ABCMdb

PMID: 10220340

Guinamard R, Akabas MH
Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel.
Biochemistry. 1999 Apr 27;38(17):5528-37., 1999-04-27 [PubMed]

Sentences

No. Mutations Sentence Comment

101 ABCC7 p.Arg352Gln ABCC7 p.Arg352His ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys In cell-attached patches, with 140 mM NaCl in the pipet, the single-channel conductances (in picosiemens) were 6.0 ( 0.3 for the wild type (n ) 7), 5.3 ( 0.3 for R352C (n ) 11), 4.2 ( 0.1 for R352Q (n ) 10), 4.0 ( 0.2 for R352H (n ) 4), and 5.7 ( 0.2 for Q353C (n ) 8). Login to comment 106 ABCC7 p.Arg352Cys In cells expressing the R352C mutation, active channels were observed in about half of the cell-attached patches; 0.6 ( 0.3 (n ) 12 patches) channel was observed before activation, and 4.9 ( 0.8 channels were observed after application of the cAMP-activating reagents. Login to comment 110 ABCC7 p.Arg352Cys Following activation with cAMP, cells transfected with wild-type CFTR had a conductance of 12 ( 2 nS (n ) 6) and cells transfected with R352C had a conductance of 2 ( 0.3 nS (n ) 8) (Figure 2A). Login to comment 113 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys The single-channel conductances (in picosiemens) were 6.2 ( 0.5 for the wild type (n ) 6)2 (Figure 3B), 5.9 ( 0.3 for R352C (n ) 5), 4.2 ( 0.1 for R352Q (n ) 8), and 5.7 ( 0.3 for Q353C (n ) 8). Login to comment 114 ABCC7 p.Arg352His For the R352H mutant, the single-channel conductance was sensitive to the pH of the bath solution; the single-channel conductance at pH 7.2 was 3.7 ( 0.1 pS (n ) 6), and at pH 5.4, it was 4.3 ( 0.1 pS (n ) 5) (Figure 4). Login to comment 128 ABCC7 p.Arg352Gln Similarly, for the R352Q mutant in the 10-fold NaCl gradient in the FIGURE 2: Whole-cell and single-channel current-voltage relationships for the wild type and Arg352 mutants in symmetrical Cl--containing solutions. Login to comment 129 ABCC7 p.Arg352Cys (A) Average whole-cell current-voltage relationships obtained from cells expressing either wild-type CFTR (b) or the R352C mutant (O) in symmetrical Cl--containing solutions. Login to comment 130 ABCC7 p.Arg352Cys Note the much lower level of current in the R352C mutant than in the wild type. Login to comment 131 ABCC7 p.Arg352Gln ABCC7 p.Arg352His ABCC7 p.Arg352Cys (B) Average single-channel current-voltage relationships for the wild type (O) and the R352C (0), R352Q (3), and R352H (bath pH of 7.2) (]) mutants. Login to comment 132 ABCC7 p.Arg352Cys The symbols for the wild type and R352C are indistinguishable at several voltages. Login to comment 133 ABCC7 p.Arg352Gln ABCC7 p.Arg352His The symbols for R352Q and R352H overlap at several voltages. Login to comment 136 ABCC7 p.Arg352His (C and D) Single-channel recordings from inside-out patches obtained from cells expressing either (C) wild-type CFTR or (D) the R352H mutant (bath pH of 7.2). Login to comment 149 ABCC7 p.Arg352His FIGURE 4: Reversal potential of the R352H mutant which is shifted by the protonation state of the histidine. Login to comment 150 ABCC7 p.Arg352His Single-channel recordings for R352H at various applied voltages in a 10-fold NaCl gradient as described in Figure 3A except the bath pH was 5.4 (A) and 7.2 (B). Login to comment 151 ABCC7 p.Arg352His (C) Single-channel current-voltage relationships for R352H with the bath pH at pH 5.4 (O) and pH 7.2 (4). Login to comment 158 ABCC7 p.Arg352His To determine the role of the positive charge at position 352, we substituted histidine for Arg352. Login to comment 160 ABCC7 p.Arg352His Maintaining the positive charge at position 352 with the R352H mutation and a bath pH of 5.4 gave a reversal potential Erev of -50.4 ( 1.2 mV (n ) 7) (Figure 4), which was not significantly different from that of the wild type. Login to comment 164 ABCC7 p.Arg352Gln ABCC7 p.Arg352His ABCC7 p.Arg352Cys These uncharged substitutions also shifted the reversal potential by an amount comparable to that observed by deprotonating R352H; Erev ) -34.9 ( 2 mV (n ) 7) for R352C, and Erev ) -26.3 ( 1.9 mV (n ) 9) for R352Q (Figure 6), resulting in calculated PCl/PNa ratios of 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Login to comment 166 ABCC7 p.Arg352Glu Unfortunately, the single-channel conductance and kinetics of the R352E mutant appeared to be too small to accurately determine the reversal potential. Login to comment 168 ABCC7 p.Arg352Gln To be sure that the measured reversal potentials were not significantly affected by the low ionic strength of the cytoplasmic solution, we measured the reversal potentials for the wild type and R352Q following replacement of NaCl in the bath with sodium gluconate. Login to comment 170 ABCC7 p.Arg352Gln For the R352Q mutant, we could not clearly identify single currents at potentials more negative than the reversal potential calculated by extrapolation from the currents seen at positive potentials. Login to comment 171 ABCC7 p.Arg352Gln The reversal potential for R352Q calculated by extrapolation from the single-channel currents observed at positive potentials was -33.3 ( 1.4 mV (n ) 9) (Figure 7D, 0), close to that predicted with the Goldman-Hodgkin-Katz equation given the Cl- to Na+ permeability ratios calculated above in the 10-fold NaCl gradient and assuming that gluconate is impermeable through CFTR. Login to comment 173 ABCC7 p.Gln353Cys Mutation of the adjacent residue (Q353C) did not alter the reversal potential relative to that of the wild type [Erev ) -51.1 ( 1.7 mV (n)7)], and thus, the Cl- to Na+ permeability ratio was the same as that of the wild type (PCl/ PNa ) 36). Login to comment 176 ABCC7 p.Arg352Gln The fractional current carried by a specific ion through the channel can be inferred from changes in conductance following ion substitutions.3 Therefore, we measured the Cl- to Na+ conductance ratios in the wild type (PCl/PNa ) 36) and in the R352Q (PCl/PNa ) 4) mutant to determine whether Na+ carries a significant fraction of the current in the mutants. Login to comment 178 ABCC7 p.Arg352Gln With wild-type CFTR in inside-out patches, as was reported previously (9), substituting NMDG-Cl for NaCl in FIGURE 5: Sucrose in the cytoplasmic bath does not alter the reversal potentials determined from the outward currents for the wild type and the R352Q mutant. Login to comment 179 ABCC7 p.Arg352Gln (A) Single-channel current-voltage relationships for the wild type (b and 0) and the R352Q mutant ([ and 4) in the presence of the 10-fold NaCl gradient. Login to comment 183 ABCC7 p.Arg352Gln Note that the reversal potentials and the conductances for the wild type and the R352Q mutant were not affected by the presence or absence of sucrose. Login to comment 191 ABCC7 p.Arg352Gln In R352Q, the effects of substituting NMDG+ for Na+ were different than in the wild type. Login to comment 192 ABCC7 p.Arg352Gln For the R352Q mutant, in inside-out patches, substituting NMDG-Cl for NaCl in the bath changed the current-voltage relationship from linear in symmetrical NaCl to inwardly rectifying (Figure 7D, 2 vs O) and caused the reversal potential to shift to 8.6 ( 0.8 mV (n ) 8). Login to comment 196 ABCC7 p.Arg352Gln Thus, for the R352Q mutant, the conductance of Cl- is about 3 pS and the conductance of Na+ is about 1 pS, giving a Cl- to Na+ conductance ratio of 3, similar to the permeability ratio of 4 for this mutant. Login to comment 200 ABCC7 p.Arg352Cys Similar reversal potentials were observed for the R352C mutant: 7 ( 1 mV (n ) 8) for Br- , -13 ( 1 mV (n ) 6) for I- , and -27 ( 2 mV (n ) 5) for F- . Login to comment 209 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys (A and C) Average single-channel current-voltage relationships for the R352C (A) and R352Q (C) mutants in symmetrical 140 mM NaCl (2) and in the presence of the 10-fold NaCl gradient (O) as described in the legend of Figure 3A. Login to comment 212 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys (B and D) Single-channel recordings are shown for the R352C (B) and R352Q (D) mutants. Login to comment 217 ABCC7 p.Arg352Cys For the R352C mutant, the conductances were similar to that of the wild type (5.9 ( 0.3 pS for Cl-, 5.2 ( 0.3 pS for Br-, 5.7 ( 0.2 pS for F-, and 3.6 ( 0.2 pS for I-). Login to comment 218 ABCC7 p.Arg347Asp ABCC7 p.Arg352Cys The mechanism for the decreased I- conductance compared to those of the other halide ions for both WT and R352C is not known, although in the R347D mutant the effect is eliminated (2). Login to comment 221 ABCC7 p.Arg352His In the R352H mutant, at a cytoplasmic pH of 5.4 when the histidine is mostly protonated, PCl/PNa ) 33, similar to that of the wild type. Login to comment 225 ABCC7 p.Arg347Asp This I- -induced change appears to result from I-binding to the CFTR protein and is eliminated by the R347D mutation (2). Login to comment 227 ABCC7 p.Arg352Gln FIGURE 7: A fraction of the current is carried by Na+ in the R352Q mutant but not in the wild type. Login to comment 232 ABCC7 p.Arg352Gln (D) Single-channel current-voltage relationships for R352Q under the same ion gradients. Login to comment 235 ABCC7 p.Arg352Gln Note the decreased current at positive potentials when NMDG+ replaces Na+ in the bath for the R352Q mutant (O vs 2), whereas there is no effect of this substitution for the wild type. Login to comment 236 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Table 1: Halide Permeability Ratios (PX/PCl)a bromide chloride iodide fluoride wild type 1.39 ( 0.05 1 0.71 ( 0.02 0.25 ( 0.04 R352C 1.04 ( 0.04 1 0.54 ( 0.05 0.22 ( 0.03 a Permeability ratios for the halides relative to those for Cl- for the wild type (WT) and the R352C mutant. Login to comment 240 ABCC7 p.Arg352His Thus, for the R352H mutant, we infer that the effect of the change in cytoplasmic pH on the Cl- to Na+ permeability ratio is due to a change in the protonation state of the histidine at position 352.5 Similarly, eliminating the positive charge at position 352 by substituting the uncharged amino acids, cysteine or glutamine, reduced PCl/PNa to about 5 (Figure 6). Login to comment 244 ABCC7 p.Arg352Gln In contrast, in the R352Q mutant, substitution of NMDG+ for Na+ caused rectification of the current-voltage relationship due to a unidirectional elimination of the contribution of Na+ to the single-channel conductance. Login to comment 248 ABCC7 p.Arg352Cys We do know, however, that Arg352 is in the channel lining because a cysteine substituted for Arg352 reacted with charged, sulfhydryl-specific MTS reagents (19) and the reaction rates were voltage-dependent (21). Login to comment 259 ABCC7 p.Arg352Gln The effect of this potential appears to be greater on cations than on anions because substitution of glutamine for arginine increased the Na+ conductance from an immeasurably low level in the wild type to about 1 pS in R352Q, whereas it only lowered the Cl-conductance by about 50%, i.e., from 6 to 3 pS (Figure 7). Login to comment 261 ABCC7 p.Arg352Cys This is unlikely to be the mechanism in CFTR because Arg352 does not appear to form a high-affinity Cl-binding site because the mutation R352C does not alter the halide selectivity sequence. Login to comment 278 ABCC7 p.Arg352His In contrast, in the R352H mutant raising the pH, presumably deprotonating His352, caused a 10-fold reduction in the Cl- to Na+ permeability ratio. Login to comment 279 ABCC7 p.Arg352His The fact that substituting other uncharged residues, glutamine and cysteine, at position 352 caused similar changes in the Cl- to Na+ permeability ratio supports our inference that raising the bath pH reduces the charge selectivity of R352H by deprotonation of the histidine at position 352. ability sequence. Login to comment 283 ABCC7 p.Pro99Leu The mutation P99L also changed the halide permeability ratios (37); however, this is presumably due to effects of the mutation on protein conformation because this residue is not on the water-accessible surface of the protein (18, 19). Login to comment