Skip to main content
SpringerLink
Log in

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ∼ Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., eds. 1993. Current Protocols in Molecular Biology. Vols. 1 and 2. Wiley and Sons, NY

    Google Scholar 

  • Black, R.A., Rota, P.A., Gorodkova, N., Cramer, A., Klenk, H.-D., Kendal, A.P. 1993. Production of the M2 protein of influenza A virus in insect cells is enhanced in the presence of amantadine. J. Gen. Virol. 74:1673–1677

    Google Scholar 

  • Bron, R., Kendal, A.P., Klenk, H.-D., Wilschut, J. 1993. Role of the M2 protein in influenza virus membrane fusion: effects of amantadine and monensin on fusion kinetics. Virology 195:808–811

    Article  CAS  PubMed  Google Scholar 

  • Bukrinskaya, A.G., Vorkunova, N.K., Kornilayeva, G.V., Narmanbetova, R.A., Vorkunova, G.K. 1982. Influenza virus uncoating in infected cells and effects of rimantadine. J. Gen. Virol. 60:49–59

    Google Scholar 

  • Ciampor, F., Bayley, P.M., Nermut, M.V., Hirst, E.M., Sugrue, R.J., Hay, A. 1992a. Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology 188:14–24

    Article  CAS  PubMed  Google Scholar 

  • Ciampor, F., Thompson, C.A., Grambas, S., Hay, A.J. 1992b. Regulation of pH by the M2 protein of influenza A viruses. Virus Res. 22:247–258

    Google Scholar 

  • Grambas, S., Bennett, M.S., Hay, A.J. 1992. Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. Virology 191:541–549

    Google Scholar 

  • Grambas, S., Hay, A.J. 1992. Maturation of influenza A virus hemagglutinin-estimates of the pH encountered during transport and its regulation by the M2 protein. Virology 190:11–18

    Article  CAS  PubMed  Google Scholar 

  • Duff, K.C., Ashley, R.H. 1992. The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers. Virology 190:485–489

    Google Scholar 

  • Eisenberg, M., Hall, J.E., Mead, C.E. 1973. The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes. J. Membrane Biol. 14:143–176

    Google Scholar 

  • Eisenman, G., Horn, R. 1983. Ionic selectivity revisited: The role of kinetic and equilibrium processes in ion permeation through channels. J. Membrane Biol. 76:197–225

    Google Scholar 

  • Eisenman, G., Sandblom, J.P. 1984. Modeling the gramicidin channel. Interpretation of experimental data using rate theory. Biophys. J. 45:88–90

    Google Scholar 

  • Goldman, D. 1943. Potential, impedance and rectification in membranes. J. Gen. Physiol. 27:37–60

    Google Scholar 

  • Hay, A.J. 1992. The action of adamantanamines against influenza A viruses: inhibition of the M2 ion channel protein. Sem. Virol. 3:21–30

    Google Scholar 

  • Hay, A.J., Wolstenholme, A., Skehel, J.J., Smith, M.H. 1985. The molecular basis of the specific anti-influenza action of amantadine. EMBO J. 4:3021–3024

    Google Scholar 

  • Helenius, A. 1992. Unpacking the incoming influenza virus. Cell 69:577–578

    Google Scholar 

  • Hille, B. 1992. Ionic channels of excitable membranes. Sinauer Associates, Sunderland, MA

    Google Scholar 

  • Hodgkin, A.L., Katz, B. 1949. The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108:37–77

    Google Scholar 

  • Holsinger, L.J., Lamb, R.A. 1991. Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds. Virology 183:32–43

    CAS  PubMed  Google Scholar 

  • Holsinger, L.J., Nichani, D., Pinto, L.H., Lamb, R.A. 1994. Influenza A virus M2 protein: a structure-function analysis. J. Virol. 68:1551–1563

    Google Scholar 

  • Hunter, M., Giebisch, G. 1987. Multi-barrelled K channels in renal tubules. Nature 327:522–524

    Google Scholar 

  • Krouse, M.E., Schneider, G.T., Gage, P.W. 1986. A large anion channel has seven conductance levels. Nature 319:58–60

    Google Scholar 

  • Lamb, R.A. 1989. Genes and proteins of the influenza viruses. In: The Influenza Viruses. (R.M. Krug, editor), pp. 1–87. Plenum Press, NY

    Google Scholar 

  • Lamb, R.A., Choppin, P.W. 1979. Segment 8 of the influenza virus genome is unique in coding for two polypeptides. Proc. Natl. Acad. Sci. USA 76:4908–4912

    Google Scholar 

  • Lamb, R.A., Etkind, P.R., Choppin, P.W. 1978. Evidence for a ninth influenza viral polypeptide. Virology 91:60–78

    Google Scholar 

  • Lamb, R.A., Lai, C.-J., Choppin, P.W. 1981. Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted mRNAs code for overlapping proteins. Proc. Natl. Acad. Sci. USA 78:4170–4174

    Google Scholar 

  • Lamb, R.A., Zebedee, S.L., Richardson, C.D. 1985. Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface. Cell 40:627–633

    Article  CAS  PubMed  Google Scholar 

  • Marsh, M. 1992. Keeping the viral coat on. Curr. Biol. 2:379–381

    Google Scholar 

  • Martin, K., and Helenius, A. 1991. Nuclear transport of influenza virus ribonucleoproteins: The viral matrix protein (M1) promotes export and inhibits import. Cell 67:117–130

    Google Scholar 

  • Miller, C. 1982. Open-state substructure of single chloride channels from Torpedo electroplax. Philos. Trans. R. Soc. (Land.) Ser. B 299:401–411

    Google Scholar 

  • Neher, E., Steinback, J.H. 1978. Local anesthetics transiently block currents through single acetylcholine-receptor channels. J. Physiol. 277:153–176

    CAS  PubMed  Google Scholar 

  • Panayotov, P.P., Schlesinger, R.W. 1992. Oligomeric organization and strain-specific proteolytic modification of the virion M2 protein of influenza A H1N1 viruses. Virology 186:352–355

    Google Scholar 

  • Pinto, L.H., Holsinger, L.J., Lamb, R.A. 1992. Influenza virus M2 protein has ion channel activity. Cell 69:517–528

    Article  CAS  PubMed  Google Scholar 

  • Skehel, J.J. 1992. Influenza virus. Amantadine blocks the channel [news]. Nature 358:110–111

    Google Scholar 

  • Skehel, J.J., Hay, A.J., Armstrong, J.A. 1978. On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride. J. Gen. Virol. 38:97–110

    Google Scholar 

  • Sugrue, R.J., Bahadur, G., Zambon, M.C., Hall-Smith, M., Douglas, A.R., Hay, A.J. 1990. Specific structural alteration of the influenza hemagglutinin by amantadine. EMBO J. 9:3469–3476

    Google Scholar 

  • Sugrue, R.J., Hay, A.J. 1991. Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180:617–624

    Article  CAS  PubMed  Google Scholar 

  • Tosteson, M.T., Nibert, M.L., Fields, B.N. 1993. Ion channels induced in lipid bilayers by subvirion particles of the nonenveloped mammalian reoviruses. Proc. Natl. Acad. Sci. USA 90:10549–10552

    Google Scholar 

  • Wang, C., Takeuchi, K., Pinto, L.H., Lamb, R.A. 1993. The ion channel activity of the influenza A virus M2 protein: characterization of the amantadine block. J. Virol. 67:5585–5594

    Google Scholar 

  • Wharton, S.A., Belshe, R.B., Skehel, J.J., Hay, A.J. 1994. Role of virion M2 protein in influenza virus uncoating: specific reduction in the rate of membrane fusion between virus and liposomes by amantadine. J. Gen. Virol. 75:945–948

    Google Scholar 

  • Wiley, D.C., Skehel, J.J. 1987. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56:365–394

    Google Scholar 

  • Zebedee, S.L., Lamb, R.A. 1988. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J. Virol. 62:2762–2772

    Google Scholar 

  • Zebedee, S.L., Richardson, C.D., Lamb, R.A. 1985. Characterization of the influenza virus M2 integral membrane protein and expression at the infected-cell surface from cloned cDNA. J. Virol. 56:502–511

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory for Membrane Transport, 240 Longwood Avenue, Harvard Medical School, 02115, Boston, Massachusetts

    M. T. Tosteson

  2. Department of Neurobiology and Physiology, Northwestern University, 60208-3500, Evanston, Illinois

    L. H. Pinto

  3. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 60208-3500, Evanston, Illinois

    L. J. Holsinger & R. A. Lamb

  4. Howard Hughes Medical Institute, Northwestern University, 60208-3500, Evanston, Illinois

    R. A. Lamb

Authors
  1. M. T. Tosteson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. H. Pinto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L. J. Holsinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. A. Lamb
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

The authors would like to acknowledge very helpful comments on the manuscript by Drs. Daniel C. Tosteson, Jose A. Halperin, John Collier and Roderick MacKinnon.

Research in the authors' laboratories was supported by Public Health Service research grants AI-20201 (R.A.L) and AI-31882 (L.H.P) from the National Institute of Allergy and Infectious Diseases. R.A.L. is an Investigator of the Howard Hughes Medical Institute.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tosteson, M.T., Pinto, L.H., Holsinger, L.J. et al. Reconstitution of the influenza virus M2 ion channel in lipid bilayers. J. Membarin Biol. 142, 117–126 (1994). https://doi.org/10.1007/BF00233389

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00233389

Key words

Search

Navigation