ABCMdb

PMID: 10827976

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., [PubMed]

Sentences

No. Mutations Sentence Comment

69 ABCC7 p.Phe337Trp F337W gave only small currents after expression in BHK cells, such that its full anion selectivity sequence could not be determined. Login to comment 70 ABCC7 p.Phe337Tyr ABCC7 p.Ile344Ala ABCC7 p.Phe337Leu 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). Login to comment 71 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 77 ABCC7 p.Phe337Tyr ABCC7 p.Ile344Ala ABCC7 p.Phe337Leu ABCC7 p.Phe337Trp 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). Login to comment 78 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 81 ABCC7 p.Phe337Tyr 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. Login to comment 82 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr ABCC7 p.Ile344Ala ABCC7 p.Phe337Leu ABCC7 p.Phe337Trp 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr ABCC7 p.Ile344Ala ABCC7 p.Phe337Leu 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 124 ABCC7 p.Ile344Ala A mutation in TM6 that greatly reduced the size of a more distant putative pore-lining hydrophobic amino acid residue, I344A, had no strong effect on selectivity (Tables 1 and 2). Login to comment 125 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 157 ABCC7 p.Thr338Ala Under the macroscopic current recording conditions used here, the mutation T338A changes the halide selectivity from Eisenman sequence III to sequence I, consistent with the strengthening of lyotropic selectivity in this mutant (Linsdell et al., 1998). Login to comment 158 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr ABCC7 p.Ile344Ala ABCC7 p.Phe337Leu 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 160 ABCC7 p.Phe337Ser Consistent with this, CFTR single-channel conductance (measured with symmetrical 154 mM Clafa; ) is reduced from 7.6 afe; 0.1 pS (n afd; 12) for wild type to 1.8 afe; 0.0 pS (n afd; 7) for F337S (P. Linsdell, unpublished observations). Login to comment 165 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala How, then, might we explain the effects of the mutations F337A and F337S on anion selectivity? Login to comment 168 ABCC7 p.Phe337Tyr ABCC7 p.Phe337Leu 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. Login to comment 169 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala 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