ABCMdb

PMID: 12745925

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., [PubMed]

Sentences

No. Mutations Sentence Comment

33 ABCC7 p.Arg334Cys However, in contrast to the linear macroscopic I Á/V relationship observed for wild-type CFTR under symmetrical ionic conditions, most mutants showed some degree of inward rectification (Figure 1B); this rectification was particularly strong for R334C. Login to comment 41 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Thr339Val ABCC7 p.Arg334Cys 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 43 ABCC7 p.Lys335Ala ABCC7 p.Thr339Val ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala Other mutants studied (K335A, I336A, I340A), like T339V, had only minor effects (data not shown). Login to comment 44 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Thr339Val ABCC7 p.Arg334Cys ABCC7 p.Ile340Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala 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 49 ABCC7 p.Ser341Ala Perhaps most significantly, the relative conductance of Br( , I( and SCN( were all increased in S341A, leading to a change in the conductance sequence to Br( !/I( Â/Cl( !/SCN( !/F( (Table 3). Login to comment 59 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Thr339Val ABCC7 p.Arg334Cys ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Thr339Val ABCC7 p.Arg334Cys ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala Wild-type R334C K335A I336A F337A T338A T339V I340A S341A Cl (G(50/G'50) 1.039/0.09 (6) 4.509/0.60 (6)** 1.399/0.09 (5)** 1.519/0.14 (5)* 1.189/0.22 (6) 1.779/0.25 (8)* 1.199/0.06 (7)* 1.419/0.11 (5)* 1.809/0.18 (5)** Cl (GCl/GCl) 1.009/0.08 (6) 1.009/0.13 (6) 1.009/0.07 (5) 1.009/0.09 (5) 1.009/0.22 (6) 1.009/0.14 (8) 1.009/0.06 (7) 1.009/0.09 (5) 1.009/0.10 (5) Br 0.649/0.05 (6) 0.329/0.02 (6)** 0.669/0.05 (5) 1.079/0.10 (5)* 0.359/0.06 (6)** 0.499/0.03 (5) 0.659/0.09 (5) 0.669/0.08 (6) 1.529/0.30 (4)* I 0.299/0.05 (6) 0.749/0.02 (3)* 0.279/0.01 (4) 0.109/0.02 (4)* 0.349/0.08 (3) 0.389/0.03 (5) 0.309/0.05 (7) 0.279/0.03 (6) 1.049/0.16 (7)** F 0.379/0.04 (6) 0.329/0.04 (3) 0.349/0.03 (3) 0.709/0.10 (4)* 0.129/0.02 (3)* 0.239/0.02 (6)* 0.509/0.10 (4) 0.309/0.02 (5) 0.519/0.07 (6) SCN 0.389/0.02 (6) 0.339/0.03 (6) 0.669/0.10 (5)* 0.279/0.02 (6)* 0.399/0.04 (5) 0.269/0.02 (5)* 0.269/0.02 (4)* 0.359/0.04 (6) 0.839/0.14 (6)* Relative conductances for different anions were calculated from the slope of the macroscopic I Á/V relationship for inward versus outward currents (see Experimental procedures). Login to comment 72 ABCC7 p.Thr338Ala ABCC7 p.Lys335Ala ABCC7 p.Ile340Ala This halide selectivity sequence is changed to Eisenman sequence II in I340A, and Eisenman sequence I in both K335A and T338A (Table 2), consistent with a strengthening of lyotropic anion selectivity in these mutants. Login to comment 73 ABCC7 p.Phe337Ala ABCC7 p.Arg334Cys 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala 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 77 ABCC7 p.Arg334Cys ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala Smaller but statistically significant decreases in PSCN/PCl were observed in R334C, I336A and I340A. Login to comment 78 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Arg334Cys 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Thr339Val ABCC7 p.Arg334Cys ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala 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 102 ABCC7 p.Arg334Cys Current rectification was particularly striking in R334C (Figure 1B), consistent with the recently described role of the positive charge at this position in attracting anions to the mouth of the pore [26]. Login to comment 105 ABCC7 p.Ser341Ala As previously described by others [20], mutation of S341 was particularly associated with changes in anion relative conductance, consistent with weakened lyotropic anion binding in S341A. Login to comment 109 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Arg334Cys Lyotropic anion selectivity is disrupted in F337A and modified in R334C, T338A and S341A. Login to comment 124 ABCC7 p.Thr339Ala ABCC7 p.Thr339Val ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala In most (six of eight) cases, alanine substitution was employed; however, we have previously found that the mutants R334A [15] and T339A [22] fail to express in BHK cells, and for these residues mutants which gave adequate current expression (R334C, T339V) were studied. Login to comment