ABCC7 p.Lys946Ala
| ClinVar: |
c.2836A>T
,
p.Lys946*
?
, not provided
|
| Predicted by SNAP2: | A: D (53%), C: N (53%), D: D (75%), E: D (59%), F: D (66%), G: D (71%), H: N (57%), I: D (53%), L: D (59%), M: N (78%), N: D (59%), P: D (80%), Q: N (87%), R: N (87%), S: D (53%), T: N (53%), V: D (59%), W: D (80%), Y: D (63%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: D, T: D, V: D, W: D, Y: D, |
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[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
141 Similarly, curcumin also activated mutants K946A, K951A, S955A, and Q958A to a different extent in the presence of ATP (Fig. 4E).
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ABCC7 p.Lys946Ala 21059651:141:43
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.Lys946Ala 21059651:234:460
status: NEW314 On the other hand, K946A, K951A, S955A, and Q958A still promoted channel opening by ATP followed by curcumin (Fig. 4, E and F).
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ABCC7 p.Lys946Ala 21059651:314:19
status: NEW[hide] Regulation of Activation and Processing of the Cys... J Biol Chem. 2012 Oct 11. Wang G, Duan DD
Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3.
J Biol Chem. 2012 Oct 11., [PMID:23060444]
Abstract [show]
NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, K946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of D835, D836 or E838 of NEG2 to prevent channel activation by PKA. R764 or R766 of the S768 phosphorylation site of the R domain is proposed to promote channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. On the other hand, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity while D835R/D836R/E838R/K946D/H950D was misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing and S768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2 and the S768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.
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No. Sentence Comment
11 First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation.
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ABCC7 p.Lys946Ala 23060444:11:48
status: NEW64 In the presence of 1.5mM ATP, 50µM curcumin greatly potentiated the K946A mutant activity, which was further increased by PKA (Fig. 3A).
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ABCC7 p.Lys946Ala 23060444:64:73
status: NEW138 First, the PKA sensitivity of channel activation was significantly enhanced for K946A, D835A, D836A and E838A mutants (Fig.2).
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ABCC7 p.Lys946Ala 23060444:138:80
status: NEW139 Second, the curcumin sensitivity was also increased for K946A, K946D and D835R/D836R/E838R (Fig.3).
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ABCC7 p.Lys946Ala 23060444:139:56
status: NEW9 First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation.
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ABCC7 p.Lys946Ala 23060444:9:49
status: NEW62 In contrast, mutation of Lys-946 or Asp-836 to alanine clearly accelerated the channel activation and reduced the PKA dependence (Fig. 2, B and C).
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ABCC7 p.Lys946Ala 23060444:62:25
status: NEW68 In the presence of 1.5 mM ATP, 50 òe;M curcumin greatly potentiated the K946A mutant activity, which was further increased by PKA (Fig. 3A).
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ABCC7 p.Lys946Ala 23060444:68:76
status: NEW98 The PKA-dependent activity of CFTR mutants at the R-CL3 interface. A-C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing WT CFTR (A) and mutants K946A (B) and D836A (C) by using a ramp protocol (afe;80 mV).
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ABCC7 p.Lys946Ala 23060444:98:203
status: NEW127 Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing CFTR mutants K946A (A), K946D (B) and D835R/D836R/E838R (C) by using a ramp protocol (afe;80 mV) are shown.
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ABCC7 p.Lys946Ala 23060444:127:120
status: NEW167 First, the PKA sensitivity of channel activation was significantly enhanced for K946A, D835A, D836A, and E838A mutants (Fig. 2).
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ABCC7 p.Lys946Ala 23060444:167:80
status: NEW168 Second, the curcumin sensitivity was also increased for K946A, K946D, and D835R/ D836R/E838R (Fig. 3).
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ABCC7 p.Lys946Ala 23060444:168:56
status: NEW