ABCMdb

ABCC7 p.Lys335Ala

Predicted by SNAP2:	A: D (66%), C: D (80%), D: D (91%), E: D (75%), F: D (91%), G: D (80%), H: D (80%), I: D (80%), L: D (80%), M: D (80%), N: D (80%), P: D (91%), Q: N (53%), R: N (87%), S: D (66%), T: D (75%), V: D (80%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

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

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

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61 Mutants K335A, T338E, and T1134E were prepared with the QuickChange protocol (Stratagene, La Jolla, CA). 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
203 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 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 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 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
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
316 The overall discriminating power was approximately the same for WT, K335A, T339A, and T1134A CFTR. Login to comment
378 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 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
464 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

[hide] CFTR: covalent and noncovalent modification sugges... J Gen Physiol. 2001 Oct;118(4):407-31. Smith SS, Liu X, Zhang ZR, Sun F, Kriewall TE, McCarty NA, Dawson DC
CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.
J Gen Physiol. 2001 Oct;118(4):407-31., [PMID:11585852]

Abstract [show]
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

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No. Sentence Comment
341 However, for two mutants, R334H and K335A, the RR value clearly deviated significantly from that predicted by the general trend. Login to comment

[hide] Molecular determinants of Au(CN)(2)(-) binding and... J Physiol. 2002 Apr 1;540(Pt 1):39-47. Gong X, Burbridge SM, Cowley EA, Linsdell P
Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.
J Physiol. 2002 Apr 1;540(Pt 1):39-47., 2002-04-01 [PMID:11927667]

Abstract [show]
Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)(2)(-) anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 microM 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. Relative Au(CN)(2)(-) permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337-S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent.

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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
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
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

[hide] Mechanism of lonidamine inhibition of the CFTR chl... Br J Pharmacol. 2002 Nov;137(6):928-36. Gong X, Burbridge SM, Lewis AC, Wong PY, Linsdell P
Mechanism of lonidamine inhibition of the CFTR chloride channel.
Br J Pharmacol. 2002 Nov;137(6):928-36., [PMID:12411425]

Abstract [show]
1. The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. 2. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage-dependent block of CFTR currents, with an apparent K(d) of 58 micro M at -100 mV. 3. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl(-) concentration. 4. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 micro M(-1) s(-1) and an off-rate of 143 s(-1). 5. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. 6. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action.

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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
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

[hide] Molecular determinants and role of an anion bindin... J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4. Gong X, Linsdell P
Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore.
J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4., 2003-06-01 [PMID:12679372]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2- ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2- binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2- binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2- ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism.

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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

[hide] Extent of the selectivity filter conferred by the ... Mol Membr Biol. 2003 Jan-Mar;20(1):45-52. Gupta J, Lindsell P
Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore.
Mol Membr Biol. 2003 Jan-Mar;20(1):45-52., [PMID:12745925]

Abstract [show]
Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl(-) channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.

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No. Sentence Comment
43 Other mutants studied (K335A, I336A, I340A), like T339V, had only minor effects (data not shown). 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
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
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

[hide] Mutation-induced blocker permeability and multiion... J Gen Physiol. 2003 Dec;122(6):673-87. Epub 2003 Nov 10. Gong X, Linsdell P
Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore.
J Gen Physiol. 2003 Dec;122(6):673-87. Epub 2003 Nov 10., [PMID:14610019]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)42- acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent Kd 556 microM), kinetically fast, and weakened by extracellular Cl- ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)42- ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)42- increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42- ions to the extracellular solution. This "punchthrough" was prevented by extracellular Cl- ions, reminiscent of a "lock-in" effect. Relief of block in F337A by Pt(NO2)42- permeation was only observed for blocker concentrations above 300 microM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl- current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)42- in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42- to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42- ions within the pore promotes their permeation to the extracellular solution.

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

[hide] Direct comparison of the functional roles played b... J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25. Ge N, Muise CN, Gong X, Linsdell P
Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25., 2004-12-31 [PMID:15504721]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, little is known about the relative functional contribution of different TM regions to the pore. We have used patch clamp recording to investigate the functional consequences of point mutations throughout the six transmembrane regions in the N-terminal part of the CFTR protein (TM1-TM6). A range of specific functional assays compared the single channel conductance, anion binding, and anion selectivity properties of different channel variants. Overall, our results suggest that TM1 and -6 play dominant roles in forming the channel pore and determining its functional properties, with TM5 perhaps playing a lesser role. In contrast, TM2, -3, and -4 appear to play only minor supporting roles. These results define transmembrane regions 1 and 6 as major contributors to the CFTR channel pore and have strong implications for emerging structural models of CFTR and related ATP-binding cassette proteins.

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77 Significant, but smaller, reductions in unitary conductance were observed in G213A, V318A, S321A, V322A, K335A, and I336A (Figs. 3 and 4). Login to comment
119 Some mutants that significantly affect both unitary conductance and Au(CN)2 - block were found to be without effect on SCN- permeability (Q98A, V318A, R334K, K335A). Login to comment

[hide] Determination of the functional unit of the cystic... J Biol Chem. 2005 Jan 7;280(1):458-68. Epub 2004 Oct 25. Zhang ZR, Cui G, Liu X, Song B, Dawson DC, McCarty NA
Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore.
J Biol Chem. 2005 Jan 7;280(1):458-68. Epub 2004 Oct 25., 2005-01-07 [PMID:15504728]

Abstract [show]
The magnitudes and distributions of subconductance states were studied in chloride channels formed by the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and in CFTRs bearing amino acid substitutions in transmembrane segment 6. Within an open burst, it was possible to distinguish three distinct conductance states referred to as the full conductance, subconductance 1, and subconductance 2 states. Amino acid substitutions in transmembrane segment 6 altered the duration and probability of occurrence of these subconductance states but did not greatly alter their relative amplitudes. Results from real time measurements indicated that covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate resulted in the simultaneous modification of all three conductance levels in what appeared to be a single step, without changing the proportion of time spent in each state. This behavior suggests that at least a portion of the conduction path is common to all three conducting states. The time course for the modification of R334C-CFTR, measured in outside-out macropatches using a rapid perfusion system, was also consistent with a single modification step as if each pore contained only a single copy of the cysteine at position 334. These results are consistent with a model for the CFTR conduction pathway in which a single anion-conducting pore is formed by a single CFTR polypeptide.

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171 This observation provided an opportunity to test directly the notion that a nearby positive charge would modify the rate of modification of the cysteine at 334 by comparing the rate of modification of R334C-CFTR with the rate of modification of R334C/K335A-CFTR (Fig. 4B). Login to comment
172 The amplitude of macroscopic current was increased 2.97 Ϯ 0.24-fold by 50 ␮M MTSETϩ in R334C/K335A-CFTR. Login to comment
174 These data are compatible with the notion that the rate of modification of a cysteine at position 334 is sensitive to the local electrostatic potential, partially determined by the amino acid at position 335, although we cannot discount the possibility that the K335A mutation altered the pore structure FIG. 3. Login to comment
190 The change in electrostatic potential due to modification of one cysteine would be expected to alter the rate of modification of the remaining cysteine, as suggested by the difference in response in R334C- and R334C/ K335A-CFTR. Login to comment
194 Outside-out macropatch experiments for mutants R334C- and R334C/K335A-CFTR. Login to comment
199 The amplitude of macroscopic current was increased by 2.3-fold upon modification by MTSETϩ in this experiment. B, representative macroscopic current of R334C/K335A-CFTR; the red line is the curve fit, with ␶ ϭ 1.1 s in this experiment. Login to comment

[hide] Identification of positive charges situated at the... Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1. Zhou JJ, Fatehi M, Linsdell P
Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.
Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1., [PMID:18449561]

Abstract [show]
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.

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62 To investigate the role of charge on these residues in controlling I-V shape, neutral substitutions (R104Q, R117Q, K335A, R1128Q) were also investigated. Login to comment
91 All mutants depicted (R104Q, R117Q, K335A, R1128Q, R104E, R117E, K335E, R1128E) showed rectification ratios significantly different from wild type (asterisk P<0.05). Login to comment
116 All mutants depicted (R104Q, R117Q, K335A, R104E, R117E, K335E) significantly different from wild type (asterisk P<0.05). Login to comment
120 As shown in Fig. 8a, while Pt(NO2)4 2-induced strong inward rectification in wild type (as a result of strongly voltage-dependent current inhibition) [26], this effect was less marked in R104Q, K335A, and R1128Q. Login to comment
122 Considering the data at +80 mV, where the inhibitory effects of Pt(NO2)4 2- are strongest, suggests that Pt(NO2)4 2- inhibition is significantly weakened in R104Q, K335A, and (to a lesser extent) R1128Q but not significantly altered in K114C, R117Q, K329A, K829Q, or R899Q (Fig. 8c). Login to comment
126 In contrast, unitary current amplitude in both R104Q and K335A appeared relatively resistant to the inhibitory effects of external Pt(NO2)4 2- ions (Fig. 9a-c), again consistent with effects on macroscopic currents (Fig. 8), suggesting that positive charge at positions 104 and 335 is necessary for the full inhibitory effects of extracellular Pt(NO2)4 2- ions. Login to comment
147 b Mean fraction of control normalized current remaining in the presence of 10 mM extracellular Pt(NO2)4 2at different membrane potentials in wild type (closed circles), R104Q (closed squares), K335A (open circles), and R1128Q (open squares), quantified as the IREL in the presence of 10 mM Pt(NO2)4 2- [IREL(block)] as a fraction of that in the absence of Pt(NO2)4 2- [IREL(control)]. Login to comment
170 c Mean fractional unitary current amplitude remaining in the presence of this concentration of Pt (NO2)4 2- ions as a function of voltage, in wild type (open circles), R104Q (closed circles, left panel), and K335A (closed circles, right panel). Login to comment

[hide] CFTR: mechanism of anion conduction. Physiol Rev. 1999 Jan;79(1 Suppl):S47-75. Dawson DC, Smith SS, Mansoura MK
CFTR: mechanism of anion conduction.
Physiol Rev. 1999 Jan;79(1 Suppl):S47-75., [PMID:9922376]

Abstract [show]
CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.

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562 firmed that SCN binding was reduced but not eliminated in K335D and E CFTR and showed that simply deleting D. Structural Elements That Are Important for the charge (K335A) was without effect. Login to comment

[hide] Differential contribution of TM6 and TM12 to the p... Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13. Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PMID:22160394]

Abstract [show]
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 muM Glyb and 200 muM Glip was 0.64 +/- 0.03 (n = 7) and 0.48 +/- 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 muM Tolb was 0.40 +/- 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

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119 The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left). Login to comment
125 Mutations R334A and K335A lie in the outer vestibule of the pore of CFTR; surprisingly, the two mutations db ca 2 nA 100 ms 100 ms 2 nA Concentration (μM) 0 200 400 600 800 1000 0.2 0.4 0.6 0.8 1.0 Fractionalblock 100 ms 4 nA Fig. 2 Concentration-dependent block of WT-CFTR by Glyb, Glip and Tolb. Login to comment
133 R334A weakened block while K335A strengthened block by both blockers. Login to comment
137 On the contrary, K335A single-channel behavior is very similar to that of WT-CFTR except the single-channel conductance is slightly decreased (-0.56± 0.01 pA, n=5; see Fig. 9), as would be expected for an amino acid that affects attraction of Cl- ions. Login to comment
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
162 Although mutations R334A and K335A exhibited opposite effects on steady-state block by Glyb and Glip, neither mutation altered initial block (Fig. 5). Login to comment
163 Effects on time-dependent block by mutations R334A and K335A Fractional block by Glip200 μM V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 * ** ** ** ** ** ** * V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 1.0 * * * * * ** ** ** ** Fractional block by Glyb50 μM Fig. 4 Alanine-scanning in TM12 to identify amino acids that interact with Glyb and Glip. 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
168 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) were similar for Glyb and Glip, although the effect of R334A on Glyb was larger than for Glip and the effect of K335A was larger for Glip than Glyb (Fig. 6). 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
221 The effects of mutations R334A and K335A are indirect, likely related to the movement of chloride within the pore, or the stabilization of the outer vestibule. Login to comment
239 Hence, strong time-dependent block of macropatch currents, and the appearance of multiple drug-induced closed states in single-channel recordings, may not arise from 0.4 pA 2 s M348A c f 0.2 pA 2 s F337A c f 0.4 pA 2 s K335A c f 0.4 pA 2 s c s2 f D1152A 0.4 pA 2 s T1134A c f 0.4 pA 2 s S1141A c f s2 0.4 pA 2 s c f WT 2000 4000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 3000 9000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 6000 400 1200 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 800 1600 1000 3000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 2000 500 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 1000 4000 12000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 8000 200 600 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 400 Fig. 9 Representative single-channel traces for WT-, K335A-, F337A-, M348A-, T1134A-, S1141A-, and D1152A-CFTR (left) from excised inside-out membrane patches with symmetrical 150 mM Cl- solution, and their all-points amplitude histograms (right). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biophys J. 1998 Mar;74(3):1320-32. Mansoura MK, Smith SS, Choi AD, Richards NW, Strong TV, Drumm ML, Collins FS, Dawson DC
Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.
Biophys J. 1998 Mar;74(3):1320-32., [PMID:9512029]

Abstract [show]
We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.

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62 Expression levels Wild-type and 11 mutant CFTR constructs were used in this study: G91A, G91E, G91R, G314A, G314D, G314E, G314Q, K335R, K335A, K335D, and K335E. Login to comment
107 Neither the arginine (K335R) nor the alanine substitution (K335A) resulted in any substantial change in the permeability ratios (with the exception of SCN for K335A). Login to comment
112 The conductance ratios for anions judged by shifts in reversal potential to be most highly permeant (SCN, NO3, and Br) were all increased significantly by substituting a negatively charged residue (D or E) for K335, whereas mutants in which the charge was conserved (K335R) or removed (K335A) were identical to wtCFTR. Login to comment
113 The K335D and K335E constructs also exhibited increased conductance ratios for I, a result previously reported by Anderson et al. (1991) for K335E CFTR. Login to comment
137 Permeability Ratios Wild type 4-9 3.42 Ϯ 0.28 1.42 Ϯ 0.04 1.22 Ϯ 0.02 0.39 Ϯ 0.01 0.44 Ϯ 0.03 G91A 3-6 3.24 Ϯ 0.26 1.53 Ϯ 0.04 1.27 Ϯ 0.02 0.37 Ϯ 0.04 0.40 Ϯ 0.04 G91E 3-7 3.50 Ϯ 0.54 1.59 Ϯ 0.04 1.27 Ϯ 0.01 0.35 Ϯ 0.01 0.51 Ϯ 0.04 G91R 3-4 5.26 ؎ 0.46* 1.60 Ϯ 0.03 1.40 ؎ 0.01* 0.32 Ϯ 0.04 0.64 ؎ 0.04* G314A 3-4 2.87 Ϯ 0.17 1.45 Ϯ 0.03 1.19 Ϯ 0.02 0.31 Ϯ 0.03 0.33 Ϯ 0.03 G314D 4 3.42 Ϯ 0.34 1.44 Ϯ 0.05 1.25 Ϯ 0.04 0.33 Ϯ 0.03 0.51 Ϯ 0.05 G314E 3-4 3.72 Ϯ 0.56 1.65 ؎ 0.09* 1.35 ؎ 0.03* 0.49 Ϯ 0.04 0.53 Ϯ 0.04 G314Q 3-4 3.89 Ϯ 0.37 1.62 Ϯ 0.11 1.27 Ϯ 0.04 0.36 Ϯ 0.03 0.62 Ϯ 0.05 K335R 3-5 3.44 Ϯ 0.29 1.35 Ϯ 0.04 1.22 Ϯ 0.03 0.40 Ϯ 0.05 0.41 Ϯ 0.07 K335A 5-6 5.34 ؎ 0.58* 1.48 Ϯ 0.06 1.28 Ϯ 0.04 0.37 Ϯ 0.03 0.60 Ϯ 0.06 K335D 4-6 3.02 Ϯ 0.19 1.50 Ϯ 0.03 1.10 ؎ 0.02* 0.54 ؎ 0.04* 0.65 ؎ 0.06* K335E 5-8 3.64 Ϯ 0.21 1.48 Ϯ 0.06 1.29 Ϯ 0.03 0.46 Ϯ 0.04 1.10 ؎ 0.04* B. Conductance Ratios Wild type 4-9 0.14 Ϯ 0.02 0.75 Ϯ 0.02 0.64 Ϯ 0.02 0.52 Ϯ 0.03 0.18 Ϯ 0.03 G91A 3-6 0.14 Ϯ 0.01 0.77 Ϯ 0.02 0.61 Ϯ 0.02 0.47 Ϯ 0.02 0.19 Ϯ 0.02 G91E 3-7 0.15 Ϯ 0.03 0.73 Ϯ 0.02 0.60 Ϯ 0.01 0.50 Ϯ 0.04 0.30 Ϯ 0.02 G91R 3-4 0.14 Ϯ 0.00 0.84 Ϯ 0.01 0.63 Ϯ 0.01 0.32 ؎ 0.01* 0.14 Ϯ 0.01 G314A 3-4 0.30 Ϯ 0.09 0.89 ؎ 0.01* 0.66 Ϯ 0.01 0.48 Ϯ 0.09 0.24 Ϯ 0.01 G314D 4 0.28 Ϯ 0.05 0.82 Ϯ 0.01 0.70 Ϯ 0.02 0.49 Ϯ 0.06 0.27 Ϯ 0.03 G314E 3-4 0.62 ؎ 0.07* 1.18 ؎ 0.04* 0.84 ؎ 0.05* 0.42 Ϯ 0.05 0.29 Ϯ 0.09 G314Q 3-4 0.63 ؎ 0.02* 1.01 ؎ 0.04* 0.82 ؎ 0.03* 0.50 Ϯ 0.02 0.42 ؎ 0.02* K335R 3-5 0.14 Ϯ 0.01 0.76 Ϯ 0.03 0.61 Ϯ 0.02 0.59 Ϯ 0.06 0.16 Ϯ 0.03 K335A 6 0.20 Ϯ 0.03 0.77 Ϯ 0.02 0.61 Ϯ 0.02 0.45 Ϯ 0.03 0.21 Ϯ 0.02 K335D 4-6 0.65 ؎ 0.04* 1.25 ؎ 0.02* 0.89 ؎ 0.02* 0.61 Ϯ 0.06 0.58 ؎ 0.06* K335E 5-8 0.50 ؎ 0.06* 1.19 ؎ 0.03* 0.89 ؎ 0.02* 0.53 Ϯ 0.03 0.48 ؎ 0.03* (A) The apparent permeability ratios (PS/PCl) for each substitute anion were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz relation (noted in Materials and Methods). Login to comment
144 Similar negative results were obtained for the K335R and K335A constructs, but in the K335D and K335E mutants maximal attenuation by [SCN]o was reduced by nearly 50%, although the apparent binding affinity was unaffected. Login to comment
145 The result is consistent with the notion that the affinity of anion binding was not diminished in the K335D and K335E constructs, but the impact of binding on conduction was reduced. Login to comment
166 Similarly, neither the alanine (K335A) nor the arginine (K335R) substituted K335 constructs exhibited an alteration in sensitivity to activating conditions. Login to comment
169 TABLE 4 Quantitative analyses of the macroscopic I-V shape changes Mutant ⌬ Net charge n RR g(ϩ30)/g(-30) RR/RRWT Wild type 5 1.220 Ϯ 0.06 1.00 G91A 0 4 1.293 Ϯ 0.06 1.06 G91E -1 5 1.512 ؎ 0.10* 1.24 G91R 1 4 8.041 ؎ 0.87* 6.59 G314A 0 4 1.201 Ϯ 0.09 0.98 G314D -1 4 1.362 Ϯ 0.08 1.12 G314E -1 7 1.405 Ϯ 0.08 1.15 G314Q 0 5 1.376 Ϯ 0.10 1.13 K335R 0 4 1.209 Ϯ 0.06 0.99 K335A -1 4 1.295 Ϯ; 0.07 1.06 K335D -2 5 0.762 ؎ 0.02* 0.62 K335E -2 4 0.919 ؎ 0.02* 0.75 The slope conductance was measured at ϩ30 mV and -30 mV with respect to the reversal potential. Login to comment
173 TABLE 5 Concentration-dependent activation of wtCFTR, G91, G314, and K335 variants by IBMX in the presence of 10 ␮M forskolin Mutant n K1/2(IBMX) (mM) Wild type 15 0.35 Ϯ 0.04 G91A 5 0.42 Ϯ 0.06 G91E 8 0.51 ؎ 0.06* G91R 5 0.49 Ϯ 0.09 G314A 10 1.21 ؎ 0.11* G314D 3 1.35 ؎ 0.16* G314E 8 6.39 ؎ 1.35* G314Q 4 14.26 ؎ 6.64* K335R 4 0.46 Ϯ 0.04 K335A 2 0.35 Ϯ 0.15 K335D 7 0.87 ؎ 0.13* K335E 3 0.95 ؎ 0.07* The steady-state slope conductance was measured at -60 mV as increasing concentrations of IBMX (0.02-5.0 mM) were added to the perfusate in the continued presence of 10 mM forskolin. Login to comment
198 The results presented here are consistent with the notion that the binding of anions within the CFTR pore is a sensitive indicator of changes in pore structure whereas permeability ratios appear to be rather insensitive to similar TABLE 6 Qualitative summary of the functional consequences of mutations at G91, G314, and K335 Property G91 (TM1) K335 (TM6) G314 (TM5) G91A G91E G91R K335R K335A K335D K335E G314A G314D G314E G314Q I-V shape - - ϩϩϩ - - ϩϩ ϩ - - - - Psub/PCl - - - - - - ϩϩ - - - - gsub/gCl - - - - - ϩϩϩ ϩϩϩ ϩϩ - ϩϩϩ ϩϩϩ SCN- binding - - - - - ϩϩϩ ϩϩϩ ϩϩ - ϩϩϩϩ ϩϩϩϩ Activation - - - - - ϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩϩ ϩϩϩϩ Results are expressed as follows: -, function of the CFTR construct with the indicated substitution was indistinguishable from wild type; ϩ to ϩϩϩϩ, semiquantitative indication of the magnitude of the change in the function compared with wild type. Login to comment
108 Neither the arginine (K335R) nor the alanine substitution (K335A) resulted in any substantial change in the permeability ratios (with the exception of SCN for K335A). Login to comment
138 Permeability Ratios Wild type 4-9 3.42 afe; 0.28 1.42 afe; 0.04 1.22 afe; 0.02 0.39 afe; 0.01 0.44 afe; 0.03 G91A 3-6 3.24 afe; 0.26 1.53 afe; 0.04 1.27 afe; 0.02 0.37 afe; 0.04 0.40 afe; 0.04 G91E 3-7 3.50 afe; 0.54 1.59 afe; 0.04 1.27 afe; 0.01 0.35 afe; 0.01 0.51 afe; 0.04 G91R 3-4 5.26 d1e; 0.46* 1.60 afe; 0.03 1.40 d1e; 0.01* 0.32 afe; 0.04 0.64 d1e; 0.04* G314A 3-4 2.87 afe; 0.17 1.45 afe; 0.03 1.19 afe; 0.02 0.31 afe; 0.03 0.33 afe; 0.03 G314D 4 3.42 afe; 0.34 1.44 afe; 0.05 1.25 afe; 0.04 0.33 afe; 0.03 0.51 afe; 0.05 G314E 3-4 3.72 afe; 0.56 1.65 d1e; 0.09* 1.35 d1e; 0.03* 0.49 afe; 0.04 0.53 afe; 0.04 G314Q 3-4 3.89 afe; 0.37 1.62 afe; 0.11 1.27 afe; 0.04 0.36 afe; 0.03 0.62 afe; 0.05 K335R 3-5 3.44 afe; 0.29 1.35 afe; 0.04 1.22 afe; 0.03 0.40 afe; 0.05 0.41 afe; 0.07 K335A 5-6 5.34 d1e; 0.58* 1.48 afe; 0.06 1.28 afe; 0.04 0.37 afe; 0.03 0.60 afe; 0.06 K335D 4-6 3.02 afe; 0.19 1.50 afe; 0.03 1.10 d1e; 0.02* 0.54 d1e; 0.04* 0.65 d1e; 0.06* K335E 5-8 3.64 afe; 0.21 1.48 afe; 0.06 1.29 afe; 0.03 0.46 afe; 0.04 1.10 d1e; 0.04* B. Conductance Ratios Wild type 4-9 0.14 afe; 0.02 0.75 afe; 0.02 0.64 afe; 0.02 0.52 afe; 0.03 0.18 afe; 0.03 G91A 3-6 0.14 afe; 0.01 0.77 afe; 0.02 0.61 afe; 0.02 0.47 afe; 0.02 0.19 afe; 0.02 G91E 3-7 0.15 afe; 0.03 0.73 afe; 0.02 0.60 afe; 0.01 0.50 afe; 0.04 0.30 afe; 0.02 G91R 3-4 0.14 afe; 0.00 0.84 afe; 0.01 0.63 afe; 0.01 0.32 d1e; 0.01* 0.14 afe; 0.01 G314A 3-4 0.30 afe; 0.09 0.89 d1e; 0.01* 0.66 afe; 0.01 0.48 afe; 0.09 0.24 afe; 0.01 G314D 4 0.28 afe; 0.05 0.82 afe; 0.01 0.70 afe; 0.02 0.49 afe; 0.06 0.27 afe; 0.03 G314E 3-4 0.62 d1e; 0.07* 1.18 d1e; 0.04* 0.84 d1e; 0.05* 0.42 afe; 0.05 0.29 afe; 0.09 G314Q 3-4 0.63 d1e; 0.02* 1.01 d1e; 0.04* 0.82 d1e; 0.03* 0.50 afe; 0.02 0.42 d1e; 0.02* K335R 3-5 0.14 afe; 0.01 0.76 afe; 0.03 0.61 afe; 0.02 0.59 afe; 0.06 0.16 afe; 0.03 K335A 6 0.20 afe; 0.03 0.77 afe; 0.02 0.61 afe; 0.02 0.45 afe; 0.03 0.21 afe; 0.02 K335D 4-6 0.65 d1e; 0.04* 1.25 d1e; 0.02* 0.89 d1e; 0.02* 0.61 afe; 0.06 0.58 d1e; 0.06* K335E 5-8 0.50 d1e; 0.06* 1.19 d1e; 0.03* 0.89 d1e; 0.02* 0.53 afe; 0.03 0.48 d1e; 0.03* (A) The apparent permeability ratios (PS/PCl) for each substitute anion were calculated from the shift in reversal potential using the Goldman-Hodgkin-Katz relation (noted in Materials and Methods). Login to comment

[hide] Evidence that extracellular anions interact with a... Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023. Zhou JJ, Linsdell P
Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties.
Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023., [PMID:19448737]

Abstract [show]
Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.

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67 Similar weakening of block by internal Pt(NO2)4 2-was also observed in K114C, K329A, K335A, and R1128Q (Fig. 2), as well as R334Q (Zhou et al. 2007). Login to comment
80 Nevertheless, the apparent Cl- dependence of block was significantly reduced in both K335A and R899Q, and significantly enhanced in R117Q (Fig. 4D). Login to comment
127 The effects of external Pt(NO2)4 2are sensitive to mutations away from the pore (K892Q, R899Q) but not deep within the pore (K335A, R334Q), which would be expected to alter ion-ion interactions in the pore. Login to comment
145 Interactions between external Cl- and internal Pt(NO2)4 2are also weakened in K335A (Fig. 4D), probably as a result of a decrease in direct pore-mediated interactions in this low-conductance mutant. Login to comment

[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]

Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

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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

[hide] The cystic fibrosis transmembrane conductance regu... Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4. Broadbent SD, Ramjeesingh M, Bear CE, Argent BE, Linsdell P, Gray MA
The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor.
Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4., [PMID:25277268]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl(-) concentration ([Cl(-)]o). Patch clamp current recordings show that a rise in [Cl(-)]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl(-)]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl(-)]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl(-)]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.

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112 To explore the role of phosphorylation further, we studied the effect of deleting the R domain from CFTR (residues 634-836) [12, 7], which removes all the major PKA/PKC Table 1 Summary of the FSK stimulation of whole cell currents and Erev shifts observed with the CFTR constructs used in this study CFTR Construct n FSK Stimulation (%&#b1;SEM) Erev shift (mV&#b1;SEM) WT (50 bc;M ATP) 5 180&#b1;96 15.0&#b1;3.6 WT (100 bc;M ATP) 6 12,000&#b1;6,000 15.2&#b1;3.0 WT (300 bc;M ATP) 8 1,200&#b1;600 17.0&#b1;3.0 WT (1 mM ATP) 24 13,000&#b1;6,000 23.7&#b1;1.8 WT (1.3 mM ATP) 9 1,400&#b1;900 16.7&#b1;2.6 WT (2 mM ATP) 24 6,100&#b1;5,300 16.7&#b1;1.6 WT (5 mM ATP) 7 1,600&#b1;1,000 20.1&#b1;4.4 WT (50 bc;M ATP + 50 bc;M P-ATP) 7 224&#b1;130 15.3&#b1;1.0 WT + Genistein 4 7,600&#b1;5,200 26.1&#b1;5.4 WT + AMP-PNP 5 2,800&#b1;2,500 21.8&#b1;5.5 WT (3 mM MgCl2) 7 28,000&#b1;17,000 18.3&#b1;3.1 R104Q 5 4,600&#b1;1,600 28.6&#b1;4.7 K114C 5 12,000&#b1;6,700 29.2&#b1;3.0 R117Q 4 33,000&#b1;20,000 30.1&#b1;3.4 K329A 5 13,000&#b1;10,000 33.7&#b1;2.1 R334Q 9 13,000&#b1;6,700 27.3&#b1;2.9 K335A 5 3,200&#b1;1,500 20.8&#b1;7.1 W401G 7 2,600&#b1;1,800 18.5&#b1;4.8 Delta-R (No Stim) 5 - 25.1&#b1;2.7 Delta-R (No FSK, Genistein) 5 140&#b1;13 22.7&#b1;3.0 Delta-R (FSK, No Genistein) 4 89&#b1;14 15.6&#b1;6.0 Delta-R (FSK + Genistein) 6 639&#b1;432 25.1&#b1;4.9 Delta-R-E1371S (No FSK) 9 - 21.4&#b1;4.8 Delta-R-E1371S (FSK) 4 2,600&#b1;1,400 15.3&#b1;4.7 K892Q 7 16,000&#b1;9,500 36.8&#b1;4.8 R899E 4 1,200&#b1;400 25.0&#b1;2.7 R899K 4 1,600&#b1;900 26.6&#b1;2.9 R899Q 7 5,400&#b1;2,800 30.0&#b1;1.3 R899Q + AMP-PNP 4 72,000&#b1;50,000 15.2&#b1;2.8 R899Q-E1371Q (No FSK) 4 - 18.4&#b1;5.9 R899Q-E1371Q (FSK) 6 107&#b1;48 15.6&#b1;3.0 R1128Q 6 14,000&#b1;6,100 41.1&#b1;4.2 Y1219G 6 3,200&#b1;2,500 19.2&#b1;3.3 E1371Q (No FSK) 6 - 25.5&#b1;3.5 E1371Q (FSK) 8 -28&#b1;9 22.3&#b1;4.0 E1371Q (FSK, No ATP, No GTP) 8 270&#b1;130 19.4&#b1;4.5 E1371Q + AMP-PNP (No FSK) 4 - 24.7&#b1;6.5 E1371Q + AMP-PNP (FSK) 8 180&#b1;170 17.4&#b1;4.0 Vector Control 4 15&#b1;38 - FSK stimulation was calculated as the percentage increase in current density at -60 mV from the Erev, after 5-min exposure to 10 bc;M FSK. Login to comment
162 In support of this conclusion, the stimulating effect of [Cl- ]o cannot be due to the ion entering the pore, since mutations that do affect CFTR Cl-conductance (R104Q, R117Q, R334Q, K335A) have no effect on [Cl- ]o stimulation (Fig. 2), and the one site that is important for sensing (R899) does not affect Cl-conductance [45, 47]. Login to comment