ABCMdb

PMID: 9089437

Cheung M, Akabas MH
Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel.
J Gen Physiol. 1997 Mar;109(3):289-99., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC7 p.Arg352Cys Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter. Login to comment 19 ABCC7 p.Arg347His Mutation of Arg347 to His resulted in pH-dependent anomalous mole-fraction effects, indicating that the positive charge at this position was important and that Arg347 was at or near one of the anion-binding sites (Tabcharani et al., 1993). Login to comment 20 ABCC7 p.Arg347Glu The mutation R347E, however, had little or no effect on the halide permeability or conductance sequences (Anderson et al., 1991b). Login to comment 21 ABCC7 p.Lys335Glu In contrast, the mutation K335E changed the halide permeability and/or conductance sequences (Anderson et al., 1991b) but did not alter the anomalous mole-fraction effects (Tabcharani et al., 1993). Login to comment 71 ABCC7 p.Thr351Cys Activation of T351C mutant and effect of MTSEAϩ. Login to comment 72 ABCC7 p.Thr351Cys (A) Illustration of the activation of the CFTR-induced current in an oocyte expressing the T351C mutant under two-electrode voltage clamp. Login to comment 102 ABCC7 p.Thr351Cys 2 and 3 A for the mutant T351C. Login to comment 107 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr351Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys ABCC7 p.Ser341Cys ABCC7 p.Ile344Cys ABCC7 p.Phe337Cys We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). Login to comment 112 ABCC7 p.Leu333Cys Furthermore, the voltage dependence of the rates of reaction of MTSES- is opposite to the voltage t a b l e i i Second-order Rate Constants at Holding Potential ϭ 0 mV and Ratios of Rate Constants at V ϭ 0 mV (kmtses/kmtset) 1 2* k0,MTSES 3* k0,MTSET 4‡ k0,MTSES 5§ kES/kET Mutant (M-1 S-1) (M-1 S-1) k0,MTSET L333C 2-MEʈ 4300 51000 0.08 1 L333 68 828 0.08 1 R334 53 376 0.14 2 K335 47 455 0.1 1 F337 96 991 0.1 1 S341 72 727 0.1 1 T351 250 219 1.14 14 R352 48 324 0.15 2 Q353 213 102 2.09 26 *Columns 2, 3 are the rate constants for MTSES- and MTSETϩ at V ϭ 0 mV. Login to comment 115 ABCC7 p.Leu333Cys The same ratio is observed for the rates of reaction with L333C indicating that there is no charge selectivity for the reaction with this residue. Login to comment 116 ABCC7 p.Leu333Cys §Column 5 is the anion to cation selectivity ratio derived by dividing the data in Column 4 by the ratio obtained for the rates of reaction with L333C. Login to comment 117 ABCC7 p.Leu333Cys The ratio of the rate constants (kmtses/kmtset) for reaction with an exposed cysteine relative to the ratio of the rate constants for the reaction with L333C gives a measure of the anion to cation selectivity at the level of an exposed cysteine. Login to comment 120 ABCC7 p.Arg347Cys 2In the course of these experiments we discovered that the R347C construct that we had used previously contained a large truncation, deleting most of the R-domain to the COOH terminus. Login to comment 121 ABCC7 p.Arg347Cys In the full length CFTR construct the R347C mutant is accessible to MTSEAϩ which causes 19% inhibition, however, MTSES- and MTSETϩ do not react with this mutant. Login to comment 123 ABCC7 p.Thr351Cys Experiments illustrating data used to determine rates of reaction of the MTS reagents with the T351C mutant. Login to comment 129 ABCC7 p.Thr351Cys (B) The natural log of the rate constants, k, for MTSES- (circles) and MTSETϩ (triangles) reacting with the T351C mutant are plotted as a function of voltage. Login to comment 134 ABCC7 p.Arg352Cys ABCC7 p.Leu333Cys ABCC7 p.Ser341Cys Note that the electrical distance to the residues from L333C to S341C is close to zero and that the electrical distance to R352C is smaller than the electrical distance to the adjacent residues. Login to comment 148 ABCC7 p.Thr351Cys In Fig. 3 B the natural log of the rate constants for the reactions of MTSES- and MTSETϩ with the mutant T351C are plotted as a function of membrane potential. Login to comment 154 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys The distance from the extracellular end to T351C and Q353C is significantly greater than to the other residues (P Ͻ 0.05). Login to comment 155 ABCC7 p.Leu333Cys lar residue tested, L333C, the ratio of the rates of reaction of MTSES-/MTSETϩ is also 0.08 (Table II, column 4); this suggests that there is no charge selectivity for access of the MTS reagents to this residue from the extracellular solution. Login to comment 156 ABCC7 p.Leu333Cys To account for this difference in the intrinsic rates of reaction of the two MTS reagents, we divided the ratio of the rates at a given residue by the ratio of the rates of reaction with L333C. Login to comment 162 ABCC7 p.Thr351Cys The major site of charge selectivity appears to be in the region of T351C and Q353C where the anion to cation selectivity rises to between 15 and 25 (Fig. 5). Login to comment 173 ABCC7 p.Arg352Cys We now show that based on the measured electrical distances R352C appears to be closer to the extracellular end of the channel than either of the adjacent residues. Login to comment 180 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys By measuring the relative rates of reaction of anionic and cationic MTS reagents with water-exposed cysteines in and flanking the M6 segment we have shown that a major determinant of anion selectivity occurs near the cytoplasmic end of the channel; access of the negatively charged MTSES- to T351C and Q353C is favored over the positively charged MTSETϩ (Fig. 5). Login to comment 183 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys Consistent with this, the reaction rate constants for the reaction of MTSES- with T351C and Q353C are larger than the rates with other channel-lining residues (Table II, column 2). Login to comment 185 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys The arginine that lies between T351C and Q353C, Arg352, appears to be a major determinant of the anion selectivity in this region; when cysteine is substituted for the arginine at position 352 the selectivity is similar to that observed in the rest of the channel (Fig. 5). Login to comment 186 ABCC7 p.Arg352Cys If other residues in this region were the main determinants of anion selectivity, then, the anion selectivity of the R352C mutant should have been similar to that of the adjacent residues. Login to comment 187 ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys Based on our measurements of electrical distance, R352C is closer to the extracellular end of the channel than T351C and Q353C (Fig. 4, see below). Login to comment 188 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys Thus, ions passing from the extracellular end of the channel would first encounter Arg352, which we infer forms part of the charge-selectivity filter, before they could reach T351C or Q353C; thereby accounting for the greater anion selectivity we observed at these residues. Login to comment 191 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys The increase in the reaction rate constants for MTSES- with the mutants T351C and Q353C (Table II, column 2) is consistent with these residues being near an anion binding site which increases the dwell time of MTSES- in this region of the channel thereby effectively increasing the reaction rate constants. Login to comment 192 ABCC7 p.Arg352Cys A further suggestion that Arg352 is important in charge selectivity is the increase in the reaction rate of the cationic MTSETϩ with the R352C mutant as compared to the adjacent residues (Table II, column 3). Login to comment 193 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Removing the positive charge in the R352C mutant may increase the ability of cations to enter this region near the cytoplasmic end of the channel, thereby accounting for the increase rate of reaction of MTSETϩ at R352C compared to the adjacent residues. Login to comment 200 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys The ability of the cationic MTS reagents to move past the anion-selectivity filter, i.e., to react with T351C and Q353C, is consistent with the lack of ideal anion selectivity that has been reported by others. Login to comment 204 ABCC7 p.Lys335Glu ABCC7 p.Lys95Asp Based on the effects of the mutations K95D and K335E on halide selectivity sequences, Anderson et al. (1991b) concluded that Lys95 and Lys335 were determinants of halide selectivity. Login to comment 205 ABCC7 p.Arg347Glu ABCC7 p.Arg1030Glu Curiously, neither of these mutations nor the mutations R347E and R1030E were reported to alter the Cl- to Naϩ permeability ratio (PCl/PNa), and the latter two mutations had minimal effects on halide permeability or conductance ratios (Anderson et al., 1991b). Login to comment 206 ABCC7 p.Lys335Glu Furthermore, the K335E mutation had no effect on anomalous mole-fraction effects suggesting that Lys335 is not part of an anion binding site in the channel (Tabcharani et al., 1993). Login to comment 210 ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys Note the marked increase in anion selectivity at the residues T351C and Q353C. Login to comment 234 ABCC7 p.Arg352Cys ABCC7 p.Thr351Cys The electrical distances from the extracellular end of the channel to these three residues, with T351C being more cytoplasmic than R352C, is also inconsistent with an ␣-helical secondary structure (Fig. 4). Login to comment