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Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

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Summary

Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels

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References

  • Beyer, E.C. 1990. Molecular cloning and developmental expression of two chick embryo gap junction proteins. J. Biol. Chem 265:14439–14443

    Google Scholar 

  • Beyer, E.C., Paul, D.L., Goodenough, D.A. 1987. Connexin43, a protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol 105:2621–2629

    Google Scholar 

  • Beyer, E.C., Paul, D.L., Goodenough, D.A. 1990. Connexin family of gap junction proteins. J. Membrane Biol 116:187–194

    Google Scholar 

  • Burt, J.M., Spray, D.C. 1988. Single-channel events and gating behavior of the cardiac gap junction channel. Proc. Natl. Acad. Sci. USA 85:3431–3434

    Google Scholar 

  • Chamley-Campbell, J., Campbell, G.R., Ross, R. 1979. The smooth muscle cell in culture. Physiol. Rev 59:1–61

    Google Scholar 

  • Chomczynski, P., Sacchi, N. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  • Crow, D.S., Beyer, E.C., Paul, D., Kobe, S.S., Lau, A.F. 1990. Phosphorylation of connexin43 gap junction protein in uninfected and RSV-transformed mammalian fibroblasts. Mol. Cell. Biol 10:1754–1763

    Google Scholar 

  • Ebihara, L., Beyer, E.C., Swenson, K.I., Paul, D.L., Goodenough, D.A. 1989. Cloning and expression of a Xenopus embryonic gap junction protein. Science 243:1194–1195

    Google Scholar 

  • Eghbali, B., Kessler, J.A., Spray, D.C. 1990. Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32. Proc. Natl. Acad. Sci. USA 87:1328–1331

    Google Scholar 

  • Filson, A.J., Azarnia, R., Beyer, E.C., Loewenstein, W.R., Brugge, J.S. 1990. Tyrosine phosphorylation of a gap-junction protein correlates with inhibition of cell-to-cell communication. Cell Growth Diff 1:666–668

    Google Scholar 

  • Fishman, G.I., Eddy, R.L., Shows, T.B., Rosenthal, L., Leinwand, L.A. 1991. The human connexin gene family of gap junction proteins: Distinct chromosomal locations but similar structures. Genomics 10:250–256

    Google Scholar 

  • Fishman, G.I., Spray, D.C., Leinwand, L.A. 1990. Molecular characterization and functional expression of the human cardiac gap junction channel. J. Cell Biol 111:589–598

    Google Scholar 

  • Higgins, D.G., Sharp, P.M. 1988. CLUSTAL: A package for performing multiple sequence alignment on a microcomputer. Gene 73:237–244

    Article  CAS  PubMed  Google Scholar 

  • Kanter, H.L., Saffitz, J.E., Beyer, E.C. 1992. Canine cardiac myocytes express multiple gap junction proteins. Circ. Res 70:438–444

    Google Scholar 

  • Kawasaki, E.S. 1990. Amplification of RNA. In: PCR Protocols. M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, editors, pp. 21–27. Academic, San Diego

    Google Scholar 

  • Lang, L.M., Beyer, E.C., Schwartz, A.L., Gitlin, J.D. 1991. Molecular cloning of a rat uterine gap junction protein and analysis of gene expression during gestation. Am. J. Physiol 260:E787–793

    Google Scholar 

  • Larson, D.M. 1988. Intercellular junctions and junctional transfer in the blood vessel wall. In: Endothelial Cells. U.S. Ryan, editor, pp. 75–88. Cambridge University Press, Cambridge

    Google Scholar 

  • Larson, D.M., Carson, M.P., Haudenschild, C.C. 1987. Junctional transfer of small molecules in cultured bovine brain microvascular endothelial cells and pericytes. Microvasc. Res 34:184–199

    Google Scholar 

  • Larson, D.M., Fujiwara, K., Alexander, R.W., Gimbrone, M.A. 1984. Myosin in cultured vascular smooth muscle cells: Immunofluorescence and immunochemical studies of alterations in antigenic expression. J. Cell Biol 99:1582–1589

    Google Scholar 

  • Larson, D.M., Haudenschild, C.C., Beyer, E.C. 1990. Gap junction messenger RNA expression by vascular wall cells. Circ. Res 66:1074–1080

    Google Scholar 

  • Lash, J.A., Critser, A.S., Pressler, M.L. 1990. Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos. J. Biol. Chem 265:13113–13117

    Google Scholar 

  • Miller, T., Dahl, G., Werner, R. 1988. Structure of a gap junction gene: Rat connexin32. Biosci. Rep 8:455–464

    Google Scholar 

  • Moore, L.K., Beyer, E.C, Burt, J.M. 1991. Characterization of gap junction channels in A7r5 vascular smooth muscle cells. Am. J. Physiol 260:C975-C981

    Google Scholar 

  • Musil, L.S., Beyer, E.C., Goodenough, D.A. 1990a. Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning, ultrastructural localization, and post-translational phosphorylation. J. Membrane Biol 116:163–175

    Google Scholar 

  • Musil, L.S., Cunningham, B.A., Edelman, G.M., Goodenough, D.A. 1990b. Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J. Cell Biol 111:2077–2088

    Google Scholar 

  • Queen, C., Korn, L.J. 1984. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res 12:581–599

    Google Scholar 

  • Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

    CAS  PubMed  Google Scholar 

  • Segal, S.S., Daman, D.N., Duling, B.R. 1989. Propagation of vasomotor responses coordinates arteriolar responses. Am. J. Physiol 256:H832-H837

    Google Scholar 

  • Segal, S.S., Duling, B.R. 1986. Flow control among microvessels coordinated by intercellular conduction. Science 234:868–870

    Google Scholar 

  • Swenson, K.I., Jordan, J., Beyer, E.C., Paul, D.L. 1989. Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell 57:145–155

    Google Scholar 

  • Swenson, K.I., Piwnica-Worms, H., McNamee, H., Paul, D.L. 1990. Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Reg 1:989–1002

    Google Scholar 

  • Traub, O., Look, J., Dermietzel, R., Brummer, F., Hulser, D., Willecke, K. 1989. Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J. Cell Biol 108:1039–1051

    Google Scholar 

  • Veenstra, R.D. 1991. Developmental changes in regulation of embryonic chick heart gap junctions. J. Membrane Biol 119:253–265

    Google Scholar 

  • Veenstra, R.D., Berg, K., Wang, H.-Z., Westphale, E.M., Beyer, E.C. 1992. Molecular and biophysical properties of the connexins from developing chick heart. In: Gap Junctions. G.A. Zampighi and J.E. Hall, editors. Elsevier, Amsterdam (in press)

    Google Scholar 

  • Werner, R., Levine, E., Rabadan-Diehl, C., Dahl, G. 1989. Formation of hybrid cell-cell channels. Proc. Natl. Acad. Sci. USA 86:5380–5384

    Google Scholar 

  • Zhang, J.-T., Nicholson, B. 1989. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J. Cell Biol 109:3391–3401

    Google Scholar 

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Beyer, E.C., Reed, K.E., Westphale, E.M. et al. Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle. J. Membarin Biol 127, 69–76 (1992). https://doi.org/10.1007/BF00232759

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  • DOI: https://doi.org/10.1007/BF00232759

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