title: Super-resolution imaging reveals that loss of the C-terminus of connexin43 limits microtubule plus-end capture and NaV1.5 localization at the intercalated disc.
abstract: OBJECTIVE
  It is well known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein-interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant 'Cx43D378stop') led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: (i) the morphology of clusters formed by the microtubule plus-end tracking protein 'end-binding 1' (EB1), (ii) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin-rich sites, and (iii) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa.
  
  
  RESULTS
  Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa, and microtubules.
  
  
  CONCLUSIONS
  NaV1.5 and EB1 localization at the cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA.
authors.forename: Esperanza
authors.initials: E
authors.lastname: Agullo-Pascual
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA.
authors.forename: Xianming
authors.initials: X
authors.lastname: Lin
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Leo-Macias
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA.
authors.forename: Mingliang
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Office of Collaborative Science Microscopy Core, NYU-SoM, New York, NY, USA.
authors.forename: Feng-Xia
authors.initials: FX
authors.lastname: Liang
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Li
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Pfenniger
authors.affiliation: Life and Medical Sciences Institute, Molecular Genetics, University of Bonn, Bonn, Germany.
authors.forename: Indra
authors.initials: I
authors.lastname: Lübkemeier
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU-SoM, New York, NY, USA Center for Health Informatics and Bioinformatics, NYU-SoM, New York, NY, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Keegan
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU-SoM, New York, NY, USA Center for Health Informatics and Bioinformatics, NYU-SoM, New York, NY, USA.
authors.forename: David
authors.initials: D
authors.lastname: Fenyö
authors.affiliation: Life and Medical Sciences Institute, Molecular Genetics, University of Bonn, Bonn, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Willecke
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU-SoM, New York, NY, USA.
authors.forename: Eli
authors.initials: E
authors.lastname: Rothenberg
authors.affiliation: Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM), 522 First Avenue, Smilow 805, New York, NY 10016, USA mario.delmar@nyumc.org.
authors.forename: Mario
authors.initials: M
authors.lastname: Delmar
mesh_terms: D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D008297:Male; D008564:Membrane Potentials; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000737:chemistry / Q000378:metabolism*; D036103:Nanotechnology / Q000379:methods; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D012964:Sodium / Q000378:metabolism; D013997:Time Factors
chemical_list: D015820:Cadherins; C489083:Cdh2 protein, mouse; D018031:Connexin 43; C584107:EB1 protein, mouse; C543076:GJA1 protein, mouse; D008869:Microtubule-Associated Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568321:Scn5a protein, mouse; D012964:Sodium
keywords: Area composita; Connexome; Cx43; EB1; Microtubule; NaV1.5