Skip to main content
SpringerLink
Log in

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine, N-bromoacetamide, and N-bromosuccinimide

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

Summary

We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.

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.

Similar content being viewed by others

References

  • Agnew, W.S., Cooper, E.C., James, W.M., Tomiko, S.A., Rosenberg, R.L., Emerick, M.C., Correa, A.M., Zhou, J.Y. 1988. Voltage sensitive sodium channels: Molecular structure and function.In: The Molecular Biology of Ion Channels. W.S. Agnew, T. Claudio, and F.J. Sigworth, editors. pp. 329–365. Academic, New York

    Google Scholar 

  • Agnew, W.S., Tomiko, S.A., Rosenberg, R.L. 1986. Reconstitution of the sodium channel fromElectrophorus electicus.In: Ion Channel Reconstitution. C. Miller, editor. pp. 307–322. Plenum, New York

    Google Scholar 

  • Armstrong, C.M., Bezanilla, F. 1977. Inactivation of the sodium channel: II. Gating current experiments.J. Gen. Physiol. 70:567–590

    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

    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 

  • Cahalan, M.D. 1978. Local anesthetic block of sodium channels in normal and pronase treated squid giant axons.Biophys. J. 23:285–311

    PubMed  Google Scholar 

  • Cahalan, M.D., Almers, W. 1979. Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin.Biophys. J. 27:39–56

    Google Scholar 

  • Cahalan, M., Shapiro, B.I., Almers, W. 1980. Relationship between inactivation of sodium channels and block by quaternary derivatives of local anesthetics and other compounds.In: Progress in Anesthesiology, Vol. 2, Molecular Mechanisms of Anesthesia. B.R. Fink, editor. pp. 17–33. Raven, New York

    Google Scholar 

  • Cooper, E.C., Tomiko, S.A., Agnew, W.S. 1987. The reconstituted voltage sensitive sodium channel fromElectrophorus electricus: Chemical modifications that alter regulation of ion permeability.Proc. Natl. Acad. Sci. USA 84:6282–6286

    PubMed  Google Scholar 

  • Duch, D.S., Levinson, S.R. 1987. Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles.J. Membrane Biol. 98:57–68

    Google Scholar 

  • Green, W.N., Weiss, L.B., Anderson, O.S. 1987. Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block.J. Gen. Physiol. 89:841–872

    Google Scholar 

  • Hille, B. 1977. Local anesthetics: Hydrophilic and hydrophobic pathways for the drug-receptor reaction.J. Gen. Physiol. 69:497–515

    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 

  • Horn, R., Vandenberg, C.A., Lange, K. 1984. Statistical analysis of single sodium channels: Effects of N-bromoacetamide.Biophys. J. 45:323–335

    PubMed  Google Scholar 

  • Huang, J.M.C., Tanguy, J., Yeh, J.Z. 1987. Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.Biophys. J. 52:155–163

    PubMed  Google Scholar 

  • James, W., Emerick, M., Agnew, W. S. 1989. Affinity purification of the voltage-sensitive sodium channel fromE. electricus. Biochemistry (in press)

  • Khodorov, B.I. 1978. Chemicals as tools to study nerve fiber sodium channels: Effects of batrachotoxin and some local anesthetics.In: Membrane Transport Processes. Vol. 2. pp. 153–174. D.C. Tosteson, Y.A. Ovchinnikov, and R. Latorre, editors. Raven, New York

    Google Scholar 

  • Miller, C. (Editor) 1986 Ion Channel Reconstitution. Plenum, New York

    Google Scholar 

  • Moczydlowski, E., Uehara, A., Hall, S. 1986. Blocking pharmacology of batrachotoxin activated sodium channels.In: Ion Channel Reconstitution. C. Miller, editor. pp. 405–428. Plenum, New York

    Google Scholar 

  • Oxford, G.S., Yeh, J.Z. 1979. Interference with sodium inactivation gating in quid axons by internal monovalent cations.Biophys. J. 25:195a

    Google Scholar 

  • Oxford, G.S., Yeh, J.Z. 1985. Interactions of monovalent cations with sodium channels in squid axon: I. Modification of physiological inactivation gating.J. Gen. Physiol. 85:583–602

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  • Patlak, J., Horn, R. 1982. Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches.J. Gen. Physiol. 79:333–351

    Google Scholar 

  • Patkak, J., Ortiz, M. 1985. Slow currents through single sodium channels in adult rat heart.J. Gen. Physiol. 86:89–104

    PubMed  Google Scholar 

  • Patlak, J., Ortiz, M. 1986. Two modes of gating during late Na+ channel currents in frog sartorius muscle.J. Gen., Physiol. 87:305–326

    Google Scholar 

  • Ramachandran, L.K., Witkop, B. 1967. N-bromosuccinimide cleavage of peptides.Methods Enzymol. 11:283–299

    Google Scholar 

  • Rojas, E., Rudy, B. 1976. Destruction of the sodium current inactivation by a specific protease in perfused nerve fibers fromLoligo.J. Physiol. (London) 262:501–531

    Google Scholar 

  • Rosenberg, R.L., Tomiko, S.A., Agnew, W.S. 1984a. Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax ofElectrophorus electricus.Proc. Natl. Acad. Sci. USA 81:1239–1243

    PubMed  Google Scholar 

  • Rosenberg, R.L., Tomiko, S.A., Agnew, W.S. 1984b. Singlechannel properties of the reconstituted voltage-regulated Na channel isolated form the electroplax ofElectrophorus electricus.Proc. Natl. Acad. Sci. USA 81:5594–5598

    Google Scholar 

  • Salgado, V.L., Yeh, J.Z., Narahashi, T. 1985. Voltage-dependent removal of sodium inactivation by N-bromoacetamide and pronase.Biophys. J. 47:567–571

    PubMed  Google Scholar 

  • Shechter, Y., Patchornik, A., Burnstein, Y. 1976. Selective chemical cleavage of tryptophanyl peptide bonds by oxidative chlorination with N-chlorosuccinimide.Biochemistry 15:5071–5075

    PubMed  Google Scholar 

  • Shenkel, S., Cooper, E.C., Agnew, W.S., Sigworth, F.J. 1989. Purified, modified sodium channels are active in planar bilayers in the absence of hatrachotoxin.Biophys. J. 55:230 (Abstr.)

    Google Scholar 

  • Shenkel, S., Cooper, E.C., James, W.M., Agnew, W.S., Sigworth, F.J. 1989. Purified, modified voltage-sensitive sodium channels are active in planar bilayers in the absence of neurotoxin.Proc. Natl. Acad. Sci. USA (in press).

  • Shoukimas, J.J., French, R.J. 1980. Incomplete inactivation of sodium currents in nonperfused squid axon.Biophys. J. 32:857–862

    PubMed  Google Scholar 

  • Spande, T.F., Greene, N.M., Witkop, B. 1966. The reactivity towards N-bromosuccimimide of tryptophan in enzymes, zymogens, and inhibited enzymes.Biochemistry 5:1926–1932

    PubMed  Google Scholar 

  • Strichartz, G.R. 1973. The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine.J. Gen. Physiol. 62:37–57

    Google Scholar 

  • Tomiko, S.A., Rosenberg, R.L., Emerick, M.C., Agnew, W.S. 1986. Fluorescence assay for neurotoxin modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ.Biochemistry 25:2162–2174

    PubMed  Google Scholar 

  • Wang, G.K., Brodwick, M.S., Eaton, D.C., Strichartz, G.R. 1987. Inhibition of sodium currents by local anesthetics in chloramine-T-treated squid axons. The role of channel activation.J. Gen. Physiol. 89:645–667

    PubMed  Google Scholar 

  • Witkop, B. 1961. Non-enzymatic methods for the preferential and selective cleavage and modification of proteins.Adv. Prot. Chem. 16:221–322

    Google Scholar 

  • Yeh, J.Z. 1978. Sodium inactivation mechanism modulates QX-314 block of sodium channels in squid axons.Biophys. J. 24:569–574

    PubMed  Google Scholar 

  • Yeh, J.Z., McCarthy, W.A., Quant, F.N., Yamamoto, D. 1986. Single channel analysis of the action of Na channel blockers 9-aminoacridine and QX-314 in neuroblastoma cells.In: Molecular Mechanisms of Anesthesia. S.H. Roth and K.W. Miller, editors. pp. 227–241. Plenum, New York

    Google Scholar 

  • Yeh, J.Z., Narahashi, T. 1977. Kinetic analysis of pancuronium interaction with sodium channels in squid axon membranes.J. Gen. Physiol. 69:293–323

    PubMed  Google Scholar 

  • Yeh, J.Z., Tanguy, J. 1985. Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons.Biophys. J. 47:685–694

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cellular and Molecular Physiology, Yale University School of Medicine, 06510, New Haven, Connecticut

    Edward C. Cooper & William S. Agnew

Authors
  1. Edward C. Cooper
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William S. Agnew
    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

Cooper, E.C., Agnew, W.S. Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine, N-bromoacetamide, and N-bromosuccinimide. J. Membrain Biol. 111, 253–264 (1989). https://doi.org/10.1007/BF01871010

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01871010

Key Words

Search

Navigation