Skip to main content
SpringerLink
Log in

Axonal microtubules necessary for generation of sodium current in squid giant axons: I. pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein

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

Summary

Effects of the reagents suppressing or supporting axoplasmic microtubule assembly were studied on the Na ionic current of squid giant axons by perfusing the axon internally with the solution containing the reagent. Among the reagents suppressing the assembly, colchicine, vinblastine, podophyllotoxin, sulfhydryl reagents such as DTNB and NEM, and chaotropic anions such as iodide and bromide, were examined. These reagents reduced maximum Na conductance and shifted the voltage dependence of steady-state Na activation in a depolarizing direction along the voltage axis. They also made the voltage dependence less steep, but did not affect sodium inactivation appreciably. Effects on Na ionic current of reagents which support microtubule assembly (Taxol, DMSO, D2O and temperature) were opposite the effects of those agents suppressing assembly. At the same time, we demonstrated that after Na currents were partially reduced, they could be restored by internally perfusing the axon with a solution containing microtubule proteins, 260K proteins and cAMP under conditions favorable for microtubule assembly. For full restoration, it was found that the following conditions were necessary: (1) The microenvironment within the axon is suitable for microtubule assembly. (2) Tubulins incorporated into microtubules are fully tyrosinated at their C-termini. (3) A peripheral protein having a molecular weight of 260,000 daltons (260K protein) is indispensable. These results suggest that axoplasmic microtubules and 260K proteins in the structure underlying the axolemma play a role in generating Na currents in squid giant axons.

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

  • Adelman, W., Palti, Y. 1969. The effect of external potassium and long duration voltage conditioning in the amplitude of sodium currents in the giant axon of the squidLoligo pealei.J. Gen. Physiol. 54:589–606

    PubMed  Google Scholar 

  • Armstrong, C.M., Bezanilla, F., Rojas, E. 1973. Destruction of sodium conductance inactivation in squid axons perfused with pronase.J. Gen. Physiol. 62:375–391

    PubMed  Google Scholar 

  • Baker, P.F., Hodgkin, A.L., Shaw, T.I. 1962. Replacement of the axoplasm of giant nerve fibres with artificial solutions.J. Physiol. (London) 164:330–354

    Google Scholar 

  • Berlin, R.D., Caron, J.M., Huntley, R. 1982. New roles for tubulin membrane function.In: Biological Functions of Microtubules and Related Structures. H. Sakai, G.G. Borisy and H. Mohri, editors. pp. 405–424. Academic, Tokyo

    Google Scholar 

  • Bezanilla, F., Armstrong, C.M. 1977. Inactivation of the sodium channel. I. Sodium current experiments.J. Gen. Physiol. 70:549–566

    PubMed  Google Scholar 

  • Bezanilla, F., Taylor, R.E. 1978. Effect of temperatur on gating currents.Biophys. J. 21:85a

    Google Scholar 

  • Chandler, W.K., Meves, H. 1970. Slow changes in membrane permeability and long lasting action potentials in axons perfused with fluoride solutions.J. Physiol. (London) 211:707–728

    Google Scholar 

  • Cheung, W.Y. 1970. Cyclic 3′, 5′-nucleotide phosphodiesterase. Demonstration of an activator.Biochem. Biophys. Res. Commun. 38:533–538

    PubMed  Google Scholar 

  • Conti, F., Palmieri, G. 1968. Nerve fiber behavior in heavy water under voltage-clamp.Biophysik 5:71–77

    PubMed  Google Scholar 

  • Eaton, D.C., Brodwick, M.S. 1975. Effect of internal divalent cations on squid axon.Biophys. J. 15:41a

    Google Scholar 

  • Endo, S., Sakai, H., Matsumoto, G. 1979. Microtubules in squid giant axon.Cell Struct. Funct. 4:285–293

    Google Scholar 

  • Gillespie, J.I., Meves, H. 1980. The time course of sodium inactivation in squid giant axons.J. Physiol. (London) 299:289–307

    Google Scholar 

  • Himes, R.H., Burton, P.R., Kersey, R.N., Pierson, G.B. 1976. Brain tubulin polymerization in the absence of “microtubule-associated-proteins.”Proc. Natl. Acad. Sci. USA 73:4397–4399

    PubMed  Google Scholar 

  • Hodge, A.J., Adelman, W.J., Jr. 1980. The neuroplasmic network inLoligo andHermissenda neurons.J. Ultrastruct. Res. 70:220–241

    PubMed  Google Scholar 

  • Hodgkin, A.L., Huxley, A.F. 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve.J. Physiol. (London) 117:500–544

    Google Scholar 

  • Inoué, S., Sato, H. 1967. Cell motility by labile association of molecules. The nature of mitotic fibers and their role in chromosomal movement.J. Gen. Physiol. 50:259–292

    PubMed  Google Scholar 

  • Kakiuchi, S., Yamazaki, R. 1970. Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain. Studies on cyclic 3′, 5′-nucleotide phosphodiesterase (III).Biochem. Biophys. Res. Commun. 41:1104–1110

    PubMed  Google Scholar 

  • Katz, G.M., Schwartz, T.L. 1974. Temporal control of voltageclamped membranes: An examination of principles.J. Membrane Biol. 17:275–291

    Google Scholar 

  • Kimura, J.E., Meves, H. 1979. The effect of temperature on the asymmetrical charge movement in squid giant axons.J. Physiol. (London) 289:479–500

    Google Scholar 

  • Kobayashi, T., Matsumoto, G. 1982. Cytoplasmic tubulin from squid nerve fully retains C-terminal tyrosine.J. Biochem. 92:647–652

    PubMed  Google Scholar 

  • Kuriyama, R. 1975. Further studies on tubulin polymerizationin vitro.J. Biochem. 77:23–31

    Google Scholar 

  • Kuriyama, R., Sakai, H. 1974. Role of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization.J. Biochem. 761:651–654

    Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275

    PubMed  Google Scholar 

  • Marcum, J.M., Dedman, J.R., Brinkley, B.R., Means, R.R. 1978. Control of mictotubule assembly-disassembly by calcium-dependent regulator protein.Proc. Natl. Acad. Sci. USA 75:3771–3775

    PubMed  Google Scholar 

  • Margolis, R.L., Wilson, L. 1977. Addition of colchicine-tubulin complex to microtubule ends: the mechanism of substoichiometric colchicine poisoning.Proc. Natl. Acad. Sci. USA 74:3466–3470

    PubMed  Google Scholar 

  • Matsumoto, G. 1976. Transportation and maintenance of adult squid (Doryteuthis bleekeri) for physiological studies.Biol. Bull. 150:279–285

    PubMed  Google Scholar 

  • Matsumoto, G., Ichikawa, M., Tasaki, A. 1984. Axonal microtubules necessary for generation of sodium current in squid giant axons: II. Effect of colchicine upon asymmetrical displacement current.J. Membrane Biol. 77:93–99

    Google Scholar 

  • Matsumoto, G., Kobayashi, T., Sakai, H. 1979. Restoration of the excitability of squid giant axon by tubulin-tyrosine ligase and microtubule proteins.J. Biochem. (Tokyo) 86:1155–1158

    Google Scholar 

  • Matsumoto, G., Murofushi, H., Endo, S., Kobayashi, T., Sakai, H. 1982a. Tyrosinated tubulin necessary for maintenance of membrane excitability in squid giant axon.In: Structure and Function of Excitable Cells. D.C. Chang, I. Tasaki and W.J. Adelman, editors. pp. 471–483. Plenum, New York

    Google Scholar 

  • Matsumoto, G., Murofushi, H., Endo, S., Sakai, H. 1982b. Microtubules composed of tyrosinated tubulin are required for membrane excitability in squid giant axon.In: Biological Functions of Microtubules and Related Structures. H. Sakai, H. Mohri, and G.G. Borisy, editors. pp. 391–404. Academic, Tokyo

    Google Scholar 

  • Matsumoto, G., Murofushi, H., Sakai, H. 1980. The effects of reagents affecting microtubules and microfilaments on the excitation of the squid giant axon measured by the voltage-clamp method.Biomed. Res. 1:355–358

    Google Scholar 

  • Matsumoto, G., Sakai, H. 1979a. Microtubules inside the plasma membrane of squid giant axons and their possible physiological function.J. Membrane Biol. 50:1–14

    Google Scholar 

  • Matsumoto, G., Sakai, H. 1979b. Restoration of membrane excitability of squid giant axons by reagents activating tyrosine-tubulin ligase.J. Membrane Biol. 50:15–22

    Google Scholar 

  • Matsumoto, G., Shimada, J. 1980. Further improvement upon maintenance of adult squid (Doryteuthis bleekeri) in a small circular and closed-system aquarium tank.Biol. Bull. 159:319–324

    Google Scholar 

  • Matteson, D.R., Armstrong, C.M. 1982. Evidence for a population of sleepy sodium channels in squid axon at low temperature.J. Gen. Physiol. 79:739–758

    PubMed  Google Scholar 

  • Metuzals, J., Tasaki, I. 1978. Subaxolammal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability.J. Cell Biol. 78:597–621

    PubMed  Google Scholar 

  • Meves, H. 1974. The effect of holding potential on the asymmetry currents in squid giant axons.J. Physiol. (London) 243:847–867

    Google Scholar 

  • Murofushi, H. 1980. Purification and characterization of tubulin-tyrosine ligase from porcine brain.J. Biochem. 87:979–984

    PubMed  Google Scholar 

  • Murofushi, H., Minami, Y., Matsumoto, G., Sakai, H. 1983. Bundling of microtubulesin vitro by a high molecular weight protein prepared from the squid axon.J. Biochem. 93:639–650

    PubMed  Google Scholar 

  • Narahashi, T., Anderson, N.C., Moore, J.W. 1967. Comparison of tetrodotoxin and procaine in internally perfused squid giant axons.J. Gen. Physiol. 50:1413–1428

    PubMed  Google Scholar 

  • Ohtsubo, K., Sakai, H., Murofushi, H., Kuriyama, R. 1975. Electrophoretic separation of tubulin and subunits after S-sulfonation.J. Biochem. 77:17–21

    PubMed  Google Scholar 

  • Olmsted, J.B., Borisy, G.G. 1975. Ionic and nucleotide requirements for microtubule polymerizationin vitro.Biochemistry 14:2996–3005

    PubMed  Google Scholar 

  • Oxford, G.S., Wu, C.H., Narahashi, T. 1978. Removal of sodium channel inactivation in squid giant axons by N-bromoacetamide.J. Gen. Physiol. 71:227–247

    PubMed  Google Scholar 

  • Pant, H.C., Shecket, G., Gainer, H., Lasek, R.J. 1978. Neurofilament protein is phosphorylated in the squid giant axon.J. Cell Biol. 78:R23-R27

    PubMed  Google Scholar 

  • Rudy, B. 1978. Slow inactivation of sodium conductance in squid giant axons. Pronase resistance.J. Physiol. (London) 283:1–21

    Google Scholar 

  • Rudy, B. 1981. Slow inactivation of voltage-dependent channels.In: Nerve Membrane, Biochemistry and Function of Channel Proteins. G. Matsumoto and M. Kotani, editors. pp. 89–111. University of Tokyo Press, Tokyo

    Google Scholar 

  • Runge, M.S., Schlaepfer, W.W., Williams, R.C., Jr. 1981. Isolation and characterization of neurofilaments from mammalian brain.Biochemistry 20:170–175

    PubMed  Google Scholar 

  • Sakai, H. 1980. Regulation of microtubule assemblyin vitro.Biomed. Res. 1:359–375

    Google Scholar 

  • Sakai, H., Matsumoto, G. 1978. Tubulin and other proteins from squid giant axon.J. Biochem. 83:1413–1422

    PubMed  Google Scholar 

  • Sato, H., Takahashi, T.C., Sata, Y. 1980. Isotope effect of heavy water on spindle assembly and anaphase chromosome movement in dividing cells.Eur. J. Cell Biol. 22:310

    Google Scholar 

  • Schauf, C.L. 1973. Temperature dependence of the ionic current kinetics ofMyxicola giant axons.J. Physiol. (London) 235:197–205

    Google Scholar 

  • Schauf, C.L., Bullock, J.O. 1980. Solvent substitution as a probe of channel gating inMyxicola. Differential effects of D2O on some components of membrane conductance.Biophys. J. 30:295–305

    PubMed  Google Scholar 

  • Schiff, P.B., Fant, J., Horwitz, S.B. 1979. Promotion of microtubule assemblyin vitro by Taxol.Nature (London) 277:665–667

    Google Scholar 

  • Schiff, P.B., Horwitz, S.B. 1980. Taxol stabilizes microtubules in mouse fibroflast cells.Proc. Natl. Acad. Sci. USA 77:1561–1565

    PubMed  Google Scholar 

  • Shelanski, M.L., Gaskin, F., Cantor, C.R. 1973. Microtubule assembly in the absence of added nucleotides.Proc. Natl. Acad. Sci. USA 70:765–768

    PubMed  Google Scholar 

  • Sherline, P., Leung, J.T., Kipnis, D.M. 1976. Binding of colchicine to purified microtubule protein.J. Biol. Chem. 250:5481–5486

    Google Scholar 

  • Spudich, J.A., Watt, S. 1971. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin.J. Biol. Chem. 246:4866–4871

    Google Scholar 

  • Tasaki, I. 1968. Nerve Excitation, A Macromolecular Approach. Charles C. Thomas, Springfield, Mass.

    Google Scholar 

  • Tasaki, I., Singer, I., Takena, T. 1965. Effects of internal and external ionic environment on excitability of squid giant axon: A macromolecular approach.J. Gen. Physiol. 48:1095–1123

    PubMed  Google Scholar 

  • Terakawa, S., Nagano, M., Watanabe, A. 1978. Intracellular pH and plateau duration of internally perfused squid giant axons.Jpn. J. Physiol. 28:847–862

    PubMed  Google Scholar 

  • Vallee, R.B. 1982. A Taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs).J. Cell Biol. 92:435–442

    PubMed  Google Scholar 

  • Wallin, M., Larsson, H., Edstrom, A. 1977. Tubulin sulfhydryl groups and polymerizationin vitro. Effect of di- and trivalent cations.Exp. Cell Res. 107:219–225

    PubMed  Google Scholar 

  • Weber, K., Osborn, M. 1969. The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis.J. Biol. Chem. 244:4406–4412

    PubMed  Google Scholar 

  • Weisenberg, R.C., Borisy, G.G., Taylor, E.W. 1968. The colchicine-binding protein of mammalian brain and its relation to microtubules.Biochemistry 7:4466–4479

    PubMed  Google Scholar 

  • Wilson, L. 1970. Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyllotoxin.Biochemistry 9:4999–5007

    PubMed  Google Scholar 

  • Wilson, L., Bamburg, J.R., Mizel, S.B., Grisham, L.M., Creswell, K.M. 1974. Interaction of drugs with microtubule proteins.Fed. Proc. 33:158–166

    PubMed  Google Scholar 

  • Zackroff, R.V., Goldman, R.D. 1980.In vitro reassembly of squid brain intermediate filaments (neurofilaments): Purification by assembly-disassembly.Science 208:1152–1155

    PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Gen Matsumoto & Michinori Ichikawa

    Present address: Institute of Applied Physics, The University of Tsukuba, Tsukuba Science City, 305, Ibaraki, Japan

Authors and Affiliations

  1. Electrotechnical Laboratory, Tsukuba Science City, 305, Ibaraki, Japan

    Gen Matsumoto, Michinori Ichikawa, Akira Tasaki, Hiromu Murofushi & Hikoichi Sakai

  2. Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, 113, Tokyo, Japan

    Gen Matsumoto, Michinori Ichikawa, Akira Tasaki, Hiromu Murofushi & Hikoichi Sakai

Authors
  1. Gen Matsumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michinori Ichikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akira Tasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiromu Murofushi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hikoichi Sakai
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Matsumoto, G., Ichikawa, M., Tasaki, A. et al. Axonal microtubules necessary for generation of sodium current in squid giant axons: I. pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein. J. Membrain Biol. 77, 77–91 (1983). https://doi.org/10.1007/BF01925858

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01925858

Key Words

Search

Navigation