ABCMdb

PMID: 19381710

Fatehi M, Linsdell P
Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.
J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19., [PubMed]

Sentences

No. Mutations Sentence Comment

66 ABCC7 p.Ser1118Cys An example is S1118C (Fig. 2c, d). Login to comment 71 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Ile1132Cys ABCC7 p.Thr1122Cys ABCC7 p.Ile1131Cys As described previously for modification of cysteines introduced into TM6 (Fatehi and Linsdell 2008) and the extracellular loop between TMs 1 and 2 (Zhou et al. 2008), MTSET and MTSES altered the IREL-V shape in S1118C, T1121C, T1122C, G1127C, V1129C, I1131C and I1132C. Login to comment 72 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys ABCC7 p.Ala1136Cys Of 21 cysteine mutants studied, only six significantly altered I-V relationship shape in the absence of external MTS reagents (Fig. 3a), with S1118C, T1121C, T1122C, G1127C and A1136C all causing significant inward rectification and V1129C showing outward rectification. Login to comment 75 ABCC7 p.Ile1131Cys As described above (Fig. 2), six mutants had I-V relationships that were sensitive to both MTSET and MTSES and only one (I1131C) was altered by MTSET but not MTSES. Login to comment 76 ABCC7 p.Ala1136Cys There was strong overlap between changes in control I-V shape and sensitivity to MTS reagents-only A1136C showed an I-V relationship that was significantly different in shape from wild-type in the absence of MTS reagents (Fig. 3a) but insensitive to charged MTS reagents (Fig. 3b). Login to comment 82 ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys In contrast, I1131C and I1132C did not significantly affect the form of either the i-V relationship (Fig. 4b) or the I-V relationship (Fig. 3a). Login to comment 83 ABCC7 p.Val1129Cys Only one mutant-V1129C-significantly affected the magnitude of unitary currents at hyperpolarized voltages (Figs. 4, 5a), giving a decrease in unitary current amplitude of *12% at -80 mV (Fig. 5a). Login to comment 84 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys Unitary currents at depolarized voltages were significantly decreased in S1118C, T1121C, T1122C and G1127C and significantly increased in V1129C (Fig. 5b). Login to comment 85 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys This resulted in changes in the shape of the i-V relationship, causing inward rectification in the case of S1118C, T1121C, T1122C and G1127C and outward rectification in the case of V1129C (Figs. 4b, 5c). Login to comment 86 ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys No changes in i-V shape were observed for I1131C or I1132C. Login to comment 91 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Ile1132Cys ABCC7 p.Thr1122Cys ABCC7 p.Ile1131Cys Indeed, changes in unitary current amplitude were observed in S1118C, T1121C, T1122C, G1127C, V1129C, I1131C and I1132C, but not wild-type, when MTS reagents were included in the pipette solution (Fig. 6). Login to comment 98 ABCC7 p.Ser1118Cys c, d Example I-V relationships and mean rectification ratios for S1118C under the same conditions. Login to comment 114 ABCC7 p.Ile1131Cys Note that the rectification ratio in six cysteine mutants is significantly affected by both MTSET and MTSES (indicated by asterisks), whereas in a seventh (I1131C) it is significantly altered by MTSET but not by MTSES voltages as an indicator, Fig. 8 shows that MTS reagents had charge-dependent effects on i-V relationship shape that broadly mirrored those observed in the macroscopic I-V relationships (see Fig. 3c). Login to comment 118 ABCC7 p.Ser1118Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys Four mutations (S1118C, T1121C, T1122C, G1127C) led to significant decreases in unitary current amplitude (Fig. 5b), which were relatively strongly affected by MTS modification-in each case conductance was further decreased by reaction with MTSES and increased to near wild-type levels by MTSET (Fig. 9a). Login to comment 119 ABCC7 p.Val1129Cys ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys In contrast, the other three mutations (V1129C, I1131C, I1132C) led to no change or even a slight increase in unitary current amplitude (Fig. 5b) and more minor effects of MTS modification, resulting in no change or a small decrease in amplitude with MTSES and increases in amplitude to levels above wild-type with MTSET (Fig. 9b). Login to comment 124 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys Under these conditions, SCN- block was significantly strengthened in I1132C (at hyperpolarized and depolarized voltages), S1118C (at hyperpolarized voltages), T1121C and V1129C (at depolarized voltages) and I1131C (at very depolarized voltages only) Fig. 4 Single-channel currents carried by cysteine mutant forms of CFTR. Login to comment 125 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Thr1122Cys a Example single-channel currents carried by wild-type, S1118C, T1121C, T1122C and V1129C, at membrane potentials of ?60 (top) and -60 (bottom) mV. Login to comment 130 ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys Block was not significantly altered in either T1122C or G1127C (data not shown). Login to comment 133 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Ile1132Cys ABCC7 p.Thr1122Cys ABCC7 p.Ile1131Cys Under these conditions, SCN- permeability was significantly increased in S1118C and (to a lesser extent) T1122C and G1127C and unaltered in T1121C, V1129C, I1131C and I1132C (Fig. 11). Login to comment 158 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys Of the seven mutants that were functionally modified by MTS reagents, five (S1118C, T1121C, T1122C, G1127C, V1129C) also showed significantly altered unitary current amplitude in the absence of MTS modification (Figs. 4, 5). Login to comment 161 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Ile1132Cys ABCC7 p.Thr1122Cys ABCC7 p.Ile1131Cys a S1118C (d), T1121C (j), T1122C (), G1127C (h); b V1129C (m), I1131C (r), I1132C (.). Login to comment 168 ABCC7 p.Ala1136Cys Only one mutant that was apparently not affected by MTS reagents (A1136C) had a small effect on the shape of the I-V relationship (Fig. 3a). Login to comment 177 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys Effects of external MTS reagents on the gating of CFTR cysteine mutants (I331C, L333C) have been described (Beck et al. 2008) but would presumably not be noticed using our experimental approach. Login to comment 183 ABCC7 p.Ser1118Cys ABCC7 p.Val1129Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys We speculate that one group of reactive mutants (S1118C, T1121C, T1122C, G1127C) is located relatively deep in the pore from the outside and that the other (V1129C, Fig. 11 Thiocyanate permeability of mutants. Login to comment 185 ABCC7 p.Ser1118Cys In these examples, the current reversal potential is ?36.4 mV for wild-type and ?47.8 mV for S1118C (shown by arrows), suggesting an increased PSCN/PCl in this mutant. Login to comment 187 ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys * Significant difference from wild-type (P \ 0.05) I1131C, I1132C) represents mutations at the outermost mouth of the pore. Login to comment 188 ABCC7 p.Ser1118Cys ABCC7 p.Thr1121Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys In this scenario, charge-neutral mutations deeper in the pore (S1118C, T1121C, T1122C, G1127C) (Fig. 9a) disrupt Cl- movement in the pore in a nonelectrostatic fashion, leading to reduced unitary currents at depolarized voltages, as described previously for TM6 mutations (McDonough et al. 1994; Linsdell et al. 1998; Linsdell 2001a). Login to comment 191 ABCC7 p.Val1129Cys ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys Chloride conductance was further reduced by MTSES modification at these sites, indicating the detrimental effect of depositing a negative charge within the permeation pathway. Cysteine substitution at the outer mouth of the pore (V1129C, I1131C, I1132C) (Fig. 9b) had somewhat different effects. Login to comment 200 ABCC7 p.Ser1118Cys ABCC7 p.Gly1127Cys ABCC7 p.Thr1122Cys Mutations S1118C, T1122C and G1127C also altered the anion selectivity of CFTR, significantly increasing SCN- per- meability (Fig. 11), which is consistent with changes in pore structure and function. Login to comment 201 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala Previously, SCN- permeabil- ity was shown to be significantly decreased in S1118F but unaltered in S1118A (Zhang et al. 2000). Login to comment 214 ABCC7 p.Ser1118Cys It should be stressed that, in some cases, the cysteine mutations we used in the present study represent rather conservative mutations (especially S1118C, which effectively changes one oxygen atom to sulfur). Login to comment