ABCMdb

PMID: 10026154

Cotten JF, Welsh MJ
Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge.
J Biol Chem. 1999 Feb 26;274(9):5429-35., 1999-02-26 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Login to comment 2 ABCC7 p.Arg347Lys ABCC7 p.Arg347Lys Every Arg347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Login to comment 5 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Login to comment 6 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. Login to comment 12 ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Leu At least four CF-associated mutations have been identified at position 347 in M6: R347C, R347H, R347L, and R347P, suggesting that Arg-347 is important for CFTR structure and function (13-15).2 Early studies by Sheppard et al. (7) showed that mutation of Arg-347 to proline significantly decreased single-channel conductance with little effect on CFTR trafficking to the plasma membrane. Login to comment 14 ABCC7 p.Arg347His ABCC7 p.Arg347His Interestingly, mutation of Arg-347 to a histidine (R347H) produced a channel that displayed pH-dependent conductance and anomalous mole-fraction behavior (8). Login to comment 18 ABCC7 p.Arg347His For example, mutation of this site could lead to a change in MSD conformation and loss of an anion-binding site(s) elsewhere; protonation of His-347 might then rescue the conformation of the R347H mutant. Login to comment 24 ABCC7 p.Arg347Cys ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Lys To better understand the role of Arg-347 in CFTR structure and function, we examined the effect of mutating Arg-347 to cysteine, aspartic acid, glutamic acid, lysine, and leucine on CFTR conductance. Login to comment 25 ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Lys ABCC7 p.Arg347Leu We examined the cytosolic pH (pHc)-dependent behavior of CFTR-R347H and that of the other residue 347 mutants both with (R347C, R347D, R347E, and R347K) and without (R347L) a pHc-titratable residue. Login to comment 26 ABCC7 p.Arg347His The conductance of CFTR-R347H is pHc-dependent. Login to comment 37 ABCC7 p.Arg347His 9, Issue of February 26, pp. 5429-5435, 1999 (c) 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org tion may exist in one of two states, either protonated or deprotonated, we tested the hypothesis that CFTR-R347H may display two pHc-dependent conductance states, which it did. Login to comment 40 ABCC7 p.Arg347His ABCC7 p.Arg347Lys Moreover, like CFTR-R347H, all the other residue 347 mutants, except R347K, displayed two pHc-dependent conductance states over a similar pHc range. Login to comment 69 ABCC7 p.Arg347His RESULTS pHc-dependent Conductance of Residue 347 Mutants-To determine whether R347H exhibits two discrete conductance states, we studied single channels in excised, inside-out patches. Login to comment 74 ABCC7 p.Arg347His from R347H displaying two pHc-dependent conductance states, OL and OB. Login to comment 75 ABCC7 p.Arg347Lys Unexpectedly, all the other variants at residue 347, except R347K, showed two pHc-dependent conductance states over a similar pH range. Login to comment 76 ABCC7 p.Arg347Leu Even R347L, which does not have a titratable side chain, displayed this behavior (Fig. 1). Login to comment 77 ABCC7 p.Arg347Lys ABCC7 p.Arg347Lys The conservative substitution of Arg-347 by Lys (R347K) showed only a single conductance state and no pHc dependence. Login to comment 82 ABCC7 p.Arg347Lys The all-points histograms (Fig. 1) illustrate qualitatively that as pHc increased, each mutant except R347K spent an increased fraction of time in OL and a correspondingly decreased fraction of time in OB. Login to comment 84 ABCC7 p.Arg347Lys Wild-type CFTR and R347K possess only one predominant conductance state over the pHc range 5.5-7.3 (Fig. 1, data not shown, and Ref. 8). Login to comment 86 ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Leu Visual inspection suggested that the lifetimes of OL and OB states were also influenced by the nature of the residue at position 347: R347E and R347H tended to have longer dwell times in the OL and OB states, whereas R347L, R347C, and R347D tended to display shorter dwell times. Login to comment 89 ABCC7 p.Arg347Asp For R347D, the lifetime in the OB state was so short that a discrete OB was not apparent on the all-points histogram; instead, as pHc decreased, a shoulder developed on the OL state distribution in the all-points histogram (Fig. 1). Login to comment 90 ABCC7 p.Arg347Leu Since R347L displayed two pHc-dependent conductance states and since leucine is aliphatic and non-ionizable, the pHc dependence of residue 347 mutants cannot be attributed simply to protonation of residue 347. Login to comment 91 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Lys Single-channel Conductance of Residue 347 Mutants-To determine whether the residue at position 347 affects single-channel conductance and not merely the conductance state of the channel, we examined the I-V relationship and slope conductance of the mutants with slower pHc-dependent kinetics, R347H and R347E, as well as R347K. Login to comment 94 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Lys The single-channel conductance at pHc 6.0 of wild-type CFTR, R347K, and the OB state of R347E and R347H were all very similar (in pS): 7.7 Ϯ 0.4, 8.3 Ϯ 0.6, 7.4 Ϯ 0.4, and 6.9 Ϯ 0.2, respectively (n ϭ 3 for each). Login to comment 95 ABCC7 p.Arg347His ABCC7 p.Arg347Glu The single-channel conductance of the OL states of R347E and R347H at pHc 6.0 were also very similar (in pS): 1.5 Ϯ 0.1 and 1.6 Ϯ 0.1, respectively (n ϭ 3 and 4 for each). Login to comment 102 ABCC7 p.Arg347Glu The first explanation seems unlikely because the reciprocal lifetime of the OL state which represents the "on" rate for the proton is very slow (e.g. it is 6 ϫ 107 M -1 s-1 for R347E). Login to comment 107 ABCC7 p.Arg347His ABCC7 p.Arg347Glu Dwell-time Analysis of OL and OB States-We performed a dwell-time analysis of the lifetimes of the OL and OB states of R347E and R347H to enable more quantitative comparisons between them and to better understand their pHc dependence. Login to comment 113 ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Leu The observable pK (0 mV) for the equilibrium between OL and OB of R347E and R347H were 6.4 and 6.3, respectively. The faster kinetics of R347D, R347C, and R347L made dwell-time analysis for these mutants less reliable. Login to comment 117 ABCC7 p.Arg347Cys ABCC7 p.Arg347Asp ABCC7 p.Arg347Leu Fig. 3B shows that R347C, R347D, and R347L did not reach a peak variance over the range of pHc studied, suggesting that their apparent pK is less than 5.0-5.5. Login to comment 119 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Lys ABCC7 p.Asp924Arg Single-channel I-V relationships for R347E (OL and OB states), R347H (OL and OB states), R347K, R347D/D924R, and wild-type CFTR at pHc 6.0. n ϭ 2-4 at each data point. Login to comment 122 ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu As a control for the variance analysis, we examined the R347E mutant on which we had also done dwell-time analysis (Fig. 3A); as expected, Fig. 3B shows that the variance of R347E goes through a maximum between pHc 6.5 and 5.5. Login to comment 124 ABCC7 p.Arg347Glu Fig. 4A shows qualitatively that both conductance states of R347E were voltage-dependent. Login to comment 125 ABCC7 p.Arg347His ABCC7 p.Arg347Glu Fig. 4B shows quantitatively that at pHc 6.0 the OL and OB states for R347E and R347H were both influenced by the transmembrane voltage. Login to comment 128 ABCC7 p.Arg347His ABCC7 p.Arg347Glu The degree of voltage dependence was similar for both mutants despite the charge differences at residue 347 and yielded a ␪z of 0.25 and 0.21 for R347E and R347H, respectively. The voltage dependence was asymmetrically disposed between the rate of entry into the OB state and the rate of exit from OB (Fig. 4B). Login to comment 130 ABCC7 p.Arg347His ABCC7 p.Arg347Glu The rate of exit from the OB state (␶B -1 ) was more voltage-dependent than the rate of exit from the OL state (␶L -1 ) for both mutants (␦ ϭ 0.8 versus 1 - ␦ ϭ 0.2 for R347E and ␦ ϭ 0.7 versus 1 - ␦ ϭ 0.3 for R347H). Login to comment 134 ABCC7 p.Arg347His ABCC7 p.Arg347Glu A, dwell-time analysis in the OL and OB conductance states versus pHc for R347E and R347H. Login to comment 136 ABCC7 p.Arg347Cys ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Leu B, open-channel current variance of the R347C, R347D, R347L, and R347E mutants versus pHc. Login to comment 142 ABCC7 p.Arg347Glu A, current records from excised, inside-out membrane patch containing R347E channel. Login to comment 144 ABCC7 p.Arg347His ABCC7 p.Arg347Glu B, dwell times (pHc 6.0) in the OL state (closed symbols) or OB state (open symbols) versus voltage for R347E (left, circles) and R347H (right, squares). Login to comment 147 ABCC7 p.Arg347His ABCC7 p.Arg347Glu arises from charge movement through a voltage field and since R347E and R347H displayed similar voltage dependences and carry different charges at position 347, residue 347 is not likely moving through a transmembrane potential during interchange between OL and OB states. Login to comment 148 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg The Phenotype of R347D Is Suppressed by the D924R Mutation-The data suggest that Arg-347 and Lys-347 may stabilize the structure of the pore; in their absence, the channel "flickers" between two conductance states. Login to comment 153 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp To identify the Arg-347 interaction partner, we replaced Arg-347 with an anionic residue (R347E or R347D) and introduced an arginine residue in the place of candidate partners in a salt bridge. Login to comment 154 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg We studied the conductance properties of the following double mutants: R347D/D924R, R347D/D993R, and R347E/E1104R. Login to comment 155 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg The R347D/D993R and R347E/E1104R mutants each had two conductance states with pHc-dependent behavior (Fig. 5). Login to comment 156 ABCC7 p.Arg347Asp ABCC7 p.Asp993Arg For R347D/D993R the increased entry into the OB state was apparent as a shoulder on the amplitude histogram at pHc 5.5. Login to comment 157 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg Accordingly, for R347D/D993R and R347E/E1104R the current variance in the open state increased with decreasing pHc (Fig. 5B). Login to comment 158 ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg Qualitatively, the lifetimes of the OL and OB conductance states in the R347D/D993R and R347E/E1104R were similar to that of the R347D and R347E mutants, respectively. The amplitude of the OL state was larger for both of these double mutants as compared with the single mutants (Figs. Login to comment 160 ABCC7 p.Arg347Glu ABCC7 p.Glu1104Arg We also observed an infrequent, additional small conductance state in the R347E/E1104 mutant (see amplitude histogram in Fig. 5A); this is likely due to the E1104R mutation itself. Login to comment 161 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg In contrast to the other double mutants, the R347D/D924R mutant did not display the pHc-dependent flicker found in the R347D single mutant (Fig. 5, A and B), and there was no effect of pH on open-channel variance (Fig. 5B). Login to comment 163 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg These data suggest that the D924R mutation compensates for or rescues the phenotype of the R347D mutation. Login to comment 164 ABCC7 p.Asp924Arg This result predicts that the D924R mutation alone (with Arg at position 347) would generate an unstable channel with at least two open conductance states. Login to comment 165 ABCC7 p.Asp924Arg Fig. 6 shows that the D924R single mutant displayed multiple (ϳ3) conductance states that appeared to be pHc-independent. Login to comment 171 ABCC7 p.Arg347His ABCC7 p.Arg347Glu Additionally, the single-channel slope conductances of R347H and R347E were the same in both OB and OL states. Login to comment 177 ABCC7 p.Arg347Lys The OB and OL Conductance States-All the mutants except R347K showed two pH-dependent conductance states. Login to comment 179 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg A, single-channel current tracings from excised, inside-out membrane patches containing R347E/E1104R, R347D/D924R, and R347D/D993R. Login to comment 181 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg B, current variance of R347E/E1104R, R347D/D924R, and R347D/D993R at the indicated pHc was collected as in Fig. 3. Login to comment 199 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg Second, and more importantly, we found that a second-site complementary mutation at position 924 (D924R) largely eliminated the pHc-dependent flickering phenotype of the R347D mutation and restored current amplitude to near wild-type values. Login to comment 202 ABCC7 p.Asp924Arg As predicted, we found that the D924R mutant displayed erratic flickery, pHc-independent behavior. Login to comment 204 ABCC7 p.Asp924Arg The pHc independence of D924R is also consistent with the hypothesis that Asp-924 is the site of protonation in the residue 347 mutants. Login to comment 207 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg The studies of R347D/D924R are consistent with a salt bridge between Arg-347 and Asp-924 and thus an interaction between M6 and M8. Login to comment 209 ABCC7 p.Asp993Arg ABCC7 p.Glu1104Arg Consistent with this, mutation of D993R and E1104R in MSD2 increased the relative amplitude of the OL conductance state in the context of Arg-347 mutations. Login to comment 210 ABCC7 p.Arg347Pro ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Leu The Arg-347 residue is targeted by several CF-associated mutations, R347C, R347H, R347L, and R347P (13-15).2 Our data suggest that CF-associated as well as other mutations at residue 347 affect CFTR similarly. Login to comment