ABCMdb

ABCC7 p.Lys946Cys

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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Publications
[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
121 However, S768C could not form an inhibitory disulfide bond with V956C (inwardly facing) or K946C, possibly as a result of a long distance or a poor relative orientation (Fig. 2E). Login to comment

[hide] Functional Architecture of the Cytoplasmic Entranc... J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5. El Hiani Y, Linsdell P
Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.
J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5., [PMID:25944907]

Abstract [show]
As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.

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109 Application of MTSES (200 òe;M) following channel activation with PKA and ATP never caused an increase in macroscopic current amplitude but decreased current amplitude in K190C, R248C, R251C, R303C, K370C, K946C, R975C, K1041C, and R1048C (Figs. 2 and 3). Login to comment
110 The effect of MTSET on these MTSES-sensitive mutants was to increase (K190C and R303C), decrease (R248C, K946C, K1041C, and R1048C), or have no effect (R251C, K370C, and R975C) on macroscopic current amplitude (Figs. 2 and 3). Login to comment
114 The effects of MTS modification of side chains within the ICLs could reflect changes in Clafa; conductance (as suggested previously for R303C (19)) or in open probability (as shown previously for K946C and R975C (11)) or a combination of the two. Login to comment
116 Results with R303C, K946C, and R975C, which as described above are expected to affect predominantly Clafa; conductance (R303C) or gating (K946C and R975C), suggest that use of PPi in this way can effectively separate effects on Clafa; conductance from those on gating. Login to comment
118 In contrast, the effects of MTS reagents on both K946C and R975C were lost following PPi treatment (Fig. 5), consistent with these reagents modifying the normal gating process (reducing channel open probability) without affecting Clafa; conductance (11). Login to comment
126 As described previously (11) and consistent with results from macroscopic current recording following channel treatment with PPi (Fig. 5), application of MTSES or MTSET had no effect on current amplitude in K946C or R975C (Figs. 6 and 7). Login to comment
141 Cytoplasmic Entrance to the CFTR Channel Pore 15860 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 25ߦJUNE 19, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 4, amplitudes in unmodified channels was Cys-less b; K946C b03; R975C b0e; K370C b0e; R251C b0e; K1041C b03; R248C b0e; R1048C b0e; R303C b0e; K190C (Fig. 7A). Login to comment
164 Consistent with this, the functional effect of MTS reagents on R251C, K946C, and R975C were abolished in channels that had been treated with PPi to maintain the channels in the open state (Fig. 5). Login to comment
165 This suggests that MTS modification of these mutants may have affected channel gating, leading to a change in overall macroscopic current amplitude (Fig. 3), as previously demonstrated directly for K946C and R975C using single channel recording (11). Login to comment
170 Effects of both mutagenesis (12, 29-32) and MTS modification (including of K946C and R975C) (11) within the ICLs on channel gating have been reported previously; however, the mechanism(s) by which these manipulations of ICL structure affect channel gating is not well understood. Login to comment