title: Contribution of intracellular calcium and pH in ischemic uncoupling of cardiac gap junction channels formed of connexins 43, 40, and 45: a critical function of C-terminal domain.
abstract: Ischemia is known to inhibit gap junction (GJ) mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs) 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD) condition. 5 minutes of OGD decreased the junctional conductance (Gj) of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca(2+)]i) and pH (pHi) with potassium phosphate buffer. Clamping of either [Ca(2+)]i or pHi, through BAPTA (2 mM) or HEPES (80 mM) respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT) truncated Cx43 (Cx43-Δ257). Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249), and Cx45 (Cx45-Δ272) helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca(2+)]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India.
authors.forename: Giriraj
authors.initials: G
authors.lastname: Sahu
authors.forename: Amal Kanti
authors.initials: AK
authors.lastname: Bera
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D017629:Gap Junctions / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D009154:Mutation; D017202:Myocardial Ischemia / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D018408:Patch-Clamp Techniques; D054730:Protein Interaction Domains and Motifs / Q000235:genetics
chemical_list: D002147:Calmodulin; D017630:Connexins; D002118:Calcium
references.citation: Biophys J. 1999 Sep;77(3):1374-83
references.pmid: 10465749
references.citation: J Mol Cell Cardiol. 1999 May;31(5):991-1003
references.pmid: 10336839
references.citation: J Neurophysiol. 1993 Jan;69(1):118-27
references.pmid: 8381854
references.citation: Biochem Biophys Res Commun. 2005 Oct 7;335(4):1191-8
references.pmid: 16112650
references.citation: J Gen Physiol. 1990 Jun;95(6):1061-75
references.pmid: 2115574
references.citation: Circ Res. 2000 Feb 4;86(2):E42-9
references.pmid: 10666425
references.citation: Biochim Biophys Acta. 2004 Mar 23;1662(1-2):61-80
references.pmid: 15033579
references.citation: Circ Res. 1999 May 28;84(10):1144-55
references.pmid: 10347089
references.citation: J Biol Chem. 2000 Nov 3;275(44):34407-14
references.pmid: 10924505
references.citation: Proc Natl Acad Sci U S A. 1992 May 1;89(9):3820-4
references.pmid: 1315041
references.citation: J Electrocardiol. 2005 Oct;38(4 Suppl):55-9
references.pmid: 16226075
references.citation: Eur J Cell Biol. 2009 Sep;88(9):481-94
references.pmid: 19473725
references.citation: Pflugers Arch. 1996 Jan;431(3):379-87
references.pmid: 8584431
references.citation: Neurosci Lett. 2007 Jan 29;412(2):118-22
references.pmid: 17178192
references.citation: Stem Cell Res. 2012 Sep;9(2):59-68
references.pmid: 22677706
references.citation: Biochim Biophys Acta. 2005 Jun 10;1711(2):172-82
references.pmid: 15955302
references.citation: Proc Natl Acad Sci U S A. 1999 May 25;96(11):6495-500
references.pmid: 10339616
references.citation: Circ Res. 1987 Aug;61(2):271-9
references.pmid: 3621491
references.citation: J Physiol. 1994 Feb 15;475(1):59-67
references.pmid: 8189393
references.citation: Cardiovasc Res. 2008 Oct 1;80(1):9-19
references.pmid: 18519446
references.citation: Circ Res. 2002 May 31;90(10):1100-7
references.pmid: 12039800
references.citation: Circ Res. 2001 Apr 13;88(7):666-73
references.pmid: 11304488
references.citation: Am J Physiol Cell Physiol. 2007 Dec;293(6):C1806-13
references.pmid: 17898133
references.citation: Am J Physiol Cell Physiol. 2012 May 15;302(10):C1548-56
references.pmid: 22422398
references.citation: Adv Cardiol. 2006;42:198-212
references.pmid: 16646592
references.citation: Am J Physiol. 1988 Dec;255(6 Pt 1):C844-56
references.pmid: 2849306
references.citation: Cardiovasc Res. 2009 Dec 1;84(3):361-7
references.pmid: 19620131
references.citation: Cell Commun Adhes. 2001;8(4-6):277-81
references.pmid: 12064602
references.citation: Neuroscience. 1999;93(3):1003-16
references.pmid: 10473265
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):495-500
references.pmid: 11756680
references.citation: Circ Res. 2007 Dec 7;101(12):1283-91
references.pmid: 17932323
references.citation: J Biol Chem. 2000 Aug 25;275(34):26220-4
references.pmid: 10852921
references.citation: Circulation. 1997 Nov 18;96(10):3579-86
references.pmid: 9396458
references.citation: J Neurosurg Spine. 2007 Jul;7(1):54-60
references.pmid: 17633488
references.citation: Am J Physiol Heart Circ Physiol. 2010 Apr;298(4):H1115-25
references.pmid: 20118409
references.citation: Heart Fail Rev. 2007 Dec;12(3-4):261-6
references.pmid: 17516165
references.citation: Science. 1982 May 7;216(4546):642-4
references.pmid: 6280283
references.citation: Biophys J. 2007 Mar 15;92(6):1952-65
references.pmid: 17189315
references.citation: Cardiovasc Res. 2004 May 1;62(2):276-86
references.pmid: 15094348
references.citation: Science. 2006 May 12;312(5775):924-7
references.pmid: 16690868
references.citation: Cardiovasc Res. 2004 May 1;62(2):228-32
references.pmid: 15094343
references.citation: Biophys J. 2001 Jul;81(1):137-52
references.pmid: 11423402
references.citation: Cell Biochem Funct. 2012 Mar;30(2):89-100
references.pmid: 22392438
references.citation: PLoS One. 2011;6(10):e26379
references.pmid: 22022608
references.citation: Cardiovasc Res. 2004 May 1;62(2):233-45
references.pmid: 15094344
references.citation: J Cell Sci. 2003 Nov 15;116(Pt 22):4479-81
references.pmid: 14576341
references.citation: Circ Res. 2000 Oct 13;87(8):656-62
references.pmid: 11029400