title: The formation of the cAMP/protein kinase A-dependent annexin 2-S100A10 complex with cystic fibrosis conductance regulator protein (CFTR) regulates CFTR channel function.
abstract: Cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/protein kinase A (PKA) and ATP-regulated Cl(-) channel. CFTR is increasingly recognized as a component of multiprotein complexes and although several inhibitory proteins to CFTR have been identified, protein complexes that stimulate CFTR function remain less well characterized. We report that annexin 2 (anx 2)-S100A10 forms a functional cAMP/PKA/calcineurin (CaN)-dependent complex with CFTR. Cell stimulation with forskolin/3-isobutyl-1-methylxanthine significantly increases the amount of anx 2-S100A10 that reciprocally coimmunoprecipitates with cell surface CFTR and calyculin A. Preinhibition with PKA or CaN inhibitors attenuates the interaction. Furthermore, we find that the acetylated peptide (STVHEILCKLSLEG, Ac1-14), but not the nonacetylated equivalent N1-14, corresponding to the S100A10 binding site on anx 2, disrupts the anx 2-S100A10/CFTR complex. Analysis of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and CFTR(inh172)-sensitive currents, taken as indication of the outwardly rectifying Cl(-) channels (ORCC) and CFTR-mediated currents, respectively, showed that Ac1-14, but not N1-14, inhibits both the cAMP/PKA-dependent ORCC and CFTR activities. CaN inhibitors (cypermethrin, cyclosporin A) discriminated between ORCC/CFTR by inhibiting the CFTR(inh172)-, but not the DIDS-sensitive currents, by >70%. Furthermore, peptide Ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. Our data suggests that the anx 2-S100A10/CFTR complex is important for CFTR function across epithelia.
authors.affiliation: Academic Unit of Child Health, The University of Sheffield, Sheffield Children's Hospital, Sheffield, S10 2TH, United Kingdom.
authors.forename: Lee A
authors.initials: LA
authors.lastname: Borthwick
authors.forename: Jean
authors.initials: J
authors.lastname: McGaw
authors.forename: Gregory
authors.initials: G
authors.lastname: Conner
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Taylor
authors.forename: Volker
authors.initials: V
authors.lastname: Gerke
authors.forename: Anil
authors.initials: A
authors.lastname: Mehta
authors.forename: Louise
authors.initials: L
authors.lastname: Robson
authors.forename: Richmond
authors.initials: R
authors.lastname: Muimo
mesh_terms: D017306:Annexin A2 / Q000378:metabolism*; D019703:Calcineurin / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D009418:S100 Proteins / Q000378:metabolism*; D000099248:S100 Calcium Binding Protein A10
chemical_list: D017306:Annexin A2; D019703:Calcineurin; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10