ABCC7 p.His950Ala
| Predicted by SNAP2: | A: N (82%), C: N (78%), D: N (87%), E: N (93%), F: D (53%), G: N (82%), I: N (53%), K: N (93%), L: N (72%), M: N (66%), N: N (97%), P: N (61%), Q: N (93%), R: N (93%), S: N (97%), T: N (93%), V: N (82%), W: D (63%), Y: N (57%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: N, G: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. 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 [PMID:20952391]
Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.
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No. Sentence Comment
141 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.
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ABCC7 p.His950Ala 20952391:141:55
status: NEW145 In support of this proposal, H950A/H954A and D836A/C832A/ H774A completely prevented Fe3ϩ inhibition, which was reversed by EDTA (Fig. 4E).
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ABCC7 p.His950Ala 20952391:145:29
status: NEW203 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).
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ABCC7 p.His950Ala 20952391:203:170
status: NEW[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.
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No. Sentence Comment
140 It is very exciting that H950A was also greatly activated by curcumin after pretreatment of ATP, but subsequent PKA further increased channel activity (Fig. 4D).
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ABCC7 p.His950Ala 21059651:140:25
status: NEW146 In order to further investigate if ATP is required for the effects of curcumin on S768A and H950A, curcumin was first applied to their intracellular sides before ATP was introduced.
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ABCC7 p.His950Ala 21059651:146:92
status: NEW150 It is interesting that both H950A and S768A still needed more PKA to be fully activated in this case (Fig. 5D).
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ABCC7 p.His950Ala 21059651:150:28
status: NEW167 A-D, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing WT (A), ⌬R (B), S768A (C), and H950A (D).
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ABCC7 p.His950Ala 21059651:167:150
status: NEW193 Unlike H950R/S768R and H950D/S768D, which exerted an electrostatic interaction between the R domain and CL3, H950A, S768A, S768D, and H950R were not apparently activated by ATP only (Fig. 7C) but more sensitive to PKA than WT CFTR (Fig. 7D).
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ABCC7 p.His950Ala 21059651:193:109
status: NEW196 A and B, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing H950A (A) and S768A (B).
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ABCC7 p.His950Ala 21059651:196:116
status: NEW213 Unlike H950A or S768A/D, an apparent open probability of H950R/S768R was higher (Po(app) ϭ 0.0042) than that of WT CFTR even in the absence of ATP, and ATP binding further increased channel opening (Po(app) ϭ 0.198) (Fig. 8, D and E).
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ABCC7 p.His950Ala 21059651:213:7
status: NEW223 However, the activation time became significantly shorter for H950A, S768A, and S768D (Fig. 9, B-E).
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ABCC7 p.His950Ala 21059651:223:62
status: NEW234 Error bars, S.E. TABLE 1 Potential roles in hydrogen bonding at the CL3-R domain interface Note that mutants whose channel activity was increased by curcumin in the presence of ATP are highlighted in boldface type. Residues Role in H-bond Mutants Arg Strong donor H950R, S768R, H950R/S768R, H950R/S768D, H950D/S768R Asp Strong acceptor H950D, S768D, H950D/S768D, H950R/S768D, H950D/S768R Thr, Gln, Ser, His Donor/Acceptor H950Q, S768T, WT Ala Negative control K946A, H950A, K951A, H954A, S955A, Q958A, S737A, S768A, ⌬R Inhibition of CFTR by Ser768 JANUARY 21, 2011•VOLUME 286•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2177 matter whether cAMP was present or not in the extracellular perfusate.
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ABCC7 p.His950Ala 21059651:234:467
status: NEW235 In contrast, H950A exhibited a higher basal open probability (Po(app) ϭ 0.0120), but extracellular cAMP did not exert further influence.
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ABCC7 p.His950Ala 21059651:235:13
status: NEW240 Supporting this possibility, H950A increased basal channel opening to a higher level, possibly by weakening Fe3ϩ binding.
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ABCC7 p.His950Ala 21059651:240:29
status: NEW272 A-C, unitary currents across inside-out membrane patches excised from transfected HEK-293T cells expressing WT CFTR (A), S768A (B), and H950A (C) in the absence and presence of ATP (1.
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ABCC7 p.His950Ala 21059651:272:136
status: NEW282 However, Figs. 4 and 5 clearly demonstrate that regulation of normal channel gating by curcumin required ATP because ATP binding to the NBDs promoted channel opening of H950A and S768A mutants (Fig. 8).
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ABCC7 p.His950Ala 21059651:282:169
status: NEW293 A-D, unitary currents across cell-attached membrane patches of transfected HEK-293T cells expressing WT CFTR (A), H950A (B), S768A (C), and S768D (D).
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ABCC7 p.His950Ala 21059651:293:114
status: NEW323 It is exciting that both S768A and H950A increased PKA sensitivity no matter whether curcumin was present or not (Figs. 4-7).
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ABCC7 p.His950Ala 21059651:323:35
status: NEW342 Supporting this proposal, H950A greatly facilitated basal channel opening in the resting cell, possibly because this mutation increased sensitivity to endogenous ATP and promoted phosphorylation at some stimulatory sites primarily by weakening endogenous Fe3ϩ binding (Fig. 9).
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ABCC7 p.His950Ala 21059651:342:26
status: NEW344 Unlike H950A, a basal channel open probability of S768A and S768D was still low (Po ϭ 0.0004) (Fig. 9).
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ABCC7 p.His950Ala 21059651:344:7
status: NEW347 Despite this complex involvement, H950A, S768A, and S768D were more sensitive to forskolin than WT CFTR because they were dramatically activated soon after forskolin was introduced (Fig. 9).
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ABCC7 p.His950Ala 21059651:347:34
status: NEW