Abstract
Gap junctions (GJs) are aggregates of channels that provide for direct cytoplasmic connection between cells. Importantly, this connection is thought responsible for cell-to-cell transfer of the cardiac action potential. The GJ channels of ventricular myocytes are composed of connexin43 (Cx43). Interaction of Cx43 with zonula occludens-1 (ZO-1) is localized not only at the GJ plaque, but also to the region surrounding the GJ, the perinexus. Cx43 in the perinexus is not detectable by immunofluorescence, yet localization of Cx43/ZO-1 interaction to this region indicated the presence of Cx43. Therefore, we hypothesized that Cx43 occurs in the perinexus at a lower concentration per unit membrane than in the GJ itself, making it difficult to visualize. To overcome this, the Duolink protein–protein interaction assay was used to detect Cx43. Duolink labeling of cardiomyocytes localized Cx43 to the perinexus. Quantification demonstrated that signal in the perinexus was lower than in the GJ but significantly higher than in nonjunctional regions. Additionally, Duolink of Triton X-100-extracted cultures suggested that perinexal Cx43 is nonjunctional. Importantly, the voltage gated sodium channel Nav1.5, which is responsible for initiation of the action potential, was found to interact with perinexal Cx43 but not with ZO-1. This work provides a detailed characterization of the structure of the perinexus at the GJ edge and indicates that one of its potential functions in the heart may be in facilitating conduction of action potential.
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Baker SM, Kim N, Gumpert AM, Segretain D, Falk MM (2008) Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation. FEBS Lett 582:4039–4046
Beahm DL, Oshima A, Gaietta GM, Hand GM, Smock AE, Zucker SN, Toloue MM, Chandrasekhar A, Nicholson BJ, Sosinsky GE (2006) Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel. J Biol Chem 281:7994–8009
Beyer EC, Paul DL, Goodenough DA (1987) Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621–2629
Clausson CM, Allalou A, Weibrecht I, Mahmoudi S, Farnebo M, Landegren U, Wahlby C, Soderberg O (2011) Increasing the dynamic range of in situ PLA. Nat Methods 8:892–893
Cohen SA (1996) Immunocytochemical localization of rH1 sodium channel in adult rat heart atria and ventricle. Presence in terminal intercalated disks. Circulation 94:3083–3086
Colussi C, Berni R, Rosati J, Straino S, Vitale S, Spallotta F, Baruffi S, Bocchi L, Delucchi F, Rossi S, Savi M, Rotili D, Quaini F, Macchi E, Stilli D, Musso E, Mai A, Gaetano C, Capogrossi MC (2010) The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac arrhythmias in dystrophic mice. Cardiovasc Res 87:73–82
Decrock E, De Vuyst E, Vinken M, Van Moorhem M, Vranckx K, Wang N, Van Laeken L, De Bock M, D’Herde K, Lai CP, Rogiers V, Evans WH, Naus CC, Leybaert L (2009) Connexin 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model. Cell Death Differ 16:151–163
Delmar M, Liang FX (2012) Connexin43 and the regulation of intercalated disc function. Heart Rhythm 9:835–838
Desplantez T, Dupont E, Severs NJ, Weingart R (2007) Gap junction channels and cardiac impulse propagation. J Membr Biol 218:13–28
Evans WH, Martin PE (2002) Gap junctions: structure and function (review). Mol Membr Biol 19:121–136
Flores CE, Nannapaneni S, Davidson KG, Yasumura T, Bennett MV, Rash JE, Pereda AE (2012) Trafficking of gap junction channels at a vertebrate electrical synapse in vivo. Proc Natl Acad Sci USA 109:E573–E582
Fort AG, Murray JW, Dandachi N, Davidson MW, Dermietzel R, Wolkoff AW, Spray DC (2011) In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors. J Biol Chem 286:22875–22885
Gaietta G, Deerinck TJ, Adams SR, Bouwer J, Tour O, Laird DW, Sosinsky GE, Tsien RY, Ellisman MH (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296:503–507
Gutstein DE, Morley GE, Tamaddon H, Vaidya D, Schneider MD, Chen J, Chien KR, Stuhlmann H, Fishman GI (2001) Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ Res 88:333–339
Hunter AW, Gourdie RG (2008) The second PDZ domain of zonula occludens-1 is dispensable for targeting to connexin 43 gap junctions. Cell Commun Adhes 15:55–63
Hunter AW, Barker RJ, Zhu C, Gourdie RG (2005) Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion. Mol Biol Cell 16:5686–5698
Jansen JA, Noorman M, Musa H, Stein M, de Jong S, van der Nagel R, Hund TJ, Mohler PJ, Vos MA, van Veen TA, de Bakker JM, Delmar M, van Rijen HV (2012) Reduced heterogeneous expression of Cx43 results in decreased Nav1.5 Expression and reduced sodium current which accounts for arrhythmia vulnerability in conditional Cx43 knockout mice. Heart Rhythm 9:600–607
Jarvius J, Melin J, Goransson J, Stenberg J, Fredriksson S, Gonzalez-Rey C, Bertilsson S, Nilsson M (2006) Digital quantification using amplified single-molecule detection. Nat Methods 3:725–727
Johnson R, Hammer M, Sheridan J, Revel JP (1974) Gap junction formation between reaggregated Novikoff hepatoma cells. Proc Natl Acad Sci USA 71:4536–4540
Johnson RG, Meyer RA, Li XR, Preus DM, Tan L, Grunenwald H, Paulson AF, Laird DW, Sheridan JD (2002) Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules. Exp Cell Res 275:67–80
Johnson RG, Reynhout JK, TenBroek EM, Quade BJ, Yasumura T, Davidson KG, Sheridan JD, Rash JE (2012) Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43. Mol Biol Cell 23:71–86
Kamasawa N, Furman CS, Davidson KG, Sampson JA, Magnie AR, Gebhardt BR, Kamasawa M, Yasumura T, Zumbrunnen JR, Pickard GE, Nagy JI, Rash JE (2006) Abundance and ultrastructural diversity of neuronal gap junctions in the OFF and ON sublaminae of the inner plexiform layer of rat and mouse retina. Neuroscience 142:1093–1117
Kleber AG, Rudy Y (2004) Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol Rev 84:431–488
Koval M (2006) Pathways and control of connexin oligomerization. Trends Cell Biol 16:159–166
Kucera JP, Rohr S, Rudy Y (2002) Localization of sodium channels in intercalated disks modulates cardiac conduction. Circ Res 91:1176–1182
Lal R, John SA, Laird DW, Arnsdorf MF (1995) Heart gap junction preparations reveal hemiplaques by atomic force microscopy. Am J Physiol 268:C968–C977
Lampe PD, Cooper CD, King TJ, Burt JM (2006) Analysis of connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. J Cell Sci 119:3435–3442
Lauf U, Giepmans BN, Lopez P, Braconnot S, Chen SC, Falk MM (2002) Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells. Proc Natl Acad Sci USA 99:10446–10451
Li H, Liu TF, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG (1996) Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J Cell Biol 134:1019–1030
Lin X, Liu N, Lu J, Zhang J, Anumonwo JM, Isom LL, Fishman GI, Delmar M (2011) Subcellular heterogeneity of sodium current properties in adult cardiac ventricular myocytes. Heart Rhythm 8:1923–1930
Liu TF, Johnson RG (1999) Effects of TPA on dye transfer and dye leakage in fibroblasts transfected with a connexin 43 mutation at ser368. Methods Find Exp Clin Pharmacol 21:387–390
Maier SK, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T, Catterall WA (2002) An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc Natl Acad Sci USA 99:4073–4078
Malhotra JD, Thyagarajan V, Chen C, Isom LL (2004) Tyrosine-phosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes. J Biol Chem 279:40748–40754
Mori Y, Fishman GI, Peskin CS (2008) Ephaptic conduction in a cardiac strand model with 3D electrodiffusion. Proc Natl Acad Sci USA 105:6463–6468
Musil LS, Goodenough DA (1991) Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol 115:1357–1374
Musil LS, Goodenough DA (1993) Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74:1065–1077
Noorman M, van Rijen HV, van Veen TA, de Bakker JM, Stein M (2008) Differences in distribution of fibrosis in the ventricles underlie dominant arrhythmia vulnerability of the right ventricle in senescent mice. Neth Heart J 16:356–358
Noorman M, van der Heyden MA, van Veen TA, Cox MG, Hauer RN, de Bakker JM, van Rijen HV (2009) Cardiac cell–cell junctions in health and disease: electrical versus mechanical coupling. J Mol Cell Cardiol 47:23–31
Palatinus JA, O’Quinn MP, Barker RJ, Harris BS, Jourdan J, Gourdie RG (2011) ZO-1 determines adherens and gap junction localization at intercalated disks. Am J Physiol Heart Circ Physiol 300:H583–H594
Palatinus JA, Rhett JM, Gourdie RG (2012) The connexin43 carboxyl terminus and cardiac gap junction organization. Biochim Biophys Acta 1818:1831–1843
Petitprez S, Zmoos AF, Ogrodnik J, Balse E, Raad N, El-Haou S, Albesa M, Bittihn P, Luther S, Lehnart SE, Hatem SN, Coulombe A, Abriel H (2011) SAP97 and dystrophin macromolecular complexes determine two pools of cardiac sodium channels Nav1.5 in cardiomyocytes. Circ Res 108:294–304
Quist AP, Rhee SK, Lin H, Lal R (2000) Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 148:1063–1074
Rhett JM, Gourdie RG (2012) The perinexus: a new feature of Cx43 gap junction organization. Heart Rhythm 9:619–623
Rhett JM, Jourdan J, Gourdie RG (2011) Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1. Mol Biol Cell 22:1516–1528
Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres P, Morente M, Ruiz-Meana M, Konietzka I, Miro E, Totzeck A, Heusch G, Schulz R, Garcia-Dorado D (2006) Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res 99:93–101
Saez JC, Contreras JE, Bukauskas FF, Retamal MA, Bennett MV (2003) Gap junction hemichannels in astrocytes of the CNS. Acta Physiol Scand 179:9–22
Severs NJ, Bruce AF, Dupont E, Rothery S (2008) Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res 80:9–19
Shaw RM, Fay AJ, Puthenveedu MA, von Zastrow M, Jan YN, Jan LY (2007) Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell 128:547–560
Shintani-Ishida K, Uemura K, Yoshida K (2007) Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia. Am J Physiol Heart Circ Physiol 293:H1714–H1720
Smyth JW, Vogan JM, Buch PJ, Zhang SS, Fong TS, Hong TT, Shaw RM (2012) Actin cytoskeleton rest stops regulate anterograde traffic of connexin 43 vesicles to the plasma membrane. Circ Res 110:978–989
Solan JL, Lampe PD (2007) Key connexin 43 phosphorylation events regulate the gap junction life cycle. J Membr Biol 217:35–41
Solan JL, Marquez-Rosado L, Sorgen PL, Thornton PJ, Gafken PR, Lampe PD (2007) Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC. J Cell Biol 179:1301–1309
Stein M, van Veen TA, Remme CA, Boulaksil M, Noorman M, van Stuijvenberg L, van der Nagel R, Bezzina CR, Hauer RN, de Bakker JM, van Rijen HV (2009) Combined reduction of intercellular coupling and membrane excitability differentially affects transverse and longitudinal cardiac conduction. Cardiovasc Res 83:52–60
Tence M, Ezan P, Amigou E, Giaume C (2012) Increased interaction of connexin43 with zonula occludens-1 during inhibition of gap junctions by G protein-coupled receptor agonists. Cell Signal 24:86–98
Thompson RJ, Zhou N, MacVicar BA (2006) Ischemia opens neuronal gap junction hemichannels. Science 312:924–927
van Zeijl L, Ponsioen B, Giepmans BN, Ariaens A, Postma FR, Varnai P, Balla T, Divecha N, Jalink K, Moolenaar WH (2007) Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate. J Cell Biol 177:881–891
Veeraraghavan R, Salama ME, Poelzing S (2012) Interstitial volume modulates the conduction velocity–gap junction relationship. Am J Physiol Heart Circ Physiol 302:H278–H286
Yuan D, Wang Q, Wu D, Yu M, Zhang S, Li L, Tao L, Harris AL (2012) Monocyte–endothelial adhesion is modulated by Cx43-stimulated ATP release from monocytes. Biochem Biophys Res Commun 420:536–541
Zhu C, Barker RJ, Hunter AW, Zhang Y, Jourdan J, Gourdie RG (2005) Quantitative analysis of ZO-1 colocalization with Cx43 gap junction plaques in cultures of rat neonatal cardiomyocytes. Microsc Microanal 11:244–248
Acknowledgments
This work was supported in part by grants from the National Institutes of Heath (RO1 HL56728-10A2 to RGG, RO11DE019355-1 RGG subcontract, F30 HL095320-01 RGG mentor, and 5P20RR016434-07 RGG mentor), and an AHA Grant-in-Aid (RGG).
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Rhett, J.M., Ongstad, E.L., Jourdan, J. et al. Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus. J Membrane Biol 245, 411–422 (2012). https://doi.org/10.1007/s00232-012-9465-z
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DOI: https://doi.org/10.1007/s00232-012-9465-z