 - were affected by mutation T1134E. Login to comment 280 ABCC7 p.Lys335Glu Gx/GCl values for most anions were altered in K335E CFTR, with decreases in Gx/GCl values for large anions and increases in Gx/GCl values for small anions (Table 4). Login to comment 281 ABCC7 p.Lys335Glu ABCC7 p.Thr1134Glu T1134E CFTR exhibited a similar pattern, with changes in Gx/GCl values smaller in magnitude than those for K335E CFTR. Login to comment 283 ABCC7 p.Thr338Glu Px/PCl was increased in T338E CFTR for all anions except acetate and gluconate. Login to comment 284 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Glu 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 285 ABCC7 p.Thr338Glu Mutation T338E resulted in increased Gx/GCl values for all anions smaller than acetate, whereas Gx/GCl values for acetate, glutamate, and gluconate were decreased. Login to comment 287 ABCC7 p.Ser341Glu In contrast, Gx/GCl values for all anions were altered in S341E CFTR (Table 4). Login to comment 289 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Glu ABCC7 p.Ser341Glu 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 ABCC7 p.Ser341Ala 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 293 ABCC7 p.Ser341Glu A prediction from the disruption of SCN- binding in S341E CFTR is that this mutation may result in disruption of DPC binding as well. Login to comment 294 ABCC7 p.Ser341Glu To test this notion, we compared the blockade of CFTR macroscopic currents in oocytes expressing WT CFTR or S341E CFTR using methods established previously (50). Login to comment 296 ABCC7 p.Ser341Glu In contrast, S341E CFTR was insensitive to DPC at all voltages, even at high concentration; in fact, 0.5 mM DPC led to a slight increase in current at all potentials. Login to comment 310 ABCC7 p.Ser341Glu ABCC7 p.Ser341Glu Currents carried by WT CFTR or S341E CFTR were measured in the absence of drug and in the presence of 100 (for WT CFTR) or 500 (for S341E CFTR) ␮M DPC applied to the bathing solution. Login to comment 313 ABCC7 p.Ser341Glu S341E CFTR was not blocked by DPC, even at high concentrations. Login to comment 316 ABCC7 p.Thr1134Ala ABCC7 p.Thr339Ala ABCC7 p.Lys335Ala The overall discriminating power was approximately the same for WT, K335A, T339A, and T1134A CFTR. Login to comment 320 ABCC7 p.Thr338Glu ABCC7 p.Ser341Glu In fact, mutations T338E and S341E led to channels that could barely distinguish between monovalent anions. Login to comment 329 ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe This behavior was retained in K335F and T1134F CFTR but lost in all other mutants. Login to comment 330 ABCC7 p.Thr338Ala In contrast, mutation T338A induced significant hysteresis for all three of the large anions studied. Login to comment 357 ABCC7 p.Lys335Glu ABCC7 p.Thr338Glu Mutation K335E led to a slight decrease in GS2O3/GCl, whereas mutations T338E and S341E greatly increased GS2O3/GCl. Login to comment 358 ABCC7 p.Ser341Ala In contrast, among alanine substitution mutants, only S341A CFTR exhibited a significant effect on GS2O3/GCl. Login to comment 359 ABCC7 p.Thr338Ala 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 378 ABCC7 p.Lys335Ala When the alanine in K335A CFTR was replaced by the bulkier phenylalanine, the effects on anion selectivity were greater (Tables 2 and 4). Login to comment 388 ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Phe ABCC7 p.Lys335Phe ABCC7 p.Lys335Phe ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Thr1134Ala ABCC7 p.Thr1134Ala ABCC7 p.Thr339Ala ABCC7 p.Thr339Ala ABCC7 p.Thr1134Glu ABCC7 p.Thr1134Glu ABCC7 p.Thr338Glu ABCC7 p.Thr338Glu ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala 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 391 ABCC7 p.Leu863Ala L863A (23), as if this locus controlled the functional pore diameter. Login to comment 392 ABCC7 p.Thr338Ala 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 394 ABCC7 p.Thr338Glu ABCC7 p.Thr338Gln Unfortunately, the single-channel conductance of charge mutants at this position (e.g., T338E compared with T338Q) was not studied to determine whether electrostatic repulsion occurred due to the introduction of charge. Login to comment 396 ABCC7 p.Thr339Ala T339A CFTR expressed poorly in Chinese hamster ovary (CHO) cells (24), although it expressed well in oocytes (33; present study). Login to comment 397 ABCC7 p.Thr339Val T339V CFTR, which did express in CHO cells, exhibited limited alterations in selectivity compared with mutants at T338 (24), consistent with a non-pore lining role for T339. Login to comment 398 ABCC7 p.Thr338Ala ABCC7 p.Thr339Ala 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 400 ABCC7 p.Thr339Ala In our hands, T339A CFTR exhibited selectivity very similar to WT CFTR. Login to comment 405 ABCC7 p.Ser341Ala 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 ABCC7 p.Ser341Ala S341 also appears to be a Cl-binding site because single-channel conductance was greatly reduced in S341A CFTR (33). Login to comment 409 ABCC7 p.Ser341Glu Mutation S341E had a greater effect on the ability of the channel to discriminate between monovalent anions than did any other glutamic acid substitution (Fig. 7). Login to comment 430 ABCC7 p.Thr1134Phe Further evidence against a discrete selectivity filter is the greater than sixfold increase in relative affinity for I- exhibited by T1134F CFTR. Login to comment 438 ABCC7 p.Ser341Glu Also, it may be true that ions more closely approach a charge placed at S341 than a charge placed at T338 by virtue of the constriction, leading to greater destabilization of anion binding in S341E CFTR. Login to comment 439 ABCC7 p.Lys335Glu ABCC7 p.Ser341Glu Our results with K335E (outside the region of high discrimination) and S341E (inside the region of high discrimination) show that this interpretation is feasible. Login to comment 446 ABCC7 p.Ser341Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala 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 ABCC7 p.Thr338Ala 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 457 ABCC7 p.Thr338Glu ABCC7 p.Ser341Glu 4) T338E CFTR exhibits pronounced inward rectification in the macroscopic I-V, whereas S341E CFTR exhibits pronounced outward rectification (Fig. 5). Login to comment 464 ABCC7 p.Thr1134Ala ABCC7 p.Lys335Ala However, our data show that mutations K335A and T1134A had nearly identical effects on selectivity patterns between large and small anions, as if these amino acids occupy nearly homologous positions in TM6 and TM12. Login to comment