ABCMdb

ABCC7 p.Ser573Ala

CF databases:	c.1718C>G , p.Ser573Cys (CFTR1) ? , c.1718C>T , p.Ser573Phe (CFTR1) ? ,
Predicted by SNAP2:	A: N (82%), C: D (63%), D: N (72%), E: N (57%), F: D (85%), G: N (72%), H: D (80%), I: D (80%), K: D (59%), L: D (80%), M: D (80%), N: N (72%), P: D (59%), Q: N (61%), R: D (85%), T: N (78%), V: D (75%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: N, Q: N, R: D, T: N, V: D, W: D, Y: D,

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Publications
[hide] Mechanistic insight into control of CFTR by AMPK. J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18. Kongsuphol P, Cassidy D, Hieke B, Treharne KJ, Schreiber R, Mehta A, Kunzelmann K
Mechanistic insight into control of CFTR by AMPK.
J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18., 2009-02-27 [PMID:19095655]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.

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43 EXPERIMENTAL PROCEDURES cRNAs for CFTR and Double Electrode Voltage Clamp-Oocytes were injected with cRNA (10 ng, 47 nl of double-distilled water) encoding wtCFTR, L1430A/L1431A, S573A, S1248A, F508del-CFTR, G551D-CFTR, S768A, S737A, S768D, S737D, E1474X, and AMPK␣1. Login to comment
145 Regulation of CFTR by AMPK 5648 respectively) with the S573A but not with the S768A (compare second and third panels of Fig. 3C with the corresponding wild type result in the first panel of the figure). Login to comment

[hide] Metformin treatment of diabetes mellitus increases... Cell Physiol Biochem. 2010;25(4-5):389-96. Epub 2010 Mar 23. Kongsuphol P, Cassidy D, Romeiras F, Schreiber R, Mehta A, Kunzelmann K
Metformin treatment of diabetes mellitus increases the risk for pancreatitis in patients bearing the CFTR-mutation S573C.
Cell Physiol Biochem. 2010;25(4-5):389-96. Epub 2010 Mar 23., [PMID:20332619]

Abstract [show]
Metformin use in diabetes can cause acidosis and might be linked to pancreatitis. Here, we mechanistically focus on this relationship via a point mutation in the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7). CFTR is an ATP-hydrolyzing, cAMP/PKA-activated anion channel regulating pancreatic bicarbonate/chloride secretion across duct-facing apical membranes in epithelia. CFTR has two nucleotide binding domains (NBD1/2) which clamp two ATP molecules across their opposed, inverted interfacial surfaces which generates anion-conductance after ATP hydrolysis. Notably, CFTR mutations not causal for classical cystic fibrosis segregate with unexplained pancreatitis and one of these lies in NBD1 near its ATP-clamp (S573C; close to the Walker B aspartate D572). We recently showed that after raising [cAMP], wt-CFTR chloride-conductance, when expressed in Xenopus oocytes, remains elevated despite the presence of metformin. Yet here, we find that S573C-CFTR manifests a metformin-inhibitable whole cell chloride-conductance after cAMP elevation. In the absence of metformin, cAMP-activated S573C-CFTR also displays a reduced anion-conductance relative to wt-CFTR. Furthermore, intra-oocyte acidification inhibited wt-CFTR and abolished S573C-CFTR conductance. We conclude that defective S573C-CFTR remains both poorly conducting and inhibited by metformin and intracellular acidosis. This might explain the propensity to pancreatitis with this rare CF mutation.

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48 Materials and Methods cRNAs for CFTR and double electrode voltage clamp Oocytes were injected with cRNA (10 ng, 47 nl double-distilled water) encoding wtCFTR, S573C-CFTR, and S573A-CFTR. Login to comment
76 Phosphorylation of NBD1 is enhanced by the mutation S573A Previously we found that two serines in the regulatory (R) domain of CFTR, S737 and S768, formerly identified as inhibitory PKA sites, are phosphorylated by AMPK and are the residues essential for inhibition by AMPK [14]. Login to comment
79 We investigated a S573A-CFTR mutant and found that it behaved like wild type and critically, has lost its inhibitory metformin sensitivity when stimulated Fig. 2. Login to comment
88 Phosphorylation and activation of S573A-CFTR. Login to comment
89 Phosphorylation by AMPK of the first nucleotide binding domains (NBD1) of wtCFTR and S573A-CFTR (A). Login to comment
90 Summary of the whole cell conductances activated by IBMX (1 mM) and forskolin (20 µM) (GI/F ) obtained in Xenopus oocyte expressing S573A-CFTR (B) or S573A-CFTR (C) and effects of activators (metformin, phenformin) or an inhibitor (compound C) of AMPK. Login to comment
98 We mutated serine 573 to an alanine and found that elimination of this potential phosphorylation site did not abolish phosphorylation of NBD1 by AMPK (Fig. 3A). Login to comment
99 These results were consistent with existence of other phosphorylation sites for AMPK in NBD1, which are different from serine 573.Although in vitro phosphorylation data do not allow for quantitative assessment, it appears that AMPK- phosphorylation of the S573A mutant was somewhat augmented but this may equally be an artifact caused by slight differences relative amounts of protein. Login to comment
101 Acidification equally inhibits wtCFTR, S573A-CFTR and S573C-CFTR. Login to comment
106 Surprisingly, metformin (500 µM), when applied in the presence of acidic pH (pH 5.5), did not inhibit forskolin (20 µM) activated Cl- currents, generated by wt-CFTR, S573C-CFTR, or S573A-CFTR (Fig. 4C). Login to comment
107 Thus the S573C CFTR which had previously retained metformin-induced inhibition despite the presence of forskolin and IBMX, had now 'become wild type` just like S573A. Login to comment
108 We further tested whether intracellular acidification differentially affects whole cell currents produced by wt-CFTR, S573C-CFTR, or S573A-CFTR. Login to comment
110 We observed regular activation of wt-CFTR and S573A- Fig. 4. Login to comment
114 Summary of the whole cell conductances generated by wt-CFTR, S573C-CFTR and S573A-CFTR when activated by forskolin (20 µM) in the presence of pH 5.5, and effects of metformin (500 µM) (C). Login to comment
117 Subsequent, acidification inhibited Cl- currents produced by wt-CFTR, S573C-CFTR, or S573A-CFTR, while subsequent alkalinization did not affect whole cell Cl- currents (Fig. 5A-C). Login to comment
127 Acidosis completely inhibited currents produced by S573C-CFTR and S573A-CFTR. Login to comment