ABCMdb

ABCC7 p.Ser660Asp

CF databases:	c.1978T>A , p.Ser660Thr (CFTR1) ? , The mutation was detected by DGGE analysis and characterized by direct sequencing. We have seen it only once, in over 1000 control chromosomes from Italian population.
Predicted by SNAP2:	A: N (82%), C: N (66%), D: D (53%), E: D (53%), F: D (59%), G: N (82%), H: N (61%), I: D (59%), K: D (53%), L: D (59%), M: N (57%), N: N (87%), P: N (72%), Q: N (61%), R: D (53%), T: N (87%), V: N (53%), W: D (63%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] CFTR Cl- channel and CFTR-associated ATP channel: ... EMBO J. 1998 Feb 16;17(4):898-908. Sugita M, Yue Y, Foskett JK
CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J. 1998 Feb 16;17(4):898-908., [PMID:9463368]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.

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No. Sentence Comment
142 To examine this further, we expressed CFTR S-oct-D, which contains eight serine-to-aspartate substitutions in the R domain (S660D, S686D, S700D, S712D, S737D, S768D, S795D and S813D) (Figure 8A). Login to comment
150 To examine this further, we expressed CFTR S-oct-D, which contains eight serine-to-aspartate substitutions in the R domain (S660D, S686D, S700D, S712D, S737D, S768D, S795D and S813D) (Figure 8A). Login to comment

[hide] Regulation of the cystic fibrosis transmembrane co... J Biol Chem. 1993 Sep 25;268(27):20259-67. Rich DP, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
J Biol Chem. 1993 Sep 25;268(27):20259-67., [PMID:7690753]

Abstract [show]
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

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Sentences [show]
No. Sentence Comment
121 The single-channel open-stateprobabil- ity for wild-type CFTR (n = 14), CFTR S-Quad-A (S600A,S737A, S712A,S737A,S768A,S795A,S813A) (n = 7), or CFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D)(n = 7in ATP alone (-PKA);n = 10 inPKA and ATP (+PkX))C1-channels was determined as described under "Experimental Procedures." Login to comment
123 S795A,S813A) (n = 5). Login to comment
205 Functional analysisof serine-to-aspartate mutantsof CFTR.A, the changes in SPQ fluorescence ofHeLa cells expressing wild-type CFTR (n = 56), CFTR S-Quad-D (S600D,S737D, S795D,S813D) (n = 24), CFTR S-Hex-D (S660D,S686D,S700D, S712D,S737D,S768D,S795D,S813D)( n = 23), or virus only-infected control cells (n = 53) after substitution of NO; for Iat 0 min. Login to comment
209 B, time course of current changes inan excised, inside-outmembrane patchfrom a HeLa cell expressingCFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D) C1- chan- nels.ATP (0.88 mM)and PKA (75 m)were added tothe cytosolic(bath) side of the membrane as indicated by the burs. Login to comment
213 S737D,S795D,S813D) (n = 37), CFTR S-Oct-D (S660D,S686D,S700D, vitro or in vivo (Figs.6 and 9). Login to comment
217 S737D,S795D,S813D) (n = 37), CFTR S-Oct-D (S660D,S686D,S700D, vitro or in vivo (Figs.6 and 9). Login to comment