ABCMdb

PMID: 20332619

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., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Ser573Cys Dr. Karl Kunzelmann Department of Physiology, University of Regensburg Universitätsstraße 31, 93053 Regensburg (Germany) Tel. +49 941 943-4302, Fax +49941 943-4315 E-Mail karl.kunzelmann@vkl.uni-regensburg.de Key Words Cystic fibrosis transmembrane conductance regulator • CFTR AMP • PKA AMP-activated protein kinase • S573C • Pancreatitis • Metformin • Pancreas • Chloride secretion Abstract Metformin use in diabetes can cause acidosis and might be linked to pancreatitis. Login to comment 5 ABCC7 p.Ser573Cys 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). Login to comment 7 ABCC7 p.Ser573Cys Yet here, we find that S573C-CFTR manifests a metformin-inhibitable whole cell chloride-conductance after cAMP elevation. Login to comment 8 ABCC7 p.Ser573Cys In the absence of metformin, cAMP-activated S573C-CFTR also displays a reduced anion-conductance relative to wt-CFTR. Login to comment 9 ABCC7 p.Ser573Cys Furthermore, intra-oocyte acidification inhibited wt-CFTR and abolished S573C-CFTR conductance. Login to comment 10 ABCC7 p.Ser573Cys We conclude that defective S573C-CFTR remains both poorly conducting and inhibited by metformin and intracellular acidosis. Login to comment 39 ABCC7 p.Ser573Cys We investigated the S573C point mutation that lies within the first nucleotide binding domain of CFTR adjacent to the Walker B aspartate at D572. Login to comment 40 ABCC7 p.Ser573Cys S573C appears not to induce classical CF but instead segregates with pancreatitis of unknown etiology [20, 21]. Login to comment 41 ABCC7 p.Ser573Cys We tested the hypothesis that this was merely a chance finding by investigating S573C-CFTR function. Login to comment 44 ABCC7 p.Ser573Cys We speculated that the presence of the CFTR gene variant S573C in non-CF diabetes patients renders them more susceptible towards the development of pancreatitis and tested the potential role of metformin. Login to comment 45 ABCC7 p.Ser573Cys We therefore examined the function of S573C and other point mutants of CFTR at this site by overexpression in Xenopus oocytes. Login to comment 46 ABCC7 p.Ser573Cys The data indicate a lower channel activity of S573C-CFTR with a higher sensitivity towards metformin-induced closure. Login to comment 47 ABCC7 p.Ser573Cys Impaired Cl-channel function and regulatory function of S573C-CFTR may explain the higher incidence of this mutant in patients with pancreatitis. Login to comment 48 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala 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 60 ABCC7 p.Ser573Cys ABCC7 p.Ser573Cys ABCC7 p.Ser573Cys Results S573C attenuates CFTR whole cell Cl-conductance and is inhibited by metformin Because S573C-CFTR has been found in patients with pancreatitis, we examined the ability of both wtCFTR and S573C-CFTR to generate Cl- currents by maximal stimulation of Xenopus oocytes with IBMX (1 mM) and forskolin (2 µM). Login to comment 61 ABCC7 p.Ser573Cys Under these conditions which elevate cAMP and activate PKA, whole cell currents and conductances produced by S573C-CFTR were reduced (~20%) when compared to those produced by wt-CFTR (Fig. 1A-D).AMP-activated protein kinase (AMPK) has been shown previously to inhibit CFTR and to be relevant in vivo [13, 14, 22, 23]. Login to comment 64 ABCC7 p.Ser573Cys ABCC7 p.Ser573Cys Metformin only inhibited S573C-CFTR but not wt-CFTR (compare second bars in Fig. 1 E,F), in contrast to the more potent activator of AMPK, phenformin that inhibited both S573C-CFTR and wt-CFTR (Fig. 1E,F). Login to comment 66 ABCC7 p.Ser573Cys S573C attenuates CFTR whole cell Cl-conductance. Login to comment 67 ABCC7 p.Ser573Cys Current recordings obtained in Xenopus oocytes expressing wt-CFTR (A) or S573C-CFTR (B) showing activation of whole cell Cl- currents by stimulation with IBMX (1 mM) and forskolin (2 µM). Login to comment 69 ABCC7 p.Ser573Cys Summaries of whole cell conductances activated by IBMX and forskolin (GI/F ) in ooctyes expressing wt-CFTR (E) and S573C-CFTR (F), and effects of the activators of AMPK metformin (Met; 2 mM) and phenformin (Phen; 5 mM) or the AMPK-inhibitor compound C (CC; 80 µM). Login to comment 75 ABCC7 p.Ser573Cys ABCC7 p.Ser573Cys ABCC7 p.Ser573Cys Under these conditions it became quite obvious that S573C-CFTR has a reduced Cl-conductance compared to wtCFTR (Fig. 2C,D) and that 500 µM metformin did not inhibit wtCFTR and yet inhibited S573C-CFTR by about 25% (Fig. 2E), thus confirming the enhanced sensitivity of S573C-CFTR for inhibition by metformin. Login to comment 76 ABCC7 p.Ser573Ala 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 77 ABCC7 p.Ser573Cys Since S573 is located in the first nucleotide binding domain (NBD1) of CFTR and because inhibition by the AMPK-activator metformin is enhanced for S573C-CFTR, the present results suggest that NBD1 may contribute to regulation of CFTR byAMPK. Login to comment 79 ABCC7 p.Ser573Ala 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 80 ABCC7 p.Ser573Cys Metformin inhibits S573C-CFTR but not wt-CFTR. Login to comment 81 ABCC7 p.Ser573Cys Current recordings obtained in a Xenopus oocyte expressing wt-CFTR (A) or S573C-CFTR (B) showing activation of whole cell Cl- currents by stimulation with only forskolin (20 µM). Login to comment 83 ABCC7 p.Ser573Cys Summaries of the ration of whole cell conductances activated in the presence of metformin or under control conditions in oocytes expressing wt-CFTR and S573C-CFTR (E). Login to comment 88 ABCC7 p.Ser573Ala Phosphorylation and activation of S573A-CFTR. Login to comment 89 ABCC7 p.Ser573Ala Phosphorylation by AMPK of the first nucleotide binding domains (NBD1) of wtCFTR and S573A-CFTR (A). Login to comment 90 ABCC7 p.Ser573Ala ABCC7 p.Ser573Ala 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 95 ABCC7 p.Ser573Cys We interpreted this data to suggest that the S573C defect might lie downstream of AMPK due to perturbation of the milieu around either the adjacent Walker B aspartate (for example at D572) or near another local cysteine, as suggested by Chen et al during their pH studies [24]. Login to comment 98 ABCC7 p.Ser573Ala 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 ABCC7 p.Ser573Ala 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 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala Acidification equally inhibits wtCFTR, S573A-CFTR and S573C-CFTR. Login to comment 106 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala 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 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala 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 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala We further tested whether intracellular acidification differentially affects whole cell currents produced by wt-CFTR, S573C-CFTR, or S573A-CFTR. Login to comment 110 ABCC7 p.Ser573Ala We observed regular activation of wt-CFTR and S573A- Fig. 4. Login to comment 114 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala 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 116 ABCC7 p.Ser573Cys CFTR by forskolin (20 µM) in the presence of CCCP and at physiological pH (pH 7.5), while activation of S573C-CFTR was largely reduced (Fig. 5A-C). Login to comment 117 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala 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 118 ABCC7 p.Ser573Cys In fact acidosis completely inhibited S573C-CFTR currents. Login to comment 119 ABCC7 p.Ser573Cys Taken together the present results are consistent with a model whereby the S573C-CFTR mutation predisposes to pancreatitis because it i) may sensitize CFTR towards inappropriate regulation by AMPK, ii) is inhibited by the therapeutic biguanide metformin and iii) is inhibited potently by acidification. Login to comment 123 ABCC7 p.Ser573Cys Inhibition of wtCFTR, S573C-CFTR, and S573 A-CFTR by intracellular acidosis. Login to comment 124 ABCC7 p.Ser573Cys Current recordings obtained in a Xenopus oocyte expressing wt-CFTR (A), S573C-CFTR (B), or S573 A-CFTR (C). Login to comment 126 ABCC7 p.Ser573Cys Acidification (pH 5.5, pH 6.5) but not alkalinization (pH 8.5) inhibits Cl- currents generated by wt-CFTR, S573C-CFTR and S573 A-CFTR. Login to comment 127 ABCC7 p.Ser573Cys ABCC7 p.Ser573Ala Acidosis completely inhibited currents produced by S573C-CFTR and S573A-CFTR. Login to comment 138 ABCC7 p.Ser573Cys ABCC7 p.Ser573Cys The result of the present study pose challenging new questions with respect to the molecular mechanisms for predisposal of S573C towards AMPK- phosphorylation, the residues within NBD1 that are phosphorylated byAMPK, and how S573C within NBD1 affects pH sensitivity of CFTR. Login to comment