Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus
- 769 Downloads
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.
KeywordsConnexin43 Duolink Gap junction Hemichannel Nav1.5 Perinexus Sodium channel
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).
- 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 281:7994–8009PubMedCrossRefGoogle Scholar
- 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 87:73–82PubMedCrossRefGoogle Scholar
- 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 16:151–163PubMedCrossRefGoogle Scholar
- 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 9:600–607PubMedCrossRefGoogle Scholar
- 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 142:1093–1117PubMedCrossRefGoogle Scholar
- 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 108:294–304PubMedCrossRefGoogle Scholar
- 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 99:93–101PubMedCrossRefGoogle Scholar
- 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 83:52–60PubMedCrossRefGoogle Scholar