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. 1995 Aug 2;130(4):987–995. doi: 10.1083/jcb.130.4.987

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PMCID: PMC2199959  PMID: 7642714

Abstract

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.

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Selected References

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  1. Bennett M. V., Barrio L. C., Bargiello T. A., Spray D. C., Hertzberg E., Sáez J. C. Gap junctions: new tools, new answers, new questions. Neuron. 1991 Mar;6(3):305–320. doi: 10.1016/0896-6273(91)90241-q. [DOI] [PubMed] [Google Scholar]
  2. Beyer E. C. Molecular cloning and developmental expression of two chick embryo gap junction proteins. J Biol Chem. 1990 Aug 25;265(24):14439–14443. [PubMed] [Google Scholar]
  3. Beyer E. C., Paul D. L., Goodenough D. A. Connexin family of gap junction proteins. J Membr Biol. 1990 Jul;116(3):187–194. doi: 10.1007/BF01868459. [DOI] [PubMed] [Google Scholar]
  4. Beyer E. C., Paul D. L., Goodenough D. A. 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. doi: 10.1083/jcb.105.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beyer E. C., Steinberg T. H. Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages. J Biol Chem. 1991 May 5;266(13):7971–7974. [PubMed] [Google Scholar]
  6. Brissette J. L., Kumar N. M., Gilula N. B., Hall J. E., Dotto G. P. Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6453–6457. doi: 10.1073/pnas.91.14.6453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bruzzone R., Haefliger J. A., Gimlich R. L., Paul D. L. Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell. 1993 Jan;4(1):7–20. doi: 10.1091/mbc.4.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carel J. C., Frazier B., Ley T. J., Holers V. M. Analysis of epitope expression and the functional repertoire of recombinant complement receptor 2 (CR2/CD21) in mouse and human cells. J Immunol. 1989 Aug 1;143(3):923–930. [PubMed] [Google Scholar]
  9. Chiba H., Sawada N., Oyamada M., Kojima T., Nomura S., Ishii S., Mori M. Relationship between the expression of the gap junction protein and osteoblast phenotype in a human osteoblastic cell line during cell proliferation. Cell Struct Funct. 1993 Dec;18(6):419–426. doi: 10.1247/csf.18.419. [DOI] [PubMed] [Google Scholar]
  10. Civitelli R., Beyer E. C., Warlow P. M., Robertson A. J., Geist S. T., Steinberg T. H. Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest. 1993 May;91(5):1888–1896. doi: 10.1172/JCI116406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ek J. F., Delmar M., Perzova R., Taffet S. M. Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43. Circ Res. 1994 Jun;74(6):1058–1064. doi: 10.1161/01.res.74.6.1058. [DOI] [PubMed] [Google Scholar]
  12. Evans W. H. Assembly of gap junction intercellular communication channels. Biochem Soc Trans. 1994 Aug;22(3):788–792. doi: 10.1042/bst0220788. [DOI] [PubMed] [Google Scholar]
  13. Hennemann H., Suchyna T., Lichtenberg-Fraté H., Jungbluth S., Dahl E., Schwarz J., Nicholson B. J., Willecke K. Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung. J Cell Biol. 1992 Jun;117(6):1299–1310. doi: 10.1083/jcb.117.6.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hertzberg E. L. A detergent-independent procedure for the isolation of gap junctions from rat liver. J Biol Chem. 1984 Aug 10;259(15):9936–9943. [PubMed] [Google Scholar]
  15. Horan P. K., Melnicoff M. J., Jensen B. D., Slezak S. E. Fluorescent cell labeling for in vivo and in vitro cell tracking. Methods Cell Biol. 1990;33:469–490. doi: 10.1016/s0091-679x(08)60547-6. [DOI] [PubMed] [Google Scholar]
  16. Kren B. T., Kumar N. M., Wang S. Q., Gilula N. B., Steer C. J. Differential regulation of multiple gap junction transcripts and proteins during rat liver regeneration. J Cell Biol. 1993 Nov;123(3):707–718. doi: 10.1083/jcb.123.3.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laing J. G., Westphale E. M., Engelmann G. L., Beyer E. C. Characterization of the gap junction protein, connexin45. J Membr Biol. 1994 Apr;139(1):31–40. doi: 10.1007/BF00232672. [DOI] [PubMed] [Google Scholar]
  18. Liu S., Taffet S., Stoner L., Delmar M., Vallano M. L., Jalife J. A structural basis for the unequal sensitivity of the major cardiac and liver gap junctions to intracellular acidification: the carboxyl tail length. Biophys J. 1993 May;64(5):1422–1433. doi: 10.1016/S0006-3495(93)81508-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Loewenstein W. R., Rose B. The cell-cell channel in the control of growth. Semin Cell Biol. 1992 Feb;3(1):59–79. doi: 10.1016/s1043-4682(10)80008-x. [DOI] [PubMed] [Google Scholar]
  20. Moreno A. P., Laing J. G., Beyer E. C., Spray D. C. Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells. Am J Physiol. 1995 Feb;268(2 Pt 1):C356–C365. doi: 10.1152/ajpcell.1995.268.2.C356. [DOI] [PubMed] [Google Scholar]
  21. Musil L. S., Goodenough D. A. Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol. 1991 Dec;115(5):1357–1374. doi: 10.1083/jcb.115.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Musil L. S., Goodenough D. A. Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell. 1993 Sep 24;74(6):1065–1077. doi: 10.1016/0092-8674(93)90728-9. [DOI] [PubMed] [Google Scholar]
  23. Schiller P. C., Mehta P. P., Roos B. A., Howard G. A. Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells. Mol Endocrinol. 1992 Sep;6(9):1433–1440. doi: 10.1210/mend.6.9.1331776. [DOI] [PubMed] [Google Scholar]
  24. Schirrmacher K., Schmitz I., Winterhager E., Traub O., Brümmer F., Jones D., Bingmann D. Characterization of gap junctions between osteoblast-like cells in culture. Calcif Tissue Int. 1992 Oct;51(4):285–290. doi: 10.1007/BF00334489. [DOI] [PubMed] [Google Scholar]
  25. Shen V., Rifas L., Kohler G., Peck W. A. Prostaglandins change cell shape and increase intercellular gap junctions in osteoblasts cultured from rat fetal calvaria. J Bone Miner Res. 1986 Jun;1(3):243–249. doi: 10.1002/jbmr.5650010302. [DOI] [PubMed] [Google Scholar]
  26. Stauffer K. A. The gap junction proteins beta 1-connexin (connexin-32) and beta 2-connexin (connexin-26) can form heteromeric hemichannels. J Biol Chem. 1995 Mar 24;270(12):6768–6772. [PubMed] [Google Scholar]
  27. Stauffer K. A., Unwin N. Structure of gap junction channels. Semin Cell Biol. 1992 Feb;3(1):17–20. doi: 10.1016/s1043-4682(10)80004-2. [DOI] [PubMed] [Google Scholar]
  28. Steinberg T. H., Civitelli R., Geist S. T., Robertson A. J., Hick E., Veenstra R. D., Wang H. Z., Warlow P. M., Westphale E. M., Laing J. G. Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J. 1994 Feb 15;13(4):744–750. doi: 10.1002/j.1460-2075.1994.tb06316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Swenson K. I., Jordan J. R., Beyer E. C., Paul D. L. Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell. 1989 Apr 7;57(1):145–155. doi: 10.1016/0092-8674(89)90180-3. [DOI] [PubMed] [Google Scholar]
  30. Tomasetto C., Neveu M. J., Daley J., Horan P. K., Sager R. Specificity of gap junction communication among human mammary cells and connexin transfectants in culture. J Cell Biol. 1993 Jul;122(1):157–167. doi: 10.1083/jcb.122.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Veenstra R. D. Voltage-dependent gating of gap junction channels in embryonic chick ventricular cell pairs. Am J Physiol. 1990 Apr;258(4 Pt 1):C662–C672. doi: 10.1152/ajpcell.1990.258.4.C662. [DOI] [PubMed] [Google Scholar]
  32. Veenstra R. D., Wang H. Z., Beyer E. C., Brink P. R. Selective dye and ionic permeability of gap junction channels formed by connexin45. Circ Res. 1994 Sep;75(3):483–490. doi: 10.1161/01.res.75.3.483. [DOI] [PubMed] [Google Scholar]
  33. Veenstra R. D., Wang H. Z., Westphale E. M., Beyer E. C. Multiple connexins confer distinct regulatory and conductance properties of gap junctions in developing heart. Circ Res. 1992 Nov;71(5):1277–1283. doi: 10.1161/01.res.71.5.1277. [DOI] [PubMed] [Google Scholar]
  34. Werner R., Levine E., Rabadan-Diehl C., Dahl G. Formation of hybrid cell-cell channels. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5380–5384. doi: 10.1073/pnas.86.14.5380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. White T. W., Bruzzone R., Wolfram S., Paul D. L., Goodenough D. A. Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J Cell Biol. 1994 May;125(4):879–892. doi: 10.1083/jcb.125.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]

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