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. 1994 May 2;125(4):879–892. doi: 10.1083/jcb.125.4.879

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

PMCID: PMC2120075  PMID: 8188753

Abstract

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.

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Selected References

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  1. Barrio L. C., Suchyna T., Bargiello T., Xu L. X., Roginski R. S., Bennett M. V., Nicholson B. J. Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8410–8414. doi: 10.1073/pnas.88.19.8410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassnett S., Duncan G. The influence of pH on membrane conductance and intercellular resistance in the rat lens. J Physiol. 1988 Apr;398:507–521. doi: 10.1113/jphysiol.1988.sp017054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benedetti E. L., Dunia I., Bloemendal H. Development of junctions during differentiation of lens fibers. Proc Natl Acad Sci U S A. 1974 Dec;71(12):5073–5077. doi: 10.1073/pnas.71.12.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bennett M. V., Verselis V. K. Biophysics of gap junctions. Semin Cell Biol. 1992 Feb;3(1):29–47. doi: 10.1016/s1043-4682(10)80006-6. [DOI] [PubMed] [Google Scholar]
  6. Beyer E. C., Kistler J., Paul D. L., Goodenough D. A. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989 Feb;108(2):595–605. doi: 10.1083/jcb.108.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Chanson M., Orci L., Meda P. Extent and modulation of junctional communication between pancreatic acinar cells in vivo. Am J Physiol. 1991 Jul;261(1 Pt 1):G28–G36. doi: 10.1152/ajpgi.1991.261.1.G28. [DOI] [PubMed] [Google Scholar]
  11. Dahl G., Levine E., Rabadan-Diehl C., Werner R. Cell/cell channel formation involves disulfide exchange. Eur J Biochem. 1991 Apr 10;197(1):141–144. doi: 10.1111/j.1432-1033.1991.tb15892.x. [DOI] [PubMed] [Google Scholar]
  12. 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. doi: 10.1126/science.3035715. [DOI] [PubMed] [Google Scholar]
  13. Dahl G., Werner R., Levine E., Rabadan-Diehl C. Mutational analysis of gap junction formation. Biophys J. 1992 Apr;62(1):172–182. doi: 10.1016/S0006-3495(92)81803-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dermietzel R., Spray D. C. Gap junctions in the brain: where, what type, how many and why? Trends Neurosci. 1993 May;16(5):186–192. doi: 10.1016/0166-2236(93)90151-b. [DOI] [PubMed] [Google Scholar]
  15. Ebihara L., Beyer E. C., Swenson K. I., Paul D. L., Goodenough D. A. Cloning and expression of a Xenopus embryonic gap junction protein. Science. 1989 Mar 3;243(4895):1194–1195. doi: 10.1126/science.2466337. [DOI] [PubMed] [Google Scholar]
  16. Ebihara L., Steiner E. Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes. J Gen Physiol. 1993 Jul;102(1):59–74. doi: 10.1085/jgp.102.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Emptage N. J., Duncan G., Croghan P. C. Internal acidification modulates membrane and junctional resistance in the isolated lens of the frog Rana pipiens. Exp Eye Res. 1992 Jan;54(1):33–39. doi: 10.1016/0014-4835(92)90066-2. [DOI] [PubMed] [Google Scholar]
  18. Epstein M. L., Gilula N. B. A study of communication specificity between cells in culture. J Cell Biol. 1977 Dec;75(3):769–787. doi: 10.1083/jcb.75.3.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Evans C. W., Eastwood S., Rains J., Gruijters W. T., Bullivant S., Kistler J. Gap junction formation during development of the mouse lens. Eur J Cell Biol. 1993 Apr;60(2):243–249. [PubMed] [Google Scholar]
  20. Fishman G. I., Moreno A. P., Spray D. C., Leinwand L. A. Functional analysis of human cardiac gap junction channel mutants. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3525–3529. doi: 10.1073/pnas.88.9.3525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goodenough D. A., Dick J. S., 2nd, Lyons J. E. Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freeze-substitution autoradiography and electron microscopy. J Cell Biol. 1980 Aug;86(2):576–589. doi: 10.1083/jcb.86.2.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Goodenough D. A., Paul D. L., 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. doi: 10.1083/jcb.107.5.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haefliger J. A., Bruzzone R., Jenkins N. A., Gilbert D. J., Copeland N. G., Paul D. L. Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping. J Biol Chem. 1992 Jan 25;267(3):2057–2064. [PubMed] [Google Scholar]
  24. Hennemann H., Dahl E., White J. B., Schwarz H. J., Lalley P. A., Chang S., Nicholson B. J., Willecke K. Two gap junction genes, connexin 31.1 and 30.3, are closely linked on mouse chromosome 4 and preferentially expressed in skin. J Biol Chem. 1992 Aug 25;267(24):17225–17233. [PubMed] [Google Scholar]
  25. Hennemann H., Schwarz H. J., Willecke K. Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs. Eur J Cell Biol. 1992 Feb;57(1):51–58. [PubMed] [Google Scholar]
  26. 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]
  27. Hoh J. H., John S. A., Revel J. P. Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31. J Biol Chem. 1991 Apr 5;266(10):6524–6531. [PubMed] [Google Scholar]
  28. Jiang J. X., White T. W., Goodenough D. A., Paul D. L. Molecular cloning and functional characterization of chick lens fiber connexin 45.6. Mol Biol Cell. 1994 Mar;5(3):363–373. doi: 10.1091/mbc.5.3.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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. doi: 10.1083/jcb.101.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kumar N. M., Gilula N. B. Molecular biology and genetics of gap junction channels. Semin Cell Biol. 1992 Feb;3(1):3–16. doi: 10.1016/s1043-4682(10)80003-0. [DOI] [PubMed] [Google Scholar]
  32. Kuszak J. R., Sivak J. G., Weerheim J. A. Lens optical quality is a direct function of lens sutural architecture. Invest Ophthalmol Vis Sci. 1991 Jun;32(7):2119–2129. [PubMed] [Google Scholar]
  33. 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]
  34. Lo C. W., Gilula N. B. Gap junctional communication in the post-implantation mouse embryo. Cell. 1979 Oct;18(2):411–422. doi: 10.1016/0092-8674(79)90060-6. [DOI] [PubMed] [Google Scholar]
  35. Lo W. K., Reese T. S. Multiple structural types of gap junctions in mouse lens. J Cell Sci. 1993 Sep;106(Pt 1):227–235. doi: 10.1242/jcs.106.1.227. [DOI] [PubMed] [Google Scholar]
  36. Mathias R. T., Riquelme G., Rae J. L. Cell to cell communication and pH in the frog lens. J Gen Physiol. 1991 Dec;98(6):1085–1103. doi: 10.1085/jgp.98.6.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Milks L. C., Kumar N. M., Houghten R., Unwin N., Gilula N. B. Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J. 1988 Oct;7(10):2967–2975. doi: 10.1002/j.1460-2075.1988.tb03159.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Miller T. M., Goodenough D. A. Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell. J Cell Biol. 1986 Jan;102(1):194–199. doi: 10.1083/jcb.102.1.194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Moreno A. P., Fishman G. I., Spray D. C. Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin43 gap junction channels. Biophys J. 1992 Apr;62(1):51–53. doi: 10.1016/S0006-3495(92)81775-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Musil L. S., Beyer E. C., Goodenough D. A. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Membr Biol. 1990 Jun;116(2):163–175. doi: 10.1007/BF01868674. [DOI] [PubMed] [Google Scholar]
  41. Nicholson B., Dermietzel R., Teplow D., Traub O., Willecke K., Revel J. P. Two homologous protein components of hepatic gap junctions. Nature. 1987 Oct 22;329(6141):732–734. doi: 10.1038/329732a0. [DOI] [PubMed] [Google Scholar]
  42. Paul D. L., Ebihara L., Takemoto L. J., Swenson K. I., Goodenough D. A. Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes. J Cell Biol. 1991 Nov;115(4):1077–1089. doi: 10.1083/jcb.115.4.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Paul D. L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. doi: 10.1083/jcb.103.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rae J. L., Kuszak J. R. The electrical coupling of epithelium and fibers in the frog lens. Exp Eye Res. 1983 Mar;36(3):317–326. doi: 10.1016/0014-4835(83)90114-8. [DOI] [PubMed] [Google Scholar]
  45. Rae J. L. The electrophysiology of the crystalline lens. Curr Top Eye Res. 1979;1:37–90. [PubMed] [Google Scholar]
  46. Revel J. P., Karnovsky M. J. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol. 1967 Jun;33(3):C7–C12. doi: 10.1083/jcb.33.3.c7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rubin J. B., Verselis V. K., Bennett M. V., Bargiello T. A. A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3820–3824. doi: 10.1073/pnas.89.9.3820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rup D. M., Veenstra R. D., Wang H. Z., Brink P. R., Beyer E. C. Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression. J Biol Chem. 1993 Jan 5;268(1):706–712. [PubMed] [Google Scholar]
  49. Schuetze S. M., Goodenough D. A. Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development. J Cell Biol. 1982 Mar;92(3):694–705. doi: 10.1083/jcb.92.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Spray D. C., Campos de Carvalho A., Bennett M. V. Sensitivity of gap junctional conductance to H ions in amphibian embryonic cells is independent of voltage sensitivity. Proc Natl Acad Sci U S A. 1986 May;83(10):3533–3536. doi: 10.1073/pnas.83.10.3533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Spray D. C., Harris A. L., Bennett M. V. Gap junctional conductance is a simple and sensitive function of intracellular pH. Science. 1981 Feb 13;211(4483):712–715. doi: 10.1126/science.6779379. [DOI] [PubMed] [Google Scholar]
  52. Spray D. C., Moreno A. P., Eghbali B., Chanson M., Fishman G. I. Gating of gap junction channels as revealed in cells stably transfected with wild type and mutant connexin cDNAs. Biophys J. 1992 Apr;62(1):48–50. doi: 10.1016/S0006-3495(92)81774-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Turin L., Warner A. Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo. Nature. 1977 Nov 3;270(5632):56–57. doi: 10.1038/270056a0. [DOI] [PubMed] [Google Scholar]
  55. Warner A. E., Lawrence P. A. Permeability of gap junctions at the segmental border in insect epidermis. Cell. 1982 Feb;28(2):243–252. doi: 10.1016/0092-8674(82)90342-7. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. White T. W., Bruzzone R., Goodenough D. A., Paul D. L. Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70. Mol Biol Cell. 1992 Jul;3(7):711–720. doi: 10.1091/mbc.3.7.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Willecke K., Heynkes R., Dahl E., Stutenkemper R., Hennemann H., Jungbluth S., Suchyna T., Nicholson B. J. Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung. J Cell Biol. 1991 Sep;114(5):1049–1057. doi: 10.1083/jcb.114.5.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yancey S. B., John S. A., Lal R., Austin B. J., Revel J. P. The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. J Cell Biol. 1989 Jun;108(6):2241–2254. doi: 10.1083/jcb.108.6.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhang J. T., Nicholson B. J. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J Cell Biol. 1989 Dec;109(6 Pt 2):3391–3401. doi: 10.1083/jcb.109.6.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zimmer D. B., Green C. R., Evans W. H., Gilula N. B. 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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