Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues (original) (raw)

• Journal List
• J Cell Biol
• v.108(2); 1989 Feb 1
• PMC2115444

J Cell Biol. 1989 Feb 1; 108(2): 595–605.

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Abstract

Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti- connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.

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Selected References

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• Albertini DF, Anderson E. The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J Cell Biol. 1974 Oct;63(1):234–250. [PMC free article] [PubMed] [Google Scholar]
• Beyer EC, Paul DL, Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987 Dec;105(6 Pt 1):2621–2629. [PMC free article] [PubMed] [Google Scholar]
• Brown D, Hirsch S, Gluck S. An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations. Nature. 1988 Feb 18;331(6157):622–624. [PubMed] [Google Scholar]
• Caspar DL, Goodenough DA, Makowski L, Phillips WC. Gap junction structures. I. Correlated electron microscopy and x-ray diffraction. J Cell Biol. 1977 Aug;74(2):605–628. [PMC free article] [PubMed] [Google Scholar]
• Dahl G, Miller T, Paul D, Voellmy R, Werner R. Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA. Science. 1987 Jun 5;236(4806):1290–1293. [PubMed] [Google Scholar]
• Dermietzel R, Leibstein A, Frixen U, Janssen-Timmen U, Traub O, Willecke K. Gap junctions in several tissues share antigenic determinants with liver gap junctions. EMBO J. 1984 Oct;3(10):2261–2270. [PMC free article] [PubMed] [Google Scholar]
• Fallon RF, Goodenough DA. Five-hour half-life of mouse liver gap-junction protein. J Cell Biol. 1981 Aug;90(2):521–526. [PMC free article] [PubMed] [Google Scholar]
• Fawcett DW, McNutt NS. The ultrastructure of the cat myocardium. I. Ventricular papillary muscle. J Cell Biol. 1969 Jul;42(1):1–45. [PMC free article] [PubMed] [Google Scholar]
• Forbes MS, Sperelakis N. Intercalated discs of mammalian heart: a review of structure and function. Tissue Cell. 1985;17(5):605–648. [PubMed] [Google Scholar]
• Fraser SE, Green CR, Bode HR, Gilula NB. Selective disruption of gap junctional communication interferes with a patterning process in hydra. Science. 1987 Jul 3;237(4810):49–55. [PubMed] [Google Scholar]
• Garfield RE, Daniel EE, Dukes M, Fitzgerald JD. Changes of gap junctions in myometrium of guinea pig at parturition and abortion. Can J Physiol Pharmacol. 1982 Mar;60(3):335–341. [PubMed] [Google Scholar]
• Goodenough DA, Paul DL, Jesaitis L. Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions. J Cell Biol. 1988 Nov;107(5):1817–1824. [PMC free article] [PubMed] [Google Scholar]
• Green CR, Severs NJ. A simplified method for the rapid isolation of cardiac intercalated discs. Tissue Cell. 1983;15(1):17–26. [PubMed] [Google Scholar]
• Gruijters WT, Kistler J, Bullivant S, Goodenough DA. Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions. J Cell Biol. 1987 Mar;104(3):565–572. [PMC free article] [PubMed] [Google Scholar]
• Hasty DL, Hay ED. Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast. J Cell Biol. 1977 Mar;72(3):667–686. [PMC free article] [PubMed] [Google Scholar]
• Hay ED, Revel JP. Fine structure of the developing avian cornea. Monogr Dev Biol. 1969;1:1–144. [PubMed] [Google Scholar]
• Hertzberg EL, Skibbens RV. A protein homologous to the 27,000 dalton liver gap junction protein is present in a wide variety of species and tissues. Cell. 1984 Nov;39(1):61–69. [PubMed] [Google Scholar]
• Kistler J, Kirkland B, Bullivant S. Identification of a 70,000-D protein in lens membrane junctional domains. J Cell Biol. 1985 Jul;101(1):28–35. [PMC free article] [PubMed] [Google Scholar]
• Kistler J, Christie D, Bullivant S. Homologies between gap junction proteins in lens, heart and liver. Nature. 1988 Feb 25;331(6158):721–723. [PubMed] [Google Scholar]
• Kumar NM, Gilula NB. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell Biol. 1986 Sep;103(3):767–776. [PMC free article] [PubMed] [Google Scholar]
• MacKenzie LW, Garfield RE. Hormonal control of gap junctions in the myometrium. Am J Physiol. 1985 Mar;248(3 Pt 1):C296–C308. [PubMed] [Google Scholar]
• Makowski L, Caspar DL, Phillips WC, Goodenough DA. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol. 1977 Aug;74(2):629–645. [PMC free article] [PubMed] [Google Scholar]
• Manjunath CK, Page E. Cell biology and protein composition of cardiac gap junctions. Am J Physiol. 1985 Jun;248(6 Pt 2):H783–H791. [PubMed] [Google Scholar]
• Manjunath CK, Nicholson BJ, Teplow D, Hood L, Page E, Revel JP. The cardiac gap junction protein (Mr 47,000) has a tissue-specific cytoplasmic domain of Mr 17,000 at its carboxy-terminus. Biochem Biophys Res Commun. 1987 Jan 15;142(1):228–234. [PubMed] [Google Scholar]
• McNutt NS, Fawcett DW. The ultrastructure of the cat myocardium. II. Atrial muscle. J Cell Biol. 1969 Jul;42(1):46–67. [PMC free article] [PubMed] [Google Scholar]
• Miller TM, Goodenough DA. Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell. J Cell Biol. 1986 Jan;102(1):194–199. [PMC free article] [PubMed] [Google Scholar]
• Nicholson BJ, Gros DB, Kent SB, Hood LE, Revel JP. The Mr 28,000 gap junction proteins from rat heart and liver are different but related. J Biol Chem. 1985 Jun 10;260(11):6514–6517. [PubMed] [Google Scholar]
• Nicholson B, Dermietzel R, Teplow D, Traub O, Willecke K, Revel JP. Two homologous protein components of hepatic gap junctions. Nature. 1987 Oct 22;329(6141):732–734. [PubMed] [Google Scholar]
• Paul DL. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. [PMC free article] [PubMed] [Google Scholar]
• Paul DL, Goodenough DA. Preparation, characterization, and localization of antisera against bovine MP26, an integral protein from lens fiber plasma membrane. J Cell Biol. 1983 Mar;96(3):625–632. [PMC free article] [PubMed] [Google Scholar]
• Traub O, Willecke K. Cross reaction of antibodies against liver gap junction protein (26K) with lens fiber junction protein (MIP) suggests structural homology between these tissue specific gene products. Biochem Biophys Res Commun. 1982 Dec 15;109(3):895–901. [PubMed] [Google Scholar]
• Warner AE, Guthrie SC, Gilula NB. Antibodies to gap-junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature. 1984 Sep 13;311(5982):127–131. [PubMed] [Google Scholar]
• Zervos AS, Hope J, Evans WH. Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous. J Cell Biol. 1985 Oct;101(4):1363–1370. [PMC free article] [PubMed] [Google Scholar]
• Zimmer DB, Green CR, Evans WH, Gilula NB. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J Biol Chem. 1987 Jun 5;262(16):7751–7763. [PubMed] [Google Scholar]

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