ABCMdb

PMID: 19837660

Chen JH, Cai Z, Sheppard DN
Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub ., 2009-12-18 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn ABCC7 p.Lys1250Met Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Login to comment 46 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser660Ala ABCC7 p.Lys1250Met These included (i) mouse mammary epithelial cells (C127 cells) expressing wild-type human CFTR, the CFTR variant ⌬R-S660A (13) or the CF mutant G1349D (14), (ii) Fischer rat thyroid epithelial cells expressing the CF mutant G551D (15), and (iii) NIH-3T3 cells expressing the CFTR construct K1250M (16). Login to comment 47 ABCC7 p.Lys464Ala ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn To study the CFTR variants K464A, D572N, and D1370N, we employed the vaccinia virus/bacteriophage T7 hybrid expression system to transiently express CFTR variants in HeLa cells as described previously (17, 18). Login to comment 84 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp However, for G551D and G1349D, because the number of active channels in a membrane patch was unknown, we measured NPo instead of Po. Login to comment 158 ABCC7 p.Ser660Ala To test this hypothesis, we adopted two strategies; first, we employed the CFTR construct ⌬R-S660A that deletes a large part of the RD and is not regulated by PKA-dependent phosphorylation (13) to explore separately how pHi influences the function of the RD and NBDs. Login to comment 161 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala Fig. 5A shows representative recordings of a single ⌬R-S660A-CFTR Cl-channel at different pHi, whereas Fig. 5, B-D quantifies the effects of pHi on channel gating. Consistent with previous studies (13), at pHi 7.3 the Po of ⌬R-S660A-CFTR was attenuated markedly compared with that of wild-type CFTR FIGURE 4. Login to comment 176 ABCC7 p.Ser660Ala Acidic pHi potentiates the activity of ⌬R-S660A-CFTR. Login to comment 177 ABCC7 p.Ser660Ala A, representative recordings show the effects of pHi on the activity of a single ⌬R-S660A-CFTR Cl-channel. Login to comment 178 ABCC7 p.Ser660Ala Dotted lines indicate where the channel is closed, and downward deflections correspond to channel openings. B, C, and D, effects of pHi on the Po, MBD, and IBI of ⌬R-S660A (columns) and wild-type CFTR (circles). Login to comment 179 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala Data are means Ϯ S.E. (B, n ϭ 6 for all data; C and D, ⌬R-S660A-CFTR, n ϭ 3; wild-type-CFTR, n Ն 6); asterisks indicate ⌬R-S660A-CFTR values that are significantly different from the pHi 7.3 control (p Ͻ 0.05). Login to comment 181 ABCC7 p.Ser660Ala Acidifying to pHi 6.3 potentiated the Po of wild-type CFTR 0.4-fold, but that of ⌬R-S660A-CFTR 3.3-fold (Fig. 5B). Login to comment 182 ABCC7 p.Ser660Ala Fig. 5, C and D, demonstrates that the marked potentiation of ⌬R-S660A-CFTR Po at pHi 6.3 was primarily caused by a 0.9-fold decrease in IBI, but it was also enhanced by a 1.5-fold increase of MBD. Login to comment 183 ABCC7 p.Ser660Ala Alkalinizing to pHi 8.3 decreased the Po of wild-type CFTR 0.3-fold, but was without effect on the Po of ⌬R-S660A-CFTR (Fig. 5B). Login to comment 184 ABCC7 p.Ser660Ala An explanation for this striking difference between wild-type and ⌬R-S660A-CFTR is shown in Fig. 5D. Login to comment 185 ABCC7 p.Ser660Ala Between pHi 7.3 and 8.3, the IBI of wild-type CFTR increased 0.3-fold, whereas that of ⌬R-S660A-CFTR decreased 0.5-fold (Fig. 5D). Login to comment 186 ABCC7 p.Ser660Ala Fig. 5D also reveals that the relationship between IBI and pHi is linear for wild-type CFTR, but bell-shaped for ⌬R-S660A-CFTR over the pHi range 6.3-8.3. Login to comment 187 ABCC7 p.Ser660Ala We interpret the failure of pHi 8.3 to attenuate ⌬R-S660A-CFTR channel gating to suggest that alkaline pHi might inhibit CFTR channel gating, at least in part, by modulating RD function. Login to comment 188 ABCC7 p.Ser660Ala However, the robust potentiation of ⌬R-S660A-CFTR channel gating at pHi 6.3 argues that acidic pHi likely enhances CFTR channel gating by acting on sites distinct from the RD. Login to comment 204 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn Previous studies have demonstrated that the mutations D572N- and D1370N-CFTR abolish Mg2ϩ binding to the NBDs (21, 22). Login to comment 205 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn Figs. 6C and 8 and supplemental Fig. 3, B and C, demonstrate that the gating behavior of D572N- and D1370N-CFTR Cl-channels at different pHi diverges from that of wild-type CFTR in several important respects. Login to comment 206 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn First, at pHi 7.3, the Po of D572N-CFTR was the same as wild-type CFTR, whereas that of D1370N-CFTR was reduced (Fig. 6C). Login to comment 207 ABCC7 p.Asp572Asn Second, at pHi 6.3, the Po of D572N-CFTR was potentiated markedly because IBI was decreased 0.7-fold and MBD was increased 1.4-fold (Figs. 6C and 8 and supplemental Fig. 3B). Login to comment 208 ABCC7 p.Asp1370Asn By contrast, for D1370N-CFTR at pHi 6.3, gating behavior and, hence, Po were unchanged (Fig. 6C and supplemental Fig. 3C). Login to comment 209 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn Third, in striking contrast to wild-type CFTR, at pHi 8.3 D572N-CFTR channel gating was enhanced because MBD was increased 0.6-fold and IBI decreased 0.3-fold, whereas that of D1370N-CFTR was unaltered (Figs. 2 and 8 and supplemental Fig. 3, B and C). Login to comment 210 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn As a result, at pHi 8.3, the Po of wild-type CFTR decreased, that of D1370N-CFTR was unchanged, whereas that of D572N-CFTR increased (Fig. 6C). Login to comment 212 ABCC7 p.Asp572Asn First, the potentiation of D572N-CFTR channel activity at pHi 8.3 suggests that the binding of Mg2ϩ ions at site 1 is essential for the inhibition of CFTR channel gating at alkaline pHi. Login to comment 213 ABCC7 p.Asp1370Asn Second the lack of effect of pHi on D1370N argues that the pHi sensitivity of CFTR channel gating is dependent on Mg2ϩ binding to site 2. Login to comment 218 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn B and C, effects of pHi on the Po of wild-type (WT), D572N-, and D1370N-CFTR in the presence of ATP (3 mM in B or 1 mM in C). Login to comment 219 ABCC7 p.Asp1370Asn ABCC7 p.Asp572Asn In B, wild-type CFTR data were acquired in the presence (circles) and absence (columns) of Mg2ϩ (3 mM), whereas in C, wild-type, D572N-, and D1370N-CFTR data were acquired in the continuous presence of Mg2ϩ (3 mM). Login to comment 224 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Of note, the CF mutations G551D and G1349D perturb severely CFTR channel gating (14, 33). Login to comment 225 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Using CFTR constructs bearing site-directed mutations, we examined the roles of the residues Lys-464, Lys-1250, Gly-551, and Gly-1349 in determining the pHi sensitivity of CFTR channel gating. Consistent with previous studies (14, 33), G1349D-CFTR and especially G551D-CFTR attenuated strongly CFTR channel gating at pHi 7.3, with brief, poorly resolved channel openings separated by very long-lasting closures (Fig. 7, A and C). Login to comment 226 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Fig. 7, A-D, demonstrates that the gating behavior and, hence, NPo of G551Dand G1349D-CFTR were unaffected by pHi. Login to comment 227 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Because tight dimerization of the NBDs is a prerequisite for channel opening (28) and because G551D and G1349D perturb severely channel gating (14, 33), we speculate that the effects of pHi on CFTR channel gating are dependent on the formation of an NBD1:NBD2 dimer. Login to comment 229 ABCC7 p.Lys464Ala ABCC7 p.Lys1250Met At pHi 7.3, bursts of K464A-CFTR channel openings were separated by prolonged channel closures, whereas dramatically prolonged bursts of K1250M-CFTR channel openings were separated by very long-lived channel closures (Figs. 7, E and G, and 8). Login to comment 230 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Although Po values of K464A-CFTR were less than those of wild-type CFTR at all pHi values tested, the effects of acidic and alkaline pHi on K464A- and wild-type CFTR were similar (Fig. 7, E and F). Login to comment 231 ABCC7 p.Lys464Ala This suggests that K464A-CFTR does not change the pHi sensitivity of CFTR. Login to comment 232 ABCC7 p.Lys1250Met By contrast, the pHi sensitivity of K1250M-CFTR differed strikingly from that of wild-type CFTR. Login to comment 233 ABCC7 p.Lys1250Met At pHi 6.3, the Po of K1250M-CFTR was reduced because MBD was decreased 0.7-fold, whereas IBI was unchanged (Figs. 7, G and H, and 8). Login to comment 234 ABCC7 p.Lys1250Met At pHi 8.3, the Po of K1250M-CFTR was increased, albeit slightly, as a result of a small increase in MBD and no change in IBI (Figs. 7H and 8). Login to comment 235 ABCC7 p.Lys1250Met Fig. 7H also reveals that the Po of K1250M-CFTR was similar to that of wild-type CFTR at pHi 8.3, but greatly diminished at pHi 7.3 and 6.3. Login to comment 236 ABCC7 p.Lys1250Met Thus, the pHi sensitivity of K1250M-CFTR is the converse of that of wild-type CFTR. Login to comment 246 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Lys464Ala ABCC7 p.Lys1250Met A, C, E, and G, representative recordings show the effects of pHi on the activity of G551D-, G1349D-, K464A-, and K1250M-CFTR Cl-channels in the presence of ATP (1 mM). Login to comment 247 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Lys1250Met ABCC7 p.Lys1250Met Dotted lines indicate where channels are closed, and downward deflections correspond to channel openings. B, D, F, and H, effects of pHi on the NPo of G551Dand G1349D-CFTR and Po of K464A- and K1250M-CFTR. Login to comment 248 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Data are means Ϯ S.E. (n ϭ 6, except G551Dand G1349D-CFTR at pHi ϭ 8.3, where n ϭ 3). Login to comment 254 ABCC7 p.Ser660Ala Second, studies of the CFTR construct ⌬R-S660A suggest that OH- ions might inhibit CFTR channel gating, at least in part, by modulating RD function. Login to comment 283 ABCC7 p.Ser660Ala In contrast to the effects of strong alkaline pHi on wild-type CFTR, our studies of the ⌬R-S660A-CFTR Cl-channel at pHi 8.3 argue that alkaline pHi inhibits CFTR channel gating, at FIGURE 8. Login to comment 284 ABCC7 p.Asp572Asn ABCC7 p.Lys1250Met Burst analysis of D572N- and K1250M-CFTR. Login to comment 285 ABCC7 p.Asp572Asn ABCC7 p.Lys1250Met A and B, MBD and IBI of D572N- and K1250M-CFTR at different pHi values; wild-type CFTR data are shown for comparison. Login to comment 286 ABCC7 p.Asp572Asn ABCC7 p.Lys1250Met Data are means Ϯ S.E. (D572N- and K1250M-CFTR, n ϭ 3; wild-type-CFTR, n Ն 6). Login to comment 290 ABCC7 p.Ser660Ala The reason why ⌬R-S660A-CFTR is not inhibited at pHi 8.3 is that its prolonged IBI is attenuated, not extended, at this pHi value. Login to comment 292 ABCC7 p.Ser660Ala ABCC7 p.Asp572Asn However, it is interesting to note that for both ⌬R-S660A-CFTR and the ATP-binding site 1 mutant D572N-CFTR, the relationship between pHi and IBI is bell-shaped, not linear, between pHi 6.3 and 8.3 (Figs. 5D and 8B). Login to comment 300 ABCC7 p.Gly551Asp ABCC7 p.Lys1250Ala Accordingly, the Walker A lysine mutant K1250A-CFTR is an ATP-dependent channel with moderate Po (16), whereas the LSGGQ motif mutant G551D-CFTR is an ATP-independent channel with extremely low Po (14, 33). Login to comment 301 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Thus, the loss of pHi sensitivity by G551Dand G1349D-CFTR suggests that correct alignment of the NBD1:NBD2 dimer is required for the potentiation of CFTR channel gating by acidic pHi and inhibition by alkaline pHi. Login to comment 311 ABCC7 p.Lys464Ala ABCC7 p.Asp572Asn Third, Hϩ ions potentiate the gating behavior of CFTR constructs bearing site-directed mutations in ATP-binding site 1 (K464A- and D572N-CFTR). Login to comment 312 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met By contrast, Hϩ ions are either without effect (D1370N-CFTR) or inhibit (K1250M-CFTR) the gating behavior of site-directed mutations in ATP-binding site 2. Login to comment 313 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp As discussed above, the failure of Hϩ ions to potentiate G551D- (site 2) and G1349D-CFTR (site 1) is likely a consequence of the profound disruption of NBD dimerization and, hence, channel gating by these constructs. Login to comment 343 ABCC7 p.Lys1250Met The pHi Sensitivity of CFTR Reveals Cross-talk between ATP Binding Sites 1 and 2-An interesting aspect of our data is the pHi sensitivity of K1250M-CFTR, which is the reverse of that of wild-type CFTR. Login to comment 345 ABCC7 p.Lys1250Met If K1250M-CFTR severely disrupts the function of site 2 (16), these hidden minor pHi effects might originate from site 1. Login to comment 346 ABCC7 p.Asp572Asn Our data also reveal that D572N-CFTR has a very unusual response to pHi: exaggerated potentiation of channel gating at acidic pHi and potentiation, not inhibition, at alkaline pHi. Login to comment 349 ABCC7 p.Pro574His ABCC7 p.Asp572Asn In support of this idea, the effects of acidic and alkaline pHi on D572N-CFTR are reminiscent of the enhanced activity of P574H-CFTR, a CF mutant affecting a residue in the Walker B-D-loop region of NBD1 (52). Login to comment