ABCC7 p.Phe337Tyr
| 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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[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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None has been submitted yet.
No. Sentence Comment
122 ------------------------------------------------------------------------------------------------------------------------ F337Y, a mutation which results in only a small change in amino acid side chain size.
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ABCC7 p.Phe337Tyr 11179391:122:122
status: NEW134 In order to determine whether the changes in relative anion conductance observed in F337S (Table 1) were associated with the change in selectivity or the change in conductance in this mutant, relative anion conductance was also examined in F337Y (Fig. 8).
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ABCC7 p.Phe337Tyr 11179391:134:240
status: NEW140 Anion conductance changes observed in F337S are not seen in F337Y Relative Cl¦, Br¦ and F¦ conductances were estimated under symmetrical ionic conditions by macroscopic current variance analysis.
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ABCC7 p.Phe337Tyr 11179391:140:60
status: NEW144 ------------------------------------------------------------------------------------------------------------------------ (compared with wild-type) in gBrÏgCl and the 710% increase in gFÏgCl, were not reproduced in F337Y (Fig. 8).
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ABCC7 p.Phe337Tyr 11179391:144:225
status: NEW246 The effects of the mutation F337S on relative anion conductance were not mimicked by another mutation, F337Y, which shows a similarly reduced Cl¦ conductance but unaltered anion selectivity (Fig. 8).
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ABCC7 p.Phe337Tyr 11179391:246:103
status: NEW[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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None has been submitted yet.
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.
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ABCC7 p.Phe337Tyr 14610019:4:145
status: NEW6 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.
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ABCC7 p.Phe337Tyr 14610019:6:105
status: NEW102 Neither of these effects were observed in another F337 mutant, F337Y, which was blocked in an apparently similar manner as wild-type (Fig. 5, C and E).
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ABCC7 p.Phe337Tyr 14610019:102:63
status: NEW105 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).
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ABCC7 p.Phe337Tyr 14610019:105:286
status: NEW106 Under these conditions, the strength of Pt(NO2)4 2- block in wild-type, F337Y, and T338A increases with increasingly negative voltages, eventually leading to a negative slope of the current-voltage relationship in the presence of blocker (Fig. 6 B).
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ABCC7 p.Phe337Tyr 14610019:106:72
status: NEW116 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).
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ABCC7 p.Phe337Tyr 14610019:116:137
status: NEW151 (E) Comparison of the mean blocking effect of 300 M intracellular Pt(NO2)4 2- on wild-type (᭺; fitted as in D) and F337Y (᭹; fitted by Eq. 2 with Kd(0) ϭ 582 M and z␦ ϭ -0.318).
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ABCC7 p.Phe337Tyr 14610019:151:130
status: NEW189 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."
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ABCC7 p.Phe337Tyr 14610019:189:66
status: NEW207 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.
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ABCC7 p.Phe337Tyr 14610019:207:82
status: NEW[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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None has been submitted yet.
No. Sentence Comment
70 The mutants F337L, F337Y, and I344A gave only modest alterations in anion permeability (Table 1) that led to only slight changes in the anion selectivity sequence (Table 2).
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ABCC7 p.Phe337Tyr 10827976:70:19
status: NEW74 As described previously for wild-type CFTR (Linsdell and Hanrahan, 1998a), the mutants F337A, F337S, and F337Y all showed negligible Naaf9; permeability (Table 1).
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ABCC7 p.Phe337Tyr 10827976:74:105
status: NEW77 Both wild-type and F337Y (Fig. 3), as well as F337L, F337W, and I344A (not shown; see Table 2), were able to select for anions that bound water molecules less strongly, consistent with the lyotropic selectivity sequence common to most classes of Clafa; channels (see Introduction).
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ABCC7 p.Phe337Tyr 10827976:77:19
status: NEW81 Thus the mutant F337Y, which substitutes a similarly sized but polar side chain, has a selectivity that is almost identical to that of wild type.
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ABCC7 p.Phe337Tyr 10827976:81:16
status: NEW116 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).
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ABCC7 p.Phe337Tyr 10827976:116:314
status: NEW122 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.
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ABCC7 p.Phe337Tyr 10827976:122:689
status: NEW158 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).
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ABCC7 p.Phe337Tyr 10827976:158:25
status: NEW165 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.
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ABCC7 p.Phe337Tyr 10827976:165:128
status: NEW168 Thus in wild-type CFTR (and to a similar extent F337L and F337Y), the bulky side chain at position 337 might impede the approach of permeating anions to a nearby anion-attracting group, ensuring relatively weak, long-distance interactions between the anion and this positive site.
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ABCC7 p.Phe337Tyr 10827976:168:58
status: NEW