ABCC7 p.His954Cys
| ClinVar: |
c.2861A>C
,
p.His954Pro
?
, not provided
|
| CF databases: |
c.2861A>C
,
p.His954Pro
(CFTR1)
?
,
|
| Predicted by SNAP2: | A: N (87%), C: N (87%), D: N (82%), E: N (93%), F: N (53%), G: N (82%), I: N (78%), K: N (93%), L: N (82%), M: N (82%), N: N (93%), P: N (61%), Q: N (93%), R: N (93%), S: N (93%), T: N (93%), V: N (82%), W: D (53%), Y: N (61%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: D, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: N, V: D, W: D, Y: N, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
155 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).
X
ABCC7 p.His954Cys 20952391:155:52
status: NEW156 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).
X
ABCC7 p.His954Cys 20952391:156:62
status: NEW157 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).
X
ABCC7 p.His954Cys 20952391:157:187
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).
X
ABCC7 p.His954Cys 20952391:227:177
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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
118 Supporting this argument, internal diamide also inhibited activity of a mutant S768C/K951C, S768C/H954C, or S768C/ S955C, and inhibition was reversed by 4-6 mM DTT (Fig. 2E).
X
ABCC7 p.His954Cys 21059651:118:98
status: NEW119 In sharp contrast, both diamide and DTT had no effect on such CFTR constructs as K951C, H954C, and S955C.
X
ABCC7 p.His954Cys 21059651:119:88
status: NEW122 Similarly, diamide also suppressed channel activity of mutants S737C/H954C, S737C/S955C, and S737C/Q958C, and suppression was reversed by DTT (Fig. 2E).
X
ABCC7 p.His954Cys 21059651:122:69
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.
X
ABCC7 p.His954Cys 21059651:125:89
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.
X
ABCC7 p.His954Cys 21059651:126:98
status: NEWX
ABCC7 p.His954Cys 21059651:126:128
status: NEW127 In contrast, the H950C/V956C mutant exhibited no X-linked band possibly because of a poor relative orientation between H954C and V956C.
X
ABCC7 p.His954Cys 21059651:127:119
status: NEW128 Therefore, a disulfide bond can be formed between H950C (or H954C) and S768C or between H954C (or H950C) and S737C.
X
ABCC7 p.His954Cys 21059651:128:60
status: NEWX
ABCC7 p.His954Cys 21059651:128:88
status: NEW143 In contrast, curcumin had no such effect on S737A and H954A mutants, suggesting that they may be weak inhibitory residues, although disulfide cross-linking of S737C to H954C strongly inhibited channel activity (Figs. 2E and 4E).
X
ABCC7 p.His954Cys 21059651:143:168
status: NEW