Journal of Cell Science, Vol 97, Issue 1 109-117, Copyright © 1990 by Company of Biologists

JOURNAL ARTICLES

Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes

DW Laird and JP Revel
Division of Biology, California Institute of Technology, Pasadena 91125.

A 43 x 10(3) Mr protein (designated connexin43 or Cx43) is a major constituent of heart gap junctions. The understanding of its arrangement in junctional membranes has been extended by means of site-directed antibodies raised against synthetic peptides of Cx43. These represent part of the first extracellular loop (EL-46), the cytoplasmic loop (CL-100), the second extracellular loop (EL-186) and carboxy-terminal sequences (CT-237 and CT-360). All of the antibodies raised reacted with their respective peptides and the Cx43 protein on Western blots. By immunoelectron microscopy two of the antibodies (CL-100 and CT-360) were shown to label the cytoplasmic surface of isolated gap junction membranes. Immunofluorescent labeling at locations of neonatal cardiac myocyte-myocyte apposition required an alkali/urea treatment when the EL-46 and EL-186 antibodies were used. Immunoblot analysis of endoproteinase Lys-C-digested gap junctions revealed that the Cx43 protein passed through the lipid bilayer four times. Alkaline phosphatase digestion of isolated junctions was used to show that the CT-360 antibody recognized many phosphorylated forms of Cx43. Our results unequivocally confirm models of the organization of Cx43 that were based on a more limited set of data and a priori considerations of the sequence.

This article has been cited by other articles:

				D. M. Pedrotty, R. Y. Klinger, N. Badie, S. Hinds, A. Kardashian, and N. Bursac Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H390 - H400. [Abstract] [Full Text] [PDF]

				X.-Q. Gong, Q. Shao, S. Langlois, D. Bai, and D. W. Laird Differential Potency of Dominant Negative Connexin43 Mutants in Oculodentodigital Dysplasia J. Biol. Chem., June 29, 2007; 282(26): 19190 - 19202. [Abstract] [Full Text] [PDF]

				X.-Q. Gong, Q. Shao, C. S. Lounsbury, D. Bai, and D. W. Laird Functional Characterization of a GJA1 Frameshift Mutation Causing Oculodentodigital Dysplasia and Palmoplantar Keratoderma J. Biol. Chem., October 20, 2006; 281(42): 31801 - 31811. [Abstract] [Full Text] [PDF]

				F. Liu, F. T. Arce, S. Ramachandran, and R. Lal Nanomechanics of Hemichannel Conformations: CONNEXIN FLEXIBILITY UNDERLYING CHANNEL OPENING AND CLOSING J. Biol. Chem., August 11, 2006; 281(32): 23207 - 23217. [Abstract] [Full Text] [PDF]

				M. A. Retamal, C. J. Cortes, L. Reuss, M. V. L. Bennett, and J. C. Saez S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: Induction by oxidant stress and reversal by reducing agents PNAS, March 21, 2006; 103(12): 4475 - 4480. [Abstract] [Full Text] [PDF]

				S. L.M.A. Beeres, D. E. Atsma, A. van der Laarse, D. A. Pijnappels, J. van Tuyn, W. E. Fibbe, A. A.F. de Vries, D. L. Ypey, E. E. van der Wall, and M. J. Schalij Human Adult Bone Marrow Mesenchymal Stem Cells Repair Experimental Conduction Block in Rat Cardiomyocyte Cultures J. Am. Coll. Cardiol., November 15, 2005; 46(10): 1943 - 1952. [Abstract] [Full Text] [PDF]

				W. Roscoe, G. I. L. Veitch, X.-Q. Gong, E. Pellegrino, D. Bai, E. McLachlan, Q. Shao, G. M. Kidder, and D. W. Laird Oculodentodigital Dysplasia-causing Connexin43 Mutants Are Non-functional and Exhibit Dominant Effects on Wild-type Connexin43 J. Biol. Chem., March 25, 2005; 280(12): 11458 - 11466. [Abstract] [Full Text] [PDF]

				J. Thimm, A. Mechler, H. Lin, S. Rhee, and R. Lal Calcium-dependent Open/Closed Conformations and Interfacial Energy Maps of Reconstituted Hemichannels J. Biol. Chem., March 18, 2005; 280(11): 10646 - 10654. [Abstract] [Full Text] [PDF]

				G. I. Veitch, J. E. I. Gittens, Q. Shao, D. W. Laird, and G. M. Kidder Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface J. Cell Sci., June 1, 2004; 117(13): 2699 - 2707. [Abstract] [Full Text] [PDF]

				Y. Guo, C. Martinez-Williams, and D. E. Rannels Gap junction-microtubule associations in rat alveolar epithelial cells Am J Physiol Lung Cell Mol Physiol, December 1, 2003; 285(6): L1213 - L1221. [Abstract] [Full Text] [PDF]

				H. Qin, Q. Shao, H. Curtis, J. Galipeau, D. J. Belliveau, T. Wang, M. A. Alaoui-Jamali, and D. W. Laird Retroviral Delivery of Connexin Genes to Human Breast Tumor Cells Inhibits in Vivo Tumor Growth by a Mechanism That Is Independent of Significant Gap Junctional Intercellular Communication J. Biol. Chem., August 2, 2002; 277(32): 29132 - 29138. [Abstract] [Full Text] [PDF]

				J. H.-C. Lin, T. Takano, M. L. Cotrina, G. Arcuino, J. Kang, S. Liu, Q. Gao, L. Jiang, F. Li, H. Lichtenberg-Frate, et al. Connexin 43 Enhances the Adhesivity and Mediates the Invasion of Malignant Glioma Cells J. Neurosci., June 1, 2002; 22(11): 4302 - 4311. [Abstract] [Full Text] [PDF]

				K Jordan, R Chodock, A. Hand, and D. Laird The origin of annular junctions: a mechanism of gap junction internalization J. Cell Sci., January 2, 2001; 114(4): 763 - 773. [Abstract] [PDF]

				X. Yin, P. T. Jedrzejewski, and J. X. Jiang Casein Kinase II Phosphorylates Lens Connexin 45.6 and Is Involved in Its Degradation J. Biol. Chem., March 15, 2000; 275(10): 6850 - 6856. [Abstract] [Full Text] [PDF]

				A. P. Quist, S. K. Rhee, H. Lin, and R. Lal Physiological Role of Gap-junctional Hemichannels: Extracellular Calcium-dependent Isosmotic Volume Regulation J. Cell Biol., March 6, 2000; 148(5): 1063 - 1074. [Abstract] [Full Text] [PDF]

				S. L. Sandow and C. E. Hill Incidence of Myoendothelial Gap Junctions in the Proximal and Distal Mesenteric Arteries of the Rat Is Suggestive of a Role in Endothelium-Derived Hyperpolarizing Factor-Mediated Responses Circ. Res., February 18, 2000; 86(3): 341 - 346. [Abstract] [Full Text] [PDF]

				S. M. Sladek, A. Westerhausen-Larson, and J. M. Roberts Endogenous Nitric Oxide Suppresses Rat Myometrial Connexin 43 Gap Junction Protein Expression during Pregnancy Biol Reprod, July 1, 1999; 61(1): 8 - 13. [Abstract] [Full Text]

				K. Jordan, J. L. Solan, M. Dominguez, M. Sia, A. Hand, P. D. Lampe, and D. W. Laird Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells Mol. Biol. Cell, June 1, 1999; 10(6): 2033 - 2050. [Abstract] [Full Text]

				D. Manthey, F. Bukauskas, C. G. Lee, C. A. Kozak, and K. Willecke Molecular Cloning and Functional Expression of the Mouse Gap Junction Gene Connexin-57 in Human HeLa Cells J. Biol. Chem., May 21, 1999; 274(21): 14716 - 14723. [Abstract] [Full Text] [PDF]

				S. C. Juneja, K. J. Barr, G. C. Enders, and G. M. Kidder Defects in the Germ Line and Gonads of Mice Lacking Connexin43 Biol Reprod, May 1, 1999; 60(5): 1263 - 1270. [Abstract] [Full Text]

				T. Woodward, M. Sia, O. Blaschuk, J. Turner, and D. Laird Deficient epithelial-fibroblast heterocellular gap junction communication can be overcome by co-culture with an intermediate cell type but not by E-cadherin transgene expression J. Cell Sci., January 12, 1998; 111(23): 3529 - 3539. [Abstract] [PDF]

				M. Obayashi, M. Yano, M. Kohno, S. Kobayashi, T. Tanigawa, K. Hironaka, T. Ryouke, and M. Matsuzaki Dose-dependent effect of ANG II-receptor antagonist on myocyte remodeling in rat cardiac hypertrophy Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1824 - H1831. [Abstract] [Full Text] [PDF]

				H.-q. Xie, D. W. Laird, T.-H. Chang, and V. W. Hu A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization J. Cell Biol., April 7, 1997; 137(1): 203 - 210. [Abstract] [Full Text] [PDF]

				P. De Sousa, S. Juneja, S Caveney, F. Houghton, T. Davies, A. Reaume, J Rossant, and G. Kidder Normal development of preimplantation mouse embryos deficient in gap junctional coupling J. Cell Sci., January 8, 1997; 110(15): 1751 - 1758. [Abstract] [PDF]

				K.-i. Yoshida, M. Inui, K. Harada, T. C. Saido, Y. Sorimachi, T. Ishihara, S.-i. Kawashima, and K. Sobue Reperfusion of Rat Heart After Brief Ischemia Induces Proteolysis of Calspectin (Nonerythroid Spectrin or Fodrin) by Calpain Circ. Res., September 1, 1995; 77(3): 603 - 610. [Abstract] [Full Text]

				S. H. Britz-Cunningham, M. M. Shah, C. W. Zuppan, and W. H. Fletcher Mutations of the Connexin43 Gap-Junction Gene in Patients with Heart Malformations and Defects of Laterality N. Engl. J. Med., May 18, 1995; 332(20): 1323 - 1330. [Abstract] [Full Text] [PDF]

				I. t. Velde, B. de Jonge, E.E. Verheijck, M.J.A. van Kempen, L. Analbers, D. Gros, and H.J. Jongsma Spatial Distribution of Connexin43, the Major Cardiac Gap Junction Protein, Visualizes the Cellular Network for Impulse Propagation From Sinoatrial Node to Atrium Circ. Res., May 1, 1995; 76(5): 802 - 811. [Abstract] [Full Text]

				A. Reaume, P. de Sousa, S Kulkarni, B. Langille, D Zhu, T. Davies, S. Juneja, G. Kidder, and J Rossant Cardiac malformation in neonatal mice lacking connexin43 Science, March 24, 1995; 267(5205): 1831 - 1834. [Abstract] [PDF]

				M. Atkinson, P. Lampe, H. Lin, R Kollander, X. Li, and D. Kiang Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells J. Cell Sci., January 9, 1995; 108(9): 3079 - 3090. [Abstract] [PDF]

				P. De Sousa, G Valdimarsson, B. Nicholson, and G. Kidder Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos Development, January 4, 1993; 117(4): 1355 - 1367. [Abstract] [PDF]

				J. Hoh, R Lal, S. John, J. Revel, and M. Arnsdorf Atomic force microscopy and dissection of gap junctions Science, September 20, 1991; 253(5026): 1405 - 1408. [Abstract] [PDF]