ABCC7 p.His950Cys
| 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
147 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.
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ABCC7 p.His950Cys 20952391:147:98
status: NEW154 These observations suggest that disulfide bond cross-linking between H950C and S832C should inhibit channel activity.
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ABCC7 p.His950Cys 20952391:154:69
status: NEW155 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).
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ABCC7 p.His950Cys 20952391:155:43
status: NEW227 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).
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ABCC7 p.His950Cys 20952391:227:120
status: NEWX
ABCC7 p.His950Cys 20952391:227:143
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
95 S768C/H950C was a representative example (Fig. 2B).
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ABCC7 p.His950Cys 21059651:95:6
status: NEW99 B-D, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing mutants H950C/S768C (B), H950C (C), and S768C (D) by using a ramp protocol (Ϯ80 mV).
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ABCC7 p.His950Cys 21059651:99:120
status: NEWX
ABCC7 p.His950Cys 21059651:99:137
status: NEW108 F, unitary currents from a H950C/S768C construct in the presence of 20 M diamide (b) and 4 mM DTT (c).
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ABCC7 p.His950Cys 21059651:108:27
status: NEW115 In contrast, both diamide and DTT had no effect on H950C and S768C CFTR constructs (Fig. 2, C and D).
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ABCC7 p.His950Cys 21059651:115:51
status: NEW116 These observations clearly suggest that a disulfide bond may be formed between S768C and H950C.
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ABCC7 p.His950Cys 21059651:116:89
status: NEW120 Furthermore, channel activity of V769C/H950C was also inhibited by diamide (Fig. 2E), suggesting that phosphorylation of Ser768 may not affect the CL3-R interface.
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ABCC7 p.His950Cys 21059651:120:39
status: NEW125 Fig. 3 demonstrates that a CFTR construct with a single cysteine S768C, S737C, H950C, or H954C exhibited a clear single band no matter whether diamide or DTT was added.
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ABCC7 p.His950Cys 21059651:125:79
status: NEW126 In sharp contrast, CFTR constructs with a cysteine pair (Cys-free background), S737C/H950C, S737C/H954C, S768C/H950C, and S768C/H954C, exhibited an additional cross-linked (X-linked) band because it was induced by diamide but was weakened by DTT.
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ABCC7 p.His950Cys 21059651:126:85
status: NEWX
ABCC7 p.His950Cys 21059651:126:111
status: NEW127 In contrast, the H950C/V956C mutant exhibited no X-linked band possibly because of a poor relative orientation between H954C and V956C.
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ABCC7 p.His950Cys 21059651:127:17
status: NEW128 Therefore, a disulfide bond can be formed between H950C (or H954C) and S768C or between H954C (or H950C) and S737C.
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ABCC7 p.His950Cys 21059651:128:50
status: NEWX
ABCC7 p.His950Cys 21059651:128:98
status: NEW129 In order to address if the disulfide bond changes the gating kinetics, a two-channel recording of the H950C/S768C construct was done.
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ABCC7 p.His950Cys 21059651:129:102
status: NEW162 Because disulfide cross-linking of S768C to H950C inhibited more channel activity than that of S768C to S955C (Fig. 2E), it is more possible for His950 to form an inhibitory H-bond with Ser768 .
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ABCC7 p.His950Cys 21059651:162:44
status: NEW244 Although thiol-specific disulfide cross-linking of S768C to H950C or nearby cysteines inserted in CL3 inhibited channel activity primarily by stopping the channel from opening, an electrostatic expulsion between S768R/D and H950R/D clearly promoted channel opening even in the absence of ATP.
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ABCC7 p.His950Cys 21059651:244:60
status: NEW288 Second, diamide-induced disulfide bond cross-linking of S768C to H950C or its neighboring cysteines, which was confirmed by the SDS-PAGE mobility (Fig. 3), inhibited channel activity (Fig. 2).
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ABCC7 p.His950Cys 21059651:288:65
status: NEW