ABCMdb

ABCC7 p.Phe337Ala

Predicted by SNAP2:	A: D (71%), C: D (66%), D: D (91%), E: D (91%), G: D (75%), H: D (75%), I: D (71%), K: D (91%), L: D (53%), M: D (66%), N: D (85%), P: D (91%), Q: D (85%), R: D (85%), S: D (85%), T: D (85%), V: D (75%), W: D (85%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N,

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Publications
[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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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

[hide] Relationship between anion binding and anion perme... J Physiol. 2001 Feb 15;531(Pt 1):51-66. Linsdell P
Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol. 2001 Feb 15;531(Pt 1):51-66., 2001-02-15 [PMID:11179391]

Abstract [show]
1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies.

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No. Sentence Comment
37 Most interestingly, two mutations which reduce amino acid side chain size at position 337, F337A and F337S, virtually abolished the normal lyotropic relationship between anion permeability and energy of hydration (Linsdell et al. 2000). 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
44 Of those mutations introduced at F337, only F337A and F337S strongly altered anion selectivity, and the effects of these two mutations were very similar (Linsdell et al. 2000). Login to comment
110 Relative Cl¦ conductance appears to be weakly correlated with the size of the amino acid side chain present at position F337, with a large side chain favouring high conductance; this correlation results mainly from the low (and similar) conductances of the two mutants which strongly disrupted normal lyotropic anion selectivity, F337A and F337S. Login to comment
132 However, of those mutations involving substitution of F337 (Fig. 5), only F337A and F337S strongly disrupted selectivity (Linsdell et al. 2000). Login to comment

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

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

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No. Sentence Comment
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

[hide] Point mutations in the pore region directly or ind... Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8. Gupta J, Linsdell P
Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.
Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8., [PMID:11889571]

Abstract [show]
The sulfonylurea glibenclamide is a relatively potent inhibitor of the CFTR Cl(-) channel. This inhibition is thought to be via an open channel block mechanism. However, nothing is known about the physical nature of the glibenclamide-binding site on CFTR. Here we show that mutations in the pore-forming 6th and 12th transmembrane regions of CFTR affect block by intracellular glibenclamide, confirming previous suggestions that glibenclamide enters the pore in order to block the channel. 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. Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. These two mutations also abolished the dependence of block on the extracellular Cl(-) concentration, which in wild-type CFTR suggests an interaction between Cl(-) and glibenclamide within the channel pore that limits block. We suggest that mutations in the 12th transmembrane region strengthen glibenclamide block not by directly altering interactions between glibenclamide and the pore walls, but indirectly by reducing interactions between Cl(-) ions and glibenclamide within the pore. This work demonstrates that glibenclamide binds within the CFTR channel pore and begins to define its intrapore binding site.

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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
106 Two mutations in TM6, F337A and T338A, significantly weakened block by glibenclamide (Figs. 2, 3); this effect was apparently independent of the extracellu- 744 Fig. 5 Extracellular Cl-dependence of the apparent affinity and voltage dependence of glibenclamide block. Login to comment
112 However, the increase in Kd(0) was modest in both cases (1.5-fold increase for F337A compared to wild-type, and 2.1-fold increase for T338A), and both mutants are still clearly blocked in a voltage-dependent manner by glibenclamide (Figs. 1, 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
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
73 Conversely, the sequence is changed to Eisenman sequence IV in R334C and Eisenman sequence V in F337A, consistent with relative loss of lyotropic anion selectivity in these mutants. Login to comment
74 Loss of lyotropic selectivity in F337A is also demonstrated by the fact that this is the only mutant studied in which selectivity for Cl( over the kosmotropic F( anion was somewhat compromised (Table 1). 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

[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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No. Sentence Comment
4 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)4 2- ions. Login to comment
5 Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Login to comment
6 Current in the presence of Pt(NO2)4 2- increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)4 2- ions to the extracellular solution. Login to comment
8 Relief of block in F337A by Pt(NO2)4 2- permeation was only observed for blocker concentrations above 300 ␮M; 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. Login to comment
9 We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)4 2in F337A, an apparent manifestation of an anomalous mole fraction effect. Login to comment
10 We suggest that the F337A mutation allows intracellular Pt(NO2)4 2to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)4 2- ions within the pore promotes their permeation to the extracellular solution. Login to comment
100 In contrast, block of F337A was poorly described by the Woodhull model (Fig. 5 B), with block of this mutant appearing to be very much more voltage dependent at negative voltages than at positive voltages (Fig. 5 B). Login to comment
101 Although estimation of the blocking effects of Pt(NO2)4 2at 0 mV membrane potential suggested a slight but significant weakening of block in F337A compared with wild-type (Fig. 5 C), direct comparison of the blocking effects of 300 ␮M Pt(NO2)4 2- on wild-type and F337A (Fig. 5 D) suggests that while block is weakened in this mutant at depolarized voltages, the block is actually stronger in F337A than in wild-type at strongly hyperpolarized voltages. Login to comment
103 The Interaction between Pt(NO2)4 and F337A-CFTR Compared with the unremarkable block of wild-type CFTR by intracellular Pt(NO2)4 2- (Figs. 1-3), block of F337A-CFTR appears complex. 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
107 However, in F337A, the current in the presence of blocker increases again at voltages more negative than around -80 mV, suggesting that as the membrane potential is made very negative blocking ions are swept from the pore and Cl- is able more easily to permeate. 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
117 Interestingly, Pt(NO2)4 2- punchthrough in F337A was observed at low (Fig. 6) but not high extracellular Cl- concentrations (Fig. 7), suggesting that extracellular Cl- ions can prevent Pt(NO2)4 2- from passing through this mutant channel. Login to comment
120 Concentration-inhibition experiments with low extracellular Cl- concentrations confirmed the multiple apparent effects of the F337A mutation on the apparent affinity of Pt(NO2)4 2- block (Fig. 8). Login to comment
121 At relatively depolarized voltages (e.g., 0 mV; Fig. 8 C), Pt(NO2)4 2- blocked wild-type more strongly than F337A (i.e., the concentration-inhibition curve for wild-type lies to the left); whereas at hyperpolarized voltages (e.g., -130 mV, Fig. 8 D), the mutant is more potently inhibited. Login to comment
122 However, these experiments also illustrate that the punchthrough mechanism that relieves block of F337A but not wild-type at strongly hyperpolarized voltages is dependent not only on voltage but also on the blocker concentration. Login to comment
128 Confirming that Pt(NO2)4 2- can itself relieve Pt(NO2)4 2- block of F337A-CFTR, increasing the concentration of blocker from 100 to 300 ␮M during an individual experiment reduced current amplitude over most of the voltage range, but anomalously increased current amplitude below about -100 mV (Fig. 9, C-E). Login to comment
131 This suggests that the ability of Pt(NO2)4 2to permeate through the F337A channel pore is dependent on its own concentration. Login to comment
132 While we have not attempted to estimate the "permeability" of Pt(NO2)4 2in F337A-CFTR, we note Figure 4. Login to comment
139 To ensure that this did not, in fact, reflect time-dependent changes in F337A current amplitude, Pt(NO2)4 2- block of F337A was also studied using a voltage-step protocol (Fig. 10). Login to comment
140 F337A-CFTR currents were practically time-independent in the absence and presence of Pt(NO2)4 2- (Fig. 10 A). 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
148 (C) Mean Kd(0) estimated from fits such as those shown in B, except for F337A where Kd(0) was calculated from the fractional current remaining (I/I0) at 0 mV (estimated by fitting a polynomial function) according to the equation Kd(0) ϭ (I (300 ␮M))/(I0 - I). Login to comment
150 (D) Comparison of the mean blocking effect of 300 ␮M intracellular Pt(NO2)4 2- on wild-type (᭺; fitted by Eq. 2 as described in Fig. 2) and F337A (᭹; fitted by a third order polynomial function of no theoretical significance). Login to comment
154 Punchthrough of Pt(NO2)4 2in F337A was blocked by extracellular Cl- ions (Fig. 7). Login to comment
157 At very negative voltages, however, Pt(NO2)4 2- block of F337A is anomalously strengthened by high extracellular Cl- concentrations (Fig. 12). Login to comment
162 Apparent Pt(NO2)4 2- unblock by permeation in F337A. Login to comment
173 Pt(NO2)4 2- punchthrough in F337A is prevented by extracellular permeant anions. Login to comment
174 (A) Example macroscopic currents carried by F337A-CFTR before (Control) and after addition of 1 mM Pt(NO2)4 2to the intracellular solution, with 150 mM chloride, nitrate or perchlorate present in the extracellular solution. Login to comment
178 Comparison of the blocking effects of intracellular Pt(NO2)4 2- on wild-type and F337A-CFTR at low extracellular Cl-concentration. Login to comment
179 (A and B) Mean fraction of control current remaining following addition of 3 ␮M (᭹), 10 ␮M (᭺), 30 ␮M (᭢), 100 ␮M (᭞), 300 ␮M (᭿), or 1 mM (ٗ) Pt(NO2)4 2to the intracellular solution, for wild-type (A) and F337A (B). Login to comment
180 (C and D) Comparison of the concentration dependence of block in wild-type (᭹) and F337A (᭺) at two different membrane potentials: 0 mV (C) and -130 mV (D). Login to comment
184 Our interest in this substance stems from the consequences of a mutation within the pore (F337A) that apparently turns the channel from being Pt(NO2)4 2- impermeable to Pt(NO2)4 2- permeable (Fig. 6) and destroys the apparent simplicity of blocking effect seen in wild-type. Login to comment
186 However, punchthrough of Pt(NO2)4 2at negative voltages suggests that this anion is capable of passing through the pore of F337A-CFTR (Figs. 6, 7, and 9-11). Login to comment
187 As described by Nimigean and Miller (2002), the punchthrough phenomenon may be able to reveal very low levels of permeability inaccessible by other experimental means, and punchthrough of Pt(NO2)4 2-was only observed under highly specific conditions (in F337A only, at voltages more negative than approximately -80 mV, low extracellular permeant anion concentration, and Pt(NO2)4 2- concentrations of at least 300 ␮M). Login to comment
188 Nevertheless, the results shown in Fig. 6 suggest that the F337A mutation confers Pt(NO2)4 2- permeability on the pore. Login to comment
189 Previously, we showed that the mutations F337A and F337S, but not F337Y, disrupted the ability of the CFTR channel pore to select between permeant anions on the basis of free energy of hydration (Linsdell et al., 2000) and suggested that F337 contributes to a lyotropic anion "selectivity filter." 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
203 In addition to allowing Pt(NO2)4 2- permeability, the F337A mutation has a complex effect on the apparent affinity of Pt(NO2)4 2- block (Figs. 5 D and 8, B and D): block appears weaker than for wild-type at positive voltages yet stronger than in wild-type at negative voltages (and then weakens again in F337A due to punchthrough; see below). Login to comment
204 The block observed in F337A is poorly fitted by conventional models that assume a single binding site (Fig. 5 B). Login to comment
205 We suggest that this reflects binding to more than one site in the F337A-CFTR pore; a low affinity site that is accessible at all voltages, and a higher affinity site that is increasingly accessed at more negative voltages. Login to comment
206 The existence of more than one Pt(NO2)4 2-binding site in the F337A pore is also supported by the apparent anomalous mole fraction dependence of Pt(NO2)4 2- permeability (Fig. 9). Login to comment
207 Since this complex blocking behavior is observed in F337A but not in wild-type or F337Y, we suggest that by allowing Pt(NO2)4 2to permeate through the pore, the F337A mutant also allows this blocker to reach a binding site which is normally inaccessible or much less easily accessed. Login to comment
209 A simple model of Pt(NO2)4 2- movement in the F337A pore is shown in Fig. 13. Login to comment
210 Even in this mutant, Pt(NO2)4 2- unblock by permeation only occurs under extreme conditions (strongly hyperpolarized voltages, low extracellular Cl- concentrations, and high Pt(NO2)4 2- concentration; Fig. 6), such that it appears that the blocker normally exits the F337A pore back into the intracellular solution. Login to comment
212 Pt(NO2)4 2- block of F337A investigated using a voltage-step protocol. Login to comment
213 (A) Example F337A-CFTR currents in an inside-out patch, recorded before current activation (Control), after full current activation with PKA and PPi, and following sequential addition of Pt(NO2)4 2to final concentrations of 100 and 300 ␮M. Login to comment
221 overcome in F337A than in wild-type, and a second barrier external to the outermost Pt(NO2)4 2-binding site depicted in Fig. 13. Login to comment
223 With the addition of a second barrier to Pt(NO2)4 2- movement in the pore (Fig. 13), our model appears able to explain the complex interaction between Pt(NO2)4 2and F337A-CFTR. Login to comment
227 At low concentrations of Pt(NO2)4 2-, the blocker returns from the high affinity site in F337A to the intracellular solution (Fig. 13 B). Login to comment
230 Mechanistically, we suggest that at concentrations Ͼ300 ␮M, the F337A pore begins to show multiple occupancy by Pt(NO2)4 2- ions, and that repulsion between simultaneously bound ions is capable of expelling ions bound to the "outer" site into the extracellular solution, relieving the high-affinity block (Fig. 13 C). Login to comment
232 Timecourse of Pt(NO2)4 2- block of F337A investigated using a voltage-step protocol. Login to comment
240 Complex effect of extracellular Cl-concentration on block of F337A-CFTR by 300 ␮M Pt(NO2)4 2-. Login to comment
246 The present results suggest that multiple Pt(NO2)4 2- ions can bind simultaneously within the F337A-CFTR pore (and perhaps also wild-type CFTR), and also that Pt(NO2)4 2-binding may be able to occur concurrently with binding of extracellular Cl- or NO3 - ions. Login to comment
250 Our results suggest that, by removing a barrier to Pt(NO2)4 2- movement in the pore, the F337A mutation allows this anion to access a relatively high affinity binding site and simultaneously exposes it to multiion pore effects that destabilize its binding at high concentrations. Login to comment
275 A pictorial model of Pt(NO2)4 2- block in F337A-CFTR. 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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76 However, the unitary conductance was drastically reduced by some mutations in TM1 (K95Q, Q98A, P99A) and TM6 (R334K, F337A) (Figs. 2-4). 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
154 However, the amplitude-independent current reversal potentials reflect an increased SCN- relative permeability (PSCN/PCl) in A96V and a diminished PSCN/PCl in F337A compared with wild type. Login to comment

[hide] Interactions between impermeant blocking ions in t... J Membr Biol. 2006 Mar;210(1):31-42. Epub 2006 Jun 22. Ge N, Linsdell P
Interactions between impermeant blocking ions in the cystic fibrosis transmembrane conductance regulator chloride channel pore: evidence for anion-induced conformational changes.
J Membr Biol. 2006 Mar;210(1):31-42. Epub 2006 Jun 22., [PMID:16794779]

Abstract [show]
It is well known that extracellular Cl(-) ions can weaken the inhibitory effects of intracellular open channel blockers in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. This effect is frequently attributed to repulsive ion-ion interactions inside the pore. However, since Cl(-) ions are permeant in CFTR, it is also possible that extracellular Cl(-) ions are directly competing with intracellular blocking ions for a common binding site; thus, this does not provide direct evidence for multiple, independent anion binding sites in the pore. To test for the possible through-space nature of ion-ion interactions inside the CFTR pore, we investigated the interaction between impermeant anions applied to either end of the pore. We found that inclusion of low concentrations of impermeant Pt(NO(2))(4) (2-) ions in the extracellular solution weaken the blocking effects of three different intracellular blockers [Pt(NO(2))(4) (2-), glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoic acid] without affecting their apparent voltage dependence. However, the effects of extracellular Pt(NO(2))(4) (2-) ions are too strong to be accounted for by simple competitive models of ion binding inside the pore. In addition, extracellular Fe(CN)(6) (3-) ions, which do not appear to enter the pore, also weaken the blocking effects of intracellular Pt(NO(2))(4) (2-) ions. In contrast to previous models that invoked interactions between anions bound concurrently inside the pore, we propose that Pt(NO(2))(4) (2-) and Fe(CN)(6) (3-) binding to an extracellularly accessible site outside of the channel permeation pathway alters the structure of an intracellular anion binding site, leading to weakened binding of intracellular blocking ions.

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13 Entry of Pt(NO2)4 2) ions from the intracellular solution into the pore of a mutant form of CFTR (F337A) accelerates the exit of otherCorrespondence to: P. Linsdell; email: paul.linsdell@dal.ca J. Membrane Biol. 210, 31-42 (2006) DOI: 10.1007/s00232-005-7028-2 Pt(NO2)4 2) ions that are already bound inside the pore (Gong & Linsdell, 2003a). Login to comment

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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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

[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
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
157 Out of 20 mutants in TM6 and 20 mutants in TM12, only two in TM6 (S341A and F337A) induced rectification in macropatch currents which were suggested to form the narrow part of the pore (see below, Fig. 7, Supplementary Fig. 3). Login to comment
158 Among the 20 single amino acid mutants of TM12 that we tested in this paper, none of them exhibited significant change in their single-channel conductance compared to WT-CFTR, while we know that mutations R334A, F337A, S341A, R347A, and R352A in TM6 all exhibited significant change in their single-channel conductance [11, 12, 29, and the present manuscript]; these data strongly suggest that TM6 and TM12 do not equally contribute to the pore of CFTR. Login to comment
166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
175 Mutation F337A caused a significant decrease in block by Glyb but a significant increase in block by Glip (Fig. 3). Login to comment
176 In contrast to the effects of mutation S341A, the reduction in block by Glyb in F337A reflected a substantial decrease in initial block without a change in the magnitude of time-dependent block (Figs. 5 and 6). Login to comment
177 Meanwhile, the increase in block of F337A by Glip reflected a clear decrease in initial block with a dramatic increase in the magnitude of time-dependent block (Figs. 5 and 6). Login to comment
183 F337C-and F337E-CFTR exhibited significantly altered reversal potential, relative permeability, and relative conductance compared to WT-CFTR (Supplementary Tables 1, 2, 3), as did F337A-, S-, Y-, and L-CFTR [24]. Login to comment
184 Both mutations S341A and F337A significantly decreased single-channel conductance (Fig. 9 and Ref. [29]). Login to comment
185 Consistent with this designation, macroscopic chloride currents in S341A exhibited inward rectification while F337A/C/E exhibited outward rectification (Fig. 7; Supplementary Fig. 1) [28, 29]. Login to comment
193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
196 From the differences in the effects of mutations S341A and F337A on block by Glyb and Glip, and the similarity of effects of mutations M348A and V350A on block by the two drugs, we can infer that both drugs bind in the pore with the sulfonylurea-linked cyclohexamide end facing toward the cytoplasm. Login to comment
201 Similar to their effects on block by Glyb, both the S341A and F337A mutations decreased the efficacy of block by Meglitinide (fractional block was 0.35±0.04 and 0.45±0.04, p<0.01, respectively). Login to comment
206 Each of the functional parameters comprising the biophysical signature of CFTR (single-channel conduc- WT S341A F337A Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP -100 -50 50 100 -2000 -1000 1000 2000 ATP ATP +Glyb 50 μM ATP +Glyb 50 μMATP +Glyb 50 μM I (pA) Vm(mV) Vm(mV) -100 -50 50 100 -2000 -1000 1000 2000 ATP I (pA) Vm (mV) -100 -50 50 100 -2000 -1000 1000 2000 50 100 I (pA) ATP Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP Vm(mV) -100 -50 50 100 -800 -400 400 800 I (pA) ATP Vm(mV) -100 -50 50 100 -800 -400 400 800 I (pA) ATP ATP +Glip 200 μMATP +Glip 200 μMATP +Glip 200 μM I (pA) Vm(mV) -100 -50 50 100 -800 -400 400 800 ATP ATP +Tolb 300 μM ATP +Tolb 300 μMATP +Tolb 300 μM Fig. 7 I-V relationships for WT-CFTR and two important mutants, from inside-out macropatches in symmetrical 150 mM Cl- solution. Data were obtained by ramping the membrane potential from VM=-100 mV to +100 mV over 300 ms. Login to comment
208 Data for each CFTR variant is from a single patch expressing WT-, S341A-, or F337A-CFTR tance, rectification, selectivity, blocker pharmacology, etc.) can be compared between wildtype and site-directed mutants to infer channel structure. Login to comment
224 At V350, M348, and S341, alanine substitutions affected block by Glyb and Glip in an identical manner; the effects of the F337A mutation were opposite for Glyb and Glip. Login to comment
225 Since these two drugs differ only at the non-sulfonylurea end of the molecular structure, it seems reasonable to conclude that it is this end of Glyb ΔFractionalblockfrom -20mVto-100mV ΔFractionalblockfrom -20mVto-100mV 0.0 0.1 0.2 0.3 0.4 0.5 * * #Glyb 0.0 0.1 0.2 0.3 0.4 0.5 * * * * * ## Glyb Vm(mV) -100 0.2 0.4 0.6 0.8 WT-Glyb50 μM F337A WT-Glyb100 μM T1142A Fractionalblock by50μMGlyb b a -200-40-60-80 Fig. 8 Voltage-dependent block of WT-CFTR and some important mutants in TM6 and TM12. Login to comment
226 a Voltage-dependence of block of WT-CFTR, F337A-CFTR, and T1142A-CFTR by 50 μM Glyb, and WT-CFTR by 100 μM Glyb, at VM=-100 mV to -20 mV. Fractional block was calculated from the steady-state currents at each potential. Login to comment
231 This conclusion is bolstered by the finding that the effects of mutations S341A and F337A on block by Glyb were the same as their effects on block by Meglitinide, which shares structure with the non-sulfonylurea end of Glyb. Login to comment
232 In conclusion with these results, for the following reasons, we believe that the narrow region in TM6 of the CFTR pore is located between F337 and S341: (1) mutations F337A/S/C/E/Y/L and S341A/E/T dramatically altered the relative permeability of different anions in the channel (Supplementary Tables 2, 3; Refs. Login to comment
235 (3) Both F337 and S341 mutations exhibited outward or inward rectification, respectively; and (4) both S341A and F337A affected block by all four sulfonylurea family blockers [8, 21, 40, 42, 50, 53]. Login to comment
238 Therefore, we cannot conclude that one part of the Glyb molecule binds exclusively to one section of the pore because: (a) mutations along the full length of the pore affected block by Tolb, and (b) mutations S341A and F337A affected block by both Tolb and Meglitinide, which represent the two disparate halves of the Glyb structure. Login to comment
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
243 The scale of the abscissa (current amplitude) in the current trace for F337A is different from the others interactions of a specific moiety with distinct binding sites in the pore [46-51]. Login to comment

[hide] Divergent CFTR orthologs respond differently to th... Am J Physiol Cell Physiol. 2012 Jan 1;302(1):C67-76. doi: 10.1152/ajpcell.00225.2011. Epub 2011 Sep 21. Stahl M, Stahl K, Brubacher MB, Forrest JN Jr
Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101.
Am J Physiol Cell Physiol. 2012 Jan 1;302(1):C67-76. doi: 10.1152/ajpcell.00225.2011. Epub 2011 Sep 21., [PMID:21940661]

Abstract [show]
Comparison of diverse orthologs is a powerful tool to study the structure and function of channel proteins. We investigated the response of human, killifish, pig, and shark cystic fibrosis transmembrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTR(inh)-172, glibenclamide, and GlyH-101. In three systems, including organ perfusion of the shark rectal gland, primary cultures of shark rectal gland tubules, and expression studies of each ortholog in cRNA microinjected Xenopus laevis oocytes, we observed fundamental differences in the sensitivity to inhibition by these channel blockers. In organ perfusion studies, shark CFTR was insensitive to inhibition by CFTR(inh)-172. This insensitivity was also seen in short-circuit current experiments with cultured rectal gland tubular epithelial cells (maximum inhibition 4 +/- 1.3%). In oocyte expression studies, shark CFTR was again insensitive to CFTR(inh)-172 (maximum inhibition 10.3 +/- 2.5% at 25 muM), pig CFTR was insensitive to glibenclamide (maximum inhibition 18.4 +/- 4.4% at 250 muM), and all orthologs were sensitive to GlyH-101. The amino acid residues considered responsible by previous site-directed mutagenesis for binding of the three inhibitors are conserved in the four CFTR isoforms studied. These experiments demonstrate a profound difference in the sensitivity of different orthologs of CFTR proteins to inhibition by CFTR blockers that cannot be explained by mutagenesis of single amino acids. We believe that the potency of the inhibitors CFTR(inh)-172, glibenclamide, and GlyH-101 on the CFTR chloride channel protein is likely dictated by the local environment and the three-dimensional structure of additional residues that form the vestibules, the chloride pore, and regulatory regions of the channel.

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

[hide] Long-range coupling between the extracellular gate... FASEB J. 2015 Nov 25. pii: fj.15-278382. Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL 4th, Kirk KL
Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
FASEB J. 2015 Nov 25. pii: fj.15-278382., [PMID:26606940]

Abstract [show]
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that: 1) all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy and 2) highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman, J. L., IV, Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

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70 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
126 F337S-CFTR and F337A-CFTR exhibited robust GOF properties that included 1) substantial currents that remained following the removal of bath ATP with a scavenger (hexokinase/glucose) and subsequent perfusion with an ATP-free solution and 2) strong activation by the poorly hydrolyzable b,g-imidoadenosine 59-triphosphate (AMP-PNP), which is a weak agonist for WT CFTR (example record in Fig. 4A, data summaries in Fig. 4C, D) (4, 43). Login to comment
152 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
153 The results for F337A and F337S were significantly different from WT by unpaired Student`s t test (P , 0.05). Login to comment
160 Identical results were obtained for F337A in separate immunoblots (not shown). Login to comment
300 The present results confirmed these earlier findings and also revealed that a subset of F337 substitutions are strong GOF mutants, notably, F337S and F337A. Login to comment
69 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
125 F337S-CFTR and F337A-CFTR exhibited robust GOF properties that included 1) substantial currents that remained following the removal of bath ATP with a scavenger (hexokinase/glucose) and subsequent perfusion with an ATP-free solution and 2) strong activation by the poorly hydrolyzable b,g-imidoadenosine 59-triphosphate (AMP-PNP), which is a weak agonist for WT CFTR (example record in Fig. 4A, data summaries in Fig. 4C, D) (4, 43). Login to comment
151 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
159 Identical results were obtained for F337A in separate immunoblots (not shown). Login to comment
299 The present results confirmed these earlier findings and also revealed that a subset of F337 substitutions are strong GOF mutants, notably, F337S and F337A. Login to comment

[hide] Molecular determinants of anion selectivity in the... Biophys J. 2000 Jun;78(6):2973-82. Linsdell P, Evagelidis A, Hanrahan JW
Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
Biophys J. 2000 Jun;78(6):2973-82., [PMID:10827976]

Abstract [show]
Ionic selectivity in many cation channels is achieved over a short region of the pore known as the selectivity filter, the molecular determinants of which have been identified in Ca(2+), Na(+), and K(+) channels. However, a filter controlling selectivity among different anions has not previously been identified in any Cl(-) channel. In fact, because Cl(-) channels are only weakly selective among small anions, and because their selectivity has proved so resistant to site-directed mutagenesis, the very existence of a discrete anion selectivity filter has been called into question. Here we show that mutation of a putative pore-lining phenylalanine residue, F337, in the sixth membrane-spanning region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, dramatically alters the relative permeabilities of different anions in the channel. Specifically, mutations that reduce the size of the amino acid side chain present at this position virtually abolish the relationship between anion permeability and hydration energy, a relationship that characterizes the anion selectivity not only of wild-type CFTR, but of most classes of Cl(-) channels. These results suggest that the pore of CFTR may indeed contain a specialized region, analogous to the selectivity filter of cation channels, at which discrimination between different permeant anions takes place. Because F337 is adjacent to another amino acid residue, T338, which also affects anion selectivity in CFTR, we suggest that selectivity is predominantly determined over a physically discrete region of the pore located near these important residues.

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No. Sentence Comment
71 In contrast, both F337A and F337S showed dramatically altered anion selectivity (Fig. 2 and Tables 1 and 2), characterized by large reductions in the relative permeability of lyotropic anions (Brafa; , Iafa; , SCNafa; , NO3 afa; ) and greatly increased permeability of the small, kosmotropic Fafa; anion. Login to comment
74 As described previously for wild-type CFTR (Linsdell and Hanrahan, 1998a), the mutants F337A, F337S, and F337Y all showed negligible Naaf9; permeability (Table 1). Login to comment
76 The altered anion selectivity of F337A and F337S led to a disruption of the relationship between anion permeability and hydration energy in these mutants (Fig. 3). Login to comment
78 In contrast, for both F337A and F337S, there was no obvious correlation between anion permeability and energy of hydration (Fig. 3), suggesting that lyotropic selectivity is greatly diminished in these mutants. Login to comment
82 In contrast, the two mutations that strongly affect selectivity, F337A and F337S, both involve a substantial reduction in amino acid side-chain volume. Login to comment
101 Note that the range of reversal potentials with different anions is greatly reduced in both F337A and F337S, indicating a reduced ability of the channel to discriminate between different anions. Login to comment
104 We used a similar approach to determine whether the altered anion selectivity of F337A and F337S was associated with any change in functional pore diameter (Table 3). Login to comment
106 The permeabilities of F337A and F337S to extracellular formate, acetate, and propanoate ions were not significantly different from those observed in wild-type CFTR, and both pyruvate and methane sulfonate were not measurably permeant in wild type, F337A, or F337S. Login to comment
111 However, the effects of the mutations F337A and F337S, which virtually abolish the normal lyotropic anion selectivity sequence (Tables 1 and 2 and Fig. 3) by decreasing the relative permeability of lyotropic anions and increasing that of kosmotropic anions (Fig. 4), support an alternative explanation, namely that selectivity is determined at a discrete region unaffected by previously studied mutations. Login to comment
116 Although we cannot rule out this possibility, we feel that the fact that mutations at two adjacent TM6 residues, F337 (this study) and T338 (Linsdell et al., 1998), significantly affect TABLE 1 Relative permeability of intracellular ions in wild-type and mutant CFTR Cld1a; channels Wild type F337A F337S F337L F337Y F337W I344A Cl 1.00 afe; 0.01 (10) 1.00 afe; 0.04 (6) 1.00 afe; 0.08 (3) 1.00 afe; 0.02 (5) 1.00 afe; 0.02 (6) 1.00 afe; 0.03 (5) 1.00 afe; 0.01 (9) Br 1.37 afe; 0.07 (8) 0.60 afe; 0.04 (4)** 0.50 afe; 0.04 (4)** 1.22 afe; 0.04 (5) 1.39 afe; 0.04 (3) 1.12 afe; 0.05 (4)* 1.74 afe; 0.01 (3)* I 0.83 afe; 0.03 (6) 0.23 afe; 0.04 (5)** 0.23 afe; 0.02 (4)** 0.39 afe; 0.01 (3)** 0.69 afe; 0.03 (7)* - 0.99 afe; 0.05 (4)* F 0.103 afe; 0.007 (9) 0.35 afe; 0.01 (4)** 0.43 afe; 0.02 (4)** 0.15 afe; 0.02 (3)* 0.095 afe; 0.009 (3) 0.081 afe; 0.009 (3) 0.075 afe; 0.012 (5)* SCN 3.55 afe; 0.26 (7) 0.97 afe; 0.05 (4)** 0.93 afe; 0.10 (5)** 2.85 afe; 0.20 (4) 3.05 afe; 0.29 (4) 4.42 afe; 0.56 (4) 3.27 afe; 0.30 (5) NO3 1.58 afe; 0.04 (10) 1.30 afe; 0.03 (3)* 1.08 afe; 0.02 (4)** 1.38 afe; 0.03 (4)* 1.43 afe; 0.04 (3) 1.62 afe; 0.03 (3) 1.71 afe; 0.06 (4) ClO4 0.25 afe; 0.01 (8) 0.19 afe; 0.00 (3)* 0.17 afe; 0.03 (4)* 0.23 afe; 0.04 (3) 0.15 afe; 0.01 (4)** - 0.24 afe; 0.02 (3) Formate 0.24 afe; 0.01 (9) 0.27 afe; 0.02 (3) 0.33 afe; 0.03 (4)* 0.35 afe; 0.02 (3)* 0.24 afe; 0.01 (3) - 0.28 afe; 0.01 (3) Acetate 0.091 afe; 0.003 (10) 0.073 afe; 0.004 (3)* 0.12 afe; 0.02 (5) - 0.092 afe; 0.014 (4) - 0.076 afe; 0.007 (3) Naaf9; 0.007 afe; 0.010 (24) 0.001 afe; 0.018 (3) 0.001 afe; 0.021 (5) - 0.002 afe; 0.004 (3) - - Relative permeabilities for different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.g., Fig. 2), according to Eq. 1 (see Materials and Methods). Login to comment
122 TABLE 2 Anion selectivity sequences for wild-type and mutant CFTR Cld1a; channels Wild-type SCNafa; b0e; NO3 afa; b0e; Brafa; b0e; Clafa; b0e; Iafa; b0e; ClO4 afa; b07; form b0e; Fafa; b0e; ace F337A NO3 afa; b0e; Clafa; c56; SCNafa; b0e; Brafa; b0e; Fafa; b0e; form c56; Iafa; b0e; ClO4 afa; b0e; ace F337S NO3 afa; b0e; Clafa; c56; SCNafa; b0e; Brafa; b0e; Fafa; b0e; form b0e; Iafa; b0e; ClO4 afa; b0e; ace F337L SCNafa; b0e; NO3 afa; b0e; Brafa; b0e; Clafa; b0e; Iafa; b0e; form b0e; ClO4 afa; b0e; Fafa; F337Y SCNafa; b0e; NO3 afa; c56; Brafa; b0e; Clafa; b0e; Iafa; b0e; form b0e; ClO4 afa; b0e; Fafa; b07; ace I344A SCNafa; b0e; Brafa; c56; NO3 afa; b0e; Clafa; b07; Iafa; b0e; form b0e; ClO4 afa; b0e; ace b07; Fafa; Sequences were derived from the relative anion permeabilities given in Table 1. form, formate; ace, acetate. Login to comment
125 Furthermore, the mutations F337A and F337S altered selectivity between different anions without disrupting the ability of the channel to select for Clafa; over Naaf9; (Table 1), supporting the hypothesis that the CFTR pore uses different mechanisms to determine lyotropic anion selectivity and anion:cation selectivity (Linsdell et al., 1998; Guinamard and Akabas, 1999). Login to comment
129 Nevertheless, it is clear that in CFTR, interactions between permeating anions and the pore do influence anion selectivity, because point mutations in the channel (F337A and F337S) disrupt the selectivity sequence. Login to comment
130 Both F337A and F337S compromise the relationship between anion permeability and hydration energy (Fig. 3), suggesting a reduction in the relative importance of anion FIGURE 3 Relationship between relative anion permeability and hydration energy for wild-type and F337-mutated CFTR. Login to comment
142 One possible explanation for the loss of the relationship between anion permeability and hydration energy in F337A and F337S is that anions are able to pass through the pores of these mutants with more of their associated waters of hydration intact than in wild type, so reducing the degree of anion dehydration required for permeation. Login to comment
151 In fact, for intracellular anions, the mutations F337A and F337S had a much stronger effect on the permeability of small anions (halides, SCNafa; , NO3 afa; ) than on larger anions (ClO4 afa; , formate, acetate), suggesting that removal of a steric barrier is not the primary effect of these mutations (Table 1). Login to comment
152 Furthermore, neither F337A nor F337S showed greatly altered permeability to extracellular organic anions (Table 3), the permeabilities of which do appear to be limited by unhydrated anion size (Linsdell et al., 1997, 1998; Linsdell and Hanrahan, 1998a). Login to comment
153 Although the relationship between the permeability of such organic anions, when present in the extracellular solution, and the actual physical dimensions of the pore, is unclear (Linsdell and Hanrahan, 1998a), the results summarized in Table 3 do not suggest a strong alteration in the functional dimensions of the pore in F337A or F337S. Login to comment
154 A reduction in the relative importance of anion dehydration in determining permeability, as is suggested in F337A and F337S, could result not only from a decrease in the degree of anion dehydration, but also from an increase in the strength of the interaction between permeating anions and the channel pore. Login to comment
158 While the mutants F337L, F337Y, and I344A maintain Eisenman sequence III, both F337A and F337S convert the channel to a relatively strong field strength sequence (Clafa; b0e; Brafa; b0e; Fafa; b0e; Iafa; ; Eisenman sequence V) (Table 2). Login to comment
159 This increase in field strength might imply that permeating anions interact more strongly with the pores of F337A and F337S than with wild-type CFTR. Login to comment
165 However, SCNafa; permeability is reduced to a similar extent in F337A (hydrophobic) and F337S (polar), but is not altered in F337Y (polar) (Table 1), suggesting that SCNafa; permeability is not influenced by hydrophobic interactions with the large, hydrophobic side chain of F337. Login to comment
166 How, then, might we explain the effects of the mutations F337A and F337S on anion selectivity? Login to comment
169 Reduction of this steric effect in both F337A and F337S would allow the permeating anion TABLE 3 Relative permeability of extracellular organic anions in wild-type and mutant CFTR Cld1a; channels Wild type F337A F337S Formate 0.129 afe; 0.007 (4) 0.157 afe; 0.013 (3) 0.112 afe; 0.002 (3) Acetate 0.038 afe; 0.007 (4) 0.026 afe; 0.008 (4) 0.029 afe; 0.013 (3) Propanoate 0.022 afe; 0.003 (4) 0.024 afe; 0.001 (3) 0.024 afe; 0.002 (3) Pyruvate b0d;0.011 (4) b0d;0.011 (3) b0d;0.011 (3) Methane sulfonate b0d;0.011 (3) b0d;0.011 (3) b0d;0.011 (2) Relative permeabilities for different organic anions present in the extracellular solution under biionic conditions were calculated as described in the legend to Table 1. Login to comment

[hide] Location of a permeant anion binding site in the c... J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12. Rubaiy HN, Linsdell P
Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12., [PMID:25673337]

Abstract [show]
In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed.

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No. Sentence Comment
32 Specifically, mutations that reduce side-chain volume (F337A, F337S) disrupt the relationship between the anion permeability and anion free energy of hydration [18]. Login to comment
41 In contrast, the F337A mutation disrupts the normal Fig. 1 Block by intracellular Au(CN)2 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of Au(CN)2 - to the intracellular (bath) solution at the concentrations stated. Login to comment
93 As well as showing tight binding of lyotropic permeant anions, CFTR also shows a lyotropic anion permeability Fig. 4 Block of F337A/ E1371Q channels by intracellular lyotropic permeant anions. Login to comment
94 a, b Example macroscopic I-V relationships for F337A/E1371Q CFTR channels recorded before (control) and after the addition of Au(CN)2 - (1 mM) or SCN- (10 mM) to the intracellular (bath) solution. c Mean KD values for Au(CN)2 - , SCN- , and C(CN)3 - (estimated at -100 mV as described in Figs. 1 and 2) compared in E1371Q, K95Q/ E1371Q, and F337A/E1371Q. Login to comment
97 This permeability sequence is disrupted by mutations at the putative narrow region of the pore, located more extracellularly in the pore than K95, in particular F337A and F337S (see ''Introduction``). Login to comment
98 As shown in Fig. 4, the F337A mutation had only a minor effect on binding of lyotropic Au(CN)2 - , SCN- and C(CN)3 - ions when compared to the K95Q mutation, suggesting that these anions can still bind relatively tightly in the pore even when lyotropic permeability selectivity is compromised. Login to comment
101 Consistent with the proposed role of F337 in controlling lyotropic anion permeability, the permeability of all anions tested in F337A/E1371Q was significantly changed relative to E1371Q (Fig. 5b), with the permeability selectivity sequence being changed to NO3 - - C SCN- C Cl- [ Br- [ F- . Login to comment
102 Again, relative permeability values for F337A/E1371Q were similar to those reported previously for F337A [18]. Login to comment
106 Note that the range of current reversal potentials was greatly reduced in F337A/E1371Q, suggesting a relative loss of permeability selectivity in this mutant. Login to comment
109 Note that the normal lyotropic relationship between relative permeability and Gh is greatly reduced in F337A/E1371Q but retained in K95Q/E1371Q and I344K/E1371Q. Login to comment
120 Conversely, a mutation that is known to have a strong effect on relative permeability-F337A (Fig. 5)-had relatively minor effects on permeant anion binding (Fig. 4). Login to comment