ABCMdb

PMID: 20952391

Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Lys978Cys Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe3؉ no matter whether NBD2 was present or not. Login to comment 25 ABCC7 p.Lys978Cys It has been demonstrated that K978C/P/S in CL3 promotes channel activity without ATP (17). Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Printed in the U.S.A. 40438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 52•DECEMBER 24, 2010 atUniversityofNorthCarolinaatChapelHill,onAugust8,2011www.jbc.orgDownloadedfrom http://www.jbc.org/content/suppl/2010/10/15/M110.161497.DC.html Supplemental Material can be found at: PKA does regulate a NBD1-NBD2 interaction (12) and that PKA can regulate ATP-independent gating in CFTR constructs with G551D (20) or without NBD2 (⌬1198) (21, 22). Login to comment 45 ABCC7 p.Ser660Ala In addition, removal of a segment 760-783 or 817-838 (NEG2) or much of the R domain (⌬708-835/ S660A) from CFTR eliminates the PKA dependence of channel activity (26-28). Login to comment 64 ABCC7 p.Val510Ala To improve expression of a Cys-free CFTR-based construct, V510A was inserted (31), and cells expressing the construct were grown for 1-2 days at 24 °C and then for 2-5 h at 37 °C before measurements. Login to comment 83 ABCC7 p.Asp1370Asn More importantly, a D1370N mutant, which removes the Mg2ϩ binding site (32), was also inhibited by Fe3ϩ (Fig. 1C), and inhibition was reversed by 5 mM EDTA (Fig. 1E). Login to comment 84 ABCC7 p.Asp1370Asn However, unlike the WT channel, the D1370N mutant exhibited less inhibition by Fe3ϩ but more reversal of inhibition by EDTA. Login to comment 97 ABCC7 p.Lys978Cys Once the channel was open, Fe3ϩ could not exert any effect on hCFTR activity. Supporting this notion, a K978C mutant from CL3 was also insensitive to Fe3ϩ for a limited time because of a high open probability of 0.84 (17). Login to comment 98 ABCC7 p.Lys978Cys As shown in Fig. 2B, the K978C mutant channel exhibited spontaneous activity when excised from the HEK-293T cell but the resulting current ran down by an unknown mechanism. Login to comment 100 ABCC7 p.Lys978Cys A similar result was also found in a K978C-⌬1198 construct (Fig. 2C). Login to comment 108 ABCC7 p.Asp1370Asn The arrows indicate the final concentrations. C, effects of Fe3ϩ (5 ϫ 10-19 M) and Fe2ϩ (5 ϫ 10-13 M) on the WT hCFTR and the D1370N mutant currents (n ϭ 4-8). Login to comment 114 ABCC7 p.Lys978Cys Once K978C reduced the activation energy for channel opening, Fe3ϩ had no influence on channel activity. Login to comment 123 ABCC7 p.Cys832Ala NEM doubled hCFTR activity (Fig. 3A), but C832A clearly stopped potentiation (Fig. 3B). Login to comment 128 ABCC7 p.Cys832Ala Fig. 3, E and F, further supports this proposal because C832A weakened inhibition by Fe3ϩ . Login to comment 129 ABCC7 p.Cys832Ala ABCC7 p.Cys832Ala This weak binding affinity seen with C832A was not due to a high open probability because the C832A mutant channel was further activated by curcumin dramatically (Fig. 3E). Login to comment 132 ABCC7 p.Glu826Ala ABCC7 p.Asp836Ala ABCC7 p.Asp835Ala ABCC7 p.Asp828Ala ABCC7 p.Glu822Ala ABCC7 p.Glu831Ala Fig. 4, B and E, indicate that only D836A dramatically prevented inhibition by Fe3ϩ , whereas E822A, E826A, D828A, E831A, and D835A did not. Login to comment 133 ABCC7 p.Asp836Ala Similarly, activity of the D836A mutant was also potentiated by curcumin dramatically (Fig. 4B). Login to comment 135 ABCC7 p.His667Arg ABCC7 p.His775Thr ABCC7 p.His784Gln In addition, H667R, H775T, and H784Q but not Cys-832 and Asp-836 are found in the R domain of the mouse CFTR channel (Fig. 4A), which was found insensitive to Fe3ϩ (Fig. 4C). Login to comment 138 ABCC7 p.His775Ala ABCC7 p.His775Ala Further investigations indicate that only H775A suppressed inhibition by Fe3ϩ and activity of the H775A mutant was also further potentiated by curcumin (data not shown). Login to comment 141 ABCC7 p.His950Arg ABCC7 p.His950Ala Fig. 4, D and E, confirm this possibility because both H950A and H950R profoundly reduced inhibition by Fe3ϩ , and their channel activity was also greatly potentiated by curcumin. Login to comment 142 ABCC7 p.His954Ala Consistent with involvement of CL3, another neighboring mutant H954A from CL3 was also amelioratory to Fe3ϩ (Fig. 4E), and its activity was increased by curcumin dramatically (data not shown). Login to comment 145 ABCC7 p.Cys832Ala ABCC7 p.Asp836Ala ABCC7 p.His950Ala ABCC7 p.His954Ala In support of this proposal, H950A/H954A and D836A/C832A/ H774A completely prevented Fe3ϩ inhibition, which was reversed by EDTA (Fig. 4E). Login to comment 147 ABCC7 p.Val510Ala ABCC7 p.His950Cys Fig. 5A shows that internal diamide (10 ␮M) suppressed ϳ30% of channel activity of a H950C/S832C/ V510A construct, and suppression was partially reversed by FIGURE 2. Login to comment 149 ABCC7 p.Lys978Cys Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the hCFTR construct (A) and the K978C mutant (B). Login to comment 151 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys The arrows indicate the final concentrations. C, fractional inhibition of the current by Fe3ϩ for mutants K978C and K978C/ ⌬1198 (n ϭ 3-9). Login to comment 154 ABCC7 p.His950Cys These observations suggest that disulfide bond cross-linking between H950C and S832C should inhibit channel activity. Login to comment 155 ABCC7 p.His950Cys ABCC7 p.His775Cys ABCC7 p.His954Cys ABCC7 p.Asp836Cys Similarly, disulfide bond cross-linking of H950C or H954C to S832C, H775C, or D836C also inhibited channel activity, whereas single cysteine mutants were not affected by diamide (Fig. 5F and supplemental Fig. S1). Login to comment 156 ABCC7 p.His954Cys ABCC7 p.Asp828Cys However, both diamide and DTT failed to exert any effect on a H954C/D828C mutant possibly because of a poor orientation or a long distance between these two engineered cysteines (Fig. 5F and supplemental Fig. S1). Login to comment 157 ABCC7 p.His954Cys Additionally, small inhibition of the current by diamide (Fig. 5F) was not due to a background noise induced by a low current amplitude in the Cys-free constructs because a WT CFTR-based H954C mutant, which contains Cys-832 in the R domain, also exhibited a clear inhibition of the current by diamide (Fig. 5D). Login to comment 162 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Although Fig. 4E indicates that both S768A and S737A mutants were much inhibited by Fe3ϩ , these sites may not be excluded as Fe3ϩ ligands. Login to comment 165 ABCC7 p.Ser768Ala However, DTT partially reversed Fe3ϩ inhibition of S768A channel activity (Fig. 6B). Login to comment 170 ABCC7 p.Cys832Ala Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the hCFTR (A and C) and the C832A mutant (B and E). Login to comment 171 ABCC7 p.Cys832Ala Effects of NEM were tested on the hCFTR channel (A and C) and the C832A mutant (B). Login to comment 174 ABCC7 p.Cys832Ala E, effect of Fe3ϩ on the C832A mutant. Login to comment 175 ABCC7 p.Cys832Ala F, fractional Fe3ϩ inhibition of the current for WT and C832A (n ϭ 4-9). Login to comment 177 ABCC7 p.Ser768Asp Regulation of CFTR by Fe3؉ 40442 not reverse Fe3ϩ inhibition found in S768D, which is equivalent to phosphorylated Ser-768 (Fig. 6, C and D). Login to comment 180 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Unlike S768A, S737A exhibited a weak DTT effect (Fig. 6D), suggesting that phosphorylated Ser-737 may not participate in Fe3ϩ binding. Login to comment 185 ABCC7 p.His775Ala In contrast, the open probability and the single channel conductance were a little higher for the H775A mutant than for the WT channel (Fig. 7C). Login to comment 203 ABCC7 p.Asp836Ala ABCC7 p.His950Ala B-D, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the D836A mutant (B), the mouse CFTR (mCFTR) (C), and the H950A mutant (D). Login to comment 215 ABCC7 p.Lys978Cys Once a K978C- or AMP-PNP-induced decrease in the activation energy overcomes an energy barrier caused by the interfacial Fe3ϩ bridge, channel activity will no longer be modulated by the metal bridge (Fig. 8B). Login to comment 227 ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.His950Cys ABCC7 p.His950Cys ABCC7 p.His954Cys A-E, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing mutants H950C/S832C/V510A (A), H950C/V510A (B), S832C/V510A (C), H954C (D), and the WT hCFTR construct (E). Login to comment 237 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp Macroscopic currents across inside-out membrane patches excised from transfected HEK293T cells expressing the hCFTR (A), S768A (B), and S768D (C) constructs. Login to comment 247 ABCC7 p.His775Ala B and D, open state probability (Po) for the WT hCFTR (B) and the H775A mutant (D) (n ϭ 3-5). Login to comment 259 ABCC7 p.Asp836Tyr In this case, D836Y is found in cystic fibrosis patients. Login to comment