The Journal of Membrane Biology

, Volume 124, Issue 1, pp 21–32 | Cite as

Channel reconstitution in liposomes and planar bilayers with HPLC-purified MIP26 of bovine lens

  • Lili Shen
  • Peter Shrager
  • Stephen J. Girsch
  • Patricia J. Donaldson
  • Camillo Peracchia


The major intrinsic protein (MIP26) of bovine lens membranes, purified by HPLC, was incorporated into liposomes and planar bilayers. Permeability of MIP26 channels was studied in liposomes by a spectrophotometric osmotic-swelling assay, and channel electrical properties were monitored in planar bilayers following liposome fusion. Particle formation in liposomes was determined by freeze fracture. MIP26 channels were permeable to KCl and sucrose. In planar bilayers, channel-conductance transitions were observed only after addition of liposomes to both chambers and with voltages greater than ±20 mV. Channel open probability decreased progressively as voltage increased, and an open probability of 50% was at 60–80 mV, indicating that the channels are voltage dependent. Histograms of single-channel current amplitudes at 80 mV showed a Gaussian distribution that peaked at 10 pA (∼120 pS), after subtraction of 1 pA baseline current. Frequency distributions of open and closed times at 80 mV were single exponential functions with time constants of 0.13 and 1.9 sec, respectively. Open time constants ranged from 0.1 to 0.3 sec, and closed time constants ranged from 1 to 7 sec. Cs+ did not decrease conductance, but reduced mean open time from 0.2 to 0.038 sec and mean closed time from 1.5 to 0.38 sec. The increase in channel flickering with Cs+ occurred in bursts. TEA affected neither conductance nor kinetics. Channel events were also observed in Na+ solutions (zero K+). These data indicate that MIP26 channels are not K+-selective channels. Channel characteristics such as: permeability to molecules larger than small ions, conductance greater than 100 pS, long open and closed time constants, etc., are similar to those of gap junction channels.

Key Words

lens MIP26 planar bilayers reconstitution single-channel recording gap junctions ion channels 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alvarez, O. 1986. How to set up a bilayer system.In: Ion Channel Reconstitution C. Miller, editor. pp. 115–130. Plenum New YorkGoogle Scholar
  2. Benedetti, E.L., Dunia, I., Bentzel, C.J., Vermorken, A.J.M., Kibbelaar, M., Bloemendal, H. 1976. A portrait of plasma membrane specializations in eye lens epithelium and fibers.Biochim. Biophys. Acta,457:353–384Google Scholar
  3. Bernardini, G., Peracchia, C. 1981. Gap junction crystallization in lens fibers after an increase in cell calcium.Invest. Ophthalmol. Vis. Sci. 21:291–299Google Scholar
  4. Beyer, E.C., Paul, D.L., Goodenough, D.A. 1990. Connexin family of gap junction proteins.J. Membrane Biol. 116:187–194Google Scholar
  5. Bok, D., Dockstader, J., Horwitz, J. 1982. Immunocytochemical localization of the lens main intrinsic polypeptide (MIP26) in communicating junctions.J. Cell Biol. 92:213–220Google Scholar
  6. Brewer, G.J. 1991. Reconstitution of lens channels between two membranes.In: Biophysics of Gap Junction Channels. C. Peracchia, editor. pp. 301–316. CRC Press, Boca Raton (FL)Google Scholar
  7. Brewer, G.J., Dong, R.G. 1990. Transjunctional lens channels reconstituted: Regulation by Ca++ and calmodulin.J. Cell Biol. 111:65aGoogle Scholar
  8. Brito, R.M.M., Vaz, L.C. 1986. Determination of the critical micelle concentration of surfactants using the fluorescent probeN-phenyl-1-naphthylamine.Anal. Biochem. 152:250–255Google Scholar
  9. Colquhoun, D., Hawkes, A.G. 1983. The principles of the stochastic interpretation of ion-channel mechanisms.In: Single-Channel Recording. B. Sakmann and E. Neher, editors. pp. 135–175, Plenum, New YorkGoogle Scholar
  10. Dunia, I., Manenti, S., Rousselet, A., Benedetti, E.L. 1987. Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.J. Cell Biol. 105:1679–1689Google Scholar
  11. Ehring, G.R., Hall, J.E. 1991. Reconstitution into planar lipid bilayers: Application to the study of lens MIP.In: Biophysics of Gap Junction Channels. C. Peracchia, editor. pp. 333–351. CRC Press, Boca Raton (FL)Google Scholar
  12. Ehring, G.R., Zampighi, G., Horwitz, J., Bok, D., Hall, J.E. 1990. Properties of channel reconstituted from the major intrinsic protein of lens fiber membranes.J. Gen. Physiol. 96:631–664Google Scholar
  13. Fitzgerald, P.G., Bok, D., Horwitz, J. 1983. Immunocytochemical localization of the main intrinsic polypeptide (MIP) in ultrathin frozen sections of rat lens.J. Cell Biol. 97:1491–1499Google Scholar
  14. Gandolfi, S.A., Duncan, G., Tomlinson, J., Maraini, G. 1990. Mammalian lens inter-fiber resistance is modulated by calcium and calmodulin.Curr. Eye Res. 9:533–541Google Scholar
  15. Girsch, S.J. 1988. Purification of lens gap junction protein by reverse-phase HPLC. p. 125. Proc. VIII Int. Congr. Eye Research, San FranciscoGoogle Scholar
  16. Girsch, S.J., Peracchia, C. 1985. Lens cell-to-cell channel protein: I. Self-assembly into liposomes and permeability regulation by calmodulin.J. Membrane Biol. 83:217–225Google Scholar
  17. Girsch, S.J., Peracchia, C. 1991. Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26.Curr. Eye Res. (in press)Google Scholar
  18. Gooden, M.M., Rintoul, D.A., Takehana, M., Takemoto, L. 1985. Major intrinsic polypeptide (MIP26) from lens membrane: Reconstitution into vesicles and inhibition of channel forming activity by peptide antiserum.Biochem. Biophys. Res. Commun. 128:993–999Google Scholar
  19. Gorin, M.B., Yancey, S.B., Cline, J., Revel, J.P., Horwitz, J. 1984. The major intrinsic protein (MIP) of the bovine lens fiber membrane: characterization and structure based on cDNA cloning.Cell 39:49–59Google Scholar
  20. Gruijters, W.T.M., Kistler, J., Bullivant, S., Goodenough, D.A. 1987. Immunologicalization of a MP7O in lens fiber 16–17 nm intercellular junctions.J. Cell Biol. 104:565–572Google Scholar
  21. Harris, A.L. 1991. Connexin32 forms ion channels in single artificial membranes.In: Biophysics of Gap Junction Channels. C. Peracchia, editor. pp. 373–389. CRC Press Boca Raton, (FL)Google Scholar
  22. Hertzberg, E.L., Gilula, N.B. 1981. Liver gap junctions and lens fiber junctions: Comparative analysis and calmodulin interaction.Cold Spring Harbor Symp. Quant. Biol. 46:639–645Google Scholar
  23. Johnson, R.G., Klukas, K.A., Tze-Hong, L., Spray, D.C. 1988. Antibodies to MP26 are localized to lens junctions, alter intercellular permeability, and demonstrate increased expression during development.In: Gap Junctions. E.L. Hertzberg and R.G. Johnson, editors. pp. 81–98. Alan R. Liss, New YorkGoogle Scholar
  24. Kistler, J., Bullivant, S. 1987. Protein processing in lens intercellular junctions: Cleavage of MP70 to MP38.Invest. Ophthalmol. Vis. Sci. 28:1687–1692Google Scholar
  25. Kistler, J., Christie, D., Bullivant, S. 1988. Homologies between gap junction proteins in lens, heart and liver.Nature 331:721–723Google Scholar
  26. Kistler, J., Kirkland, B., Bullivant, S. 1985. Identification of a 70,000-D protein in lens membrane junctional domains.J. Cell Biol. 101:28–35Google Scholar
  27. Lasalde, J.A., Zuazaga, C. 1991. Cholesterol enrichment decreases the conductance of nicotinic acetylcholine receptor channels in tissue cultured chick muscle.Biophys. J. 59: 444aGoogle Scholar
  28. Lea, J.A., Duncan, G. 1991. Lens cell communication—from the whole organ to single channels.In: Biophysics of Gap Junction Channels. C. Peracchia, editor. pp. 353–371, CRC Press, Boca Raton (FL)Google Scholar
  29. Louis, C.F., Hogan, P., Visco, L., Strasburg, G. 1990. Identity of the calmodulin-binding proteins in bovine lens plasma membrane.Exp. Eye Res. 50:495–503Google Scholar
  30. Luckey, M., Nikaido, H. 1980. Specificity of diffusion channels produced by λ phage receptor protein ofEscherichia coli.Proc. Natl. Acad. Sci. U.S.A. 77:167–171Google Scholar
  31. Malewicz, B., Kumar, V.V., Johnson, R.G., Baumann, W.J. 1990. Lipids in gap junction assembly and function.Lipids 25:419–427Google Scholar
  32. Mueller, P., Rudin, D., Tien, H.T., Wescott, W.C. 1962. Reconstitution of cell membrane structure in vitro and its transformation into an excitable system.Nature 194:979–980Google Scholar
  33. Nikaido, H., Rosenberg, E.Y. 1985. Functional reconstitution of lens gap junction proteins into proteoliposomes.J. Membrane Biol. 85:87–92Google Scholar
  34. Paul, D.L., Goodenough, D.A. 1983. Preparation, characterization and localization of antisera against bovine MP26 an integral protein from the lens fiber plasma membrane.J. Cell Biol. 96:625–632Google Scholar
  35. Peracchia, C. 1978. Calcium effects on gap junction structure and cell coupling.Nature 271:669–671Google Scholar
  36. Peracchia, C. 1988. The calmodulin hypothesis for gap junction regulation six years laterIn: Gap Junctions. E.L. Hertzberg and R.G. Johnson, editors. pp. 267–282. Alan R. Liss, New YorkGoogle Scholar
  37. Peracchia, S., Girsch, S.J. 1985. Is the cleavable C-terminal arm of cell-to-cell channel protein the channel gate?Biochem. Biophys. Res. Commun. 133:688–695Google Scholar
  38. Peracchia, C., Girsch, S.J. 1989. Calmodulin site at the C-terminus of the putative lens gap junction protein MIP26.Lens Eye Toxic. Res. 6:613–621Google Scholar
  39. Peracchia, C., Peracchia, L.L. 1980a. Gap junction dynamics: reversible effects of divalent cations.J. Cell Biol. 87:708–718Google Scholar
  40. Peracchia, C., Peracchia, L.L. 1980b. Gap junction dynamics: Reversible effects of hydrogen ions.J. Cell Biol. 87:719–727Google Scholar
  41. Sas, D.F., Sas, J., Johnson, K.R., Menko, A.S., Johnson, R.G. 1985. Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26.J. Cell Biol. 100:216–225Google Scholar
  42. Shen, L., Shrager, P., Girsch, S., Donaldson, P., Peracchia, C. 1991. Functional reconstitution of HPLC-purified lens channel protein MIP26 in liposomes and planar bilayers.Biophys J. 59:438aGoogle Scholar
  43. Spray, D.C., Saez, J.C., Brosius, D., Bennett, M.V.L., Hertzberg E.L. 1986. Isolated liver gap junctions: Gating of transjunctional currents is similar to that in intact pairs of rat hepatocytes.Proc. Natl. Acad. Sci USA 83:5494–5497Google Scholar
  44. Vallon, O., Dunia, I., Favard-Sereno, C., Hoebeke, J., Benedetti, E.L. 1985. MP26 in the bovine lens: A post-embedding immunocytochemical study.Biol. Cell 53:85–88Google Scholar
  45. Van den Eijden-van Raaij, A.J.M., de Leeuw, A.L.M., Broekhuyse, R.M. 1985. Bovine lens calmodulin. Isolation, partial characterization and calcium-independent binding to lens membrane proteins.Curr. Eye Res. 4:905–912Google Scholar
  46. Van Eldick, L.J., Hertzberg, E.L., Berdan, R.C., Gilula, N.B. 1985. Interaction of calmodulin and other calcium-modulated proteins with mammalian and arthropod junctional membrane proteins.Biochem. Biophys. Res Commun. 126:825–832Google Scholar
  47. Welsh, M.J., Aster, J.C., Ireland, M., Alcala, J., Maisel, H. 1982. Calmodulin binds to chick lens gap junction protein in a calcium-independent manner.Science 216:642–644Google Scholar
  48. Yellen, G. 1984. Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells.J. Gen. Physiol. 84:157–186Google Scholar
  49. Young, D.-L., Cohen, Z.A., Gilula, N.B. 1987. Functional assembly of gap junction conductance in lipid bilayers: Demonstration that the major 27 kDa protein forms the junctional channel.Cell 48:733–743Google Scholar
  50. Zampighi, G., Hall, J.E., Ehring, G.R., Simon, S.A. 1989. The structural organization and protein composition of lens fiber junctions.J. Cell Biol. 108:2255–2275Google Scholar
  51. Zampighi, G.A., Hall, J.E., Kreman, M. 1985. Purified lens junctional protein forms channels in planar lipid films.Proc. Natl. Acad. Sci USA 82:8468–8472Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1991

Authors and Affiliations

  • Lili Shen
    • 1
  • Peter Shrager
    • 1
  • Stephen J. Girsch
    • 1
  • Patricia J. Donaldson
    • 1
  • Camillo Peracchia
    • 1
  1. 1.Department of PhysiologyUniversity of RochesterRochester

Personalised recommendations