title: Splice cassette II of Na+,HCO3(-) cotransporter NBCn1 (slc4a7) interacts with calcineurin A: implications for transporter activity and intracellular pH control during rat artery contractions.
abstract: Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.
authors: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark.
authors: Andreas A
authors: AA
authors: Danielsen
authors: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
authors: Mark D
authors: MD
authors: Parker
authors: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark.
authors: Soojung
authors: S
authors: Lee
authors: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
authors: Walter F
authors: WF
authors: Boron
authors: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark.
authors: Christian
authors: C
authors: Aalkjaer
authors: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark; Water and Salt Research Center, Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address: eb@fi.au.dk.
authors: Ebbe
authors: E
authors: Boedtkjer
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D020013:Calcium Signaling; D016384:Consensus Sequence; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D008638:Mesenteric Arteries / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism*; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
chemical_list: D000316:Adrenergic alpha-Agonists; D065095:Calcineurin Inhibitors; D020033:Protein Isoforms; C498902:Slc4a7 protein, rat; C452850:Slc9a1 protein, rat; D027982:Sodium-Bicarbonate Symporters; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D019703:Calcineurin; C581051:PPP3CB protein, rat; D016559:Tacrolimus; D009638:Norepinephrine
references: J Biol Chem. 2001 May 18;276(20):17367-72
references: 11350981
references: Am J Physiol Heart Circ Physiol. 2009 Feb;296(2):H247-55
references: 19036852
references: Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6117-22
references: 19332797
references: J Biol Chem. 2005 May 6;280(18):17823-30
references: 15718246
references: J Biomed Sci. 2006 Jul;13(4):593-5
references: 16547769
references: J Physiol. 2012 Apr 15;590(8):1895-906
references: 22351634
references: Science. 1999 Sep 24;285(5436):2129-33
references: 10497131
references: Surgery. 1998 Apr;123(4):456-60
references: 9551073
references: J Exp Biol. 2009 Jun;212(Pt 11):1697-706
references: 19448079
references: Circ Res. 1994 Jun;74(6):1220-6
references: 7514511
references: Am J Respir Crit Care Med. 2008 Jun 1;177(11):1276-84
references: 18310478
references: Transpl Int. 2012 Mar;25(3):337-46
references: 22239125
references: Physiol Rev. 2000 Oct;80(4):1483-521
references: 11015619
references: Trends Cell Biol. 2011 Feb;21(2):91-103
references: 21115349
references: J Clin Invest. 1995 Apr;95(4):1480-9
references: 7706452
references: Trends Cardiovasc Med. 2013 Apr;23(3):59-65
references: 23266155
references: Physiol Rev. 1981 Apr;61(2):296-434
references: 7012859
references: Sci Signal. 2009 Dec 08;2(100):re9
references: 19996458
references: J Proteome Res. 2007 Sep;6(9):3501-8
references: 17683130
references: Am J Physiol Heart Circ Physiol. 2006 Jan;290(1):H172-80
references: 16126812
references: J Gen Physiol. 1994 Apr;103(4):647-63
references: 8057082
references: J Vasc Res. 2001 Jan-Feb;38(1):30-8
references: 11173992
references: Curr Pharm Des. 2012;18(10):1345-71
references: 22360557
references: Am J Physiol. 1986 Mar;250(3 Pt 1):C391-405
references: 2420193
references: Br J Pharmacol. 2009 Jun;157(4):554-64
references: 19422382
references: J Physiol. 1997 Dec 1;505 ( Pt 2):329-36
references: 9423176
references: Mol Cell Biol. 2009 May;29(10):2777-93
references: 19273587
references: Physiol Rev. 2010 Oct;90(4):1437-59
references: 20959620
references: J Gen Physiol. 1997 Nov;110(5):503-13
references: 9348323
references: J Biol Chem. 1999 Jun 4;274(23):16569-75
references: 10347222
references: J Vasc Res. 2012;49(6):479-96
references: 22907294
references: Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12631-6
references: 8901634
references: Am J Physiol. 1991 Dec;261(6 Pt 1):C1010-7
references: 1722641
references: Am J Physiol. 1993 Apr;264(4 Pt 1):C932-43
references: 8476022
references: J Biol Chem. 2002 Dec 27;277(52):50503-9
references: 12403779
references: Am J Physiol Cell Physiol. 2008 Feb;294(2):C591-603
references: 18077606
references: Cell. 2006 Nov 3;127(3):635-48
references: 17081983
references: Circ Res. 1977 Jul;41(1):19-26
references: 862138
references: Circulation. 2011 Oct 25;124(17):1819-29
references: 21947296
references: Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8606-10
references: 1528868
references: Circ Res. 2006 Mar 3;98(4):515-23
references: 16439691
references: J Exp Med. 2011 Sep 26;208(10):2125-39
references: 21930771
references: Heart Rhythm. 2011 Mar;8(3):474-7
references: 20887805
references: Nature. 2000 Jun 1;405(6786):571-5
references: 10850716
references: J Physiol. 1991 May;436:57-73
references: 2061847
references: Mol Cell Proteomics. 2007 Dec;6(12):2072-87
references: 17855441
references: J Gen Physiol. 1976 Jan;67(1):91-112
references: 1460
references: Annu Rev Physiol. 2005;67:719-58
references: 15709976
references: Prog Biochem Pharmacol. 1988;23:150-8
references: 3186769
references: Pflugers Arch. 1989 Mar;413(5):553-8
references: 2740208