Summary
The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.
Similar content being viewed by others
References
Agnew, W.S., Levinson, S.R., Brabson, J.S., Raftery, M.A. 1978. Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel fromElectrophorus electricus electroplax membranes.Proc. Natl. Acad. Sci. USA 75:2606–2610
Agnew, W.S., Moore, A.C., Levinson, S.R., Raftery, M.A. 1981. Biochemical characterization of a voltage-sensitive sodium channel protein from the electroplax ofElectrophorus electricus.In: Nerve Membrane: Biochemistry and Function of Channel Proteins. G. Matsumoto and M. Kotani, editors. pp. 25–44. University of Tokyo, Tokyo
Aurora, T.S., Li, W., Cummins, H.Z., Haines, T.H. 1985. Preparation and characterization of monodisperse unilamellar phospholipid vesicles with selected diameters of from 300 to 600 nm.Biochim. Biophys. Acta 820:250–258
Catterall, W.A. 1980. Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes.Annu. Rev. Pharmacol. Toxicol. 20:15–43
Chandler, W.K., Meves, H. 1970a. Sodium and potassium currents in squid giant axons perfused with fluoride solution.J. Physiol. (London) 211:623–652
Chandler, W.K., Meves, H. 1970b. Evidence for two types of sodium conductance in axons perfused with sodium, fluoride solution.J. Physiol. (London) 211:653–678
Chandler, W.K., Meves, H. 1970c. Rate constants associated with changes in axons perfused with sodium fluoride.J. Physiol. (London) 211:679–705
Duch, D.S. 1986. Reconstitution and functional characterization of the voltage-dependent sodium channel from eel electroplax. Thesis dissertation. University of Colorado Health Sciences Center, Denver
Duch, D.S., Levinson, S.R. 1985. Purification and reconstitution of the single, large tetrodotoxin-binding polypeptide from eel electroplax using CHAPS detergent buffers.Biophys. J. 47:192a
Duch, D.S., Levinson, S.R. 1987. Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles.J. Membrane Biol. 98:43–55
Epstein, M., Racker, E. 1978. Reconstitution of carbamylcholine dependent sodium ion flux and desensitization of the acetylcholine receptor fromTorpedo californica J. Biol. Chem. 19:6660–6662
Erdahl, W.L., Stolywho, A., Privett, O.S. 1973. Analysis of soybean lecithin by thin layer and analytical chromatography.J. Am. Oil Chem. Soc. 50:513–515
Garber, S.S., Miller, C. 1987 Single sodium channels activated by veratridine and batrachotoxin.J. Gen. Physiol. 89:459–480
Green, W.N., Weiss, L.B., Andersen, O.S. 1984. Batrachotoxin-modified sodium channels in lipid bilayers.Am., N.Y. Acad. Sci. 435:548–550
Hartshorne, R.P., Keller, B.U., Talvenheimo, J.A., Catterall, W.A., Montal, M. 1985. Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.Proc. Natl. Acad. Sci. USA 82:240–244
Hille, B. 1984. Ionic channels of excitable membranes. Sinauer Associates, Sunderland, Mass.
Kagawa, Y., Racker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation.J. Biol. Chem. 246:5477–5487
Krueger, B.K., Worley, J.F., French, R.J. 1983. Single sodium channels from rat brain incorporated into planar lipid bilayer membranes.Nature (London) 303:172–175
Kunze, D.L., Lacerda, D.L., Wilson, D.L., Brown, A.M. 1985. Cardiac Na currents and the inactivating, reopening and waiting properties of single cardiac Na channels.J. Gen. Physiol. 86:691–719
Lehmann-Horn, F., Rudel, R., Rickler, K., Lorkovic, H., Dengler, R., Hopf, H.C. 1983. Two cases of adynamia episodica hereditaria: in vitro investigation of muscle, cell membrane and contraction parameters.Muscle Nerve 6:113–121
Levinson, S.R. 1975. The purity of tritiated tetrodotoxin as determined by bioassay.Phil. Trans. R. Soc. London B 270:337–348
Levinson, S.R., Duch, D.S., Urban, B.W., Recio-Pinto, E. 1986b. The sodium channel fromElectrophorus electricus.Ann. N. Y. Acad. Sci. 479:162–178
Miller, J.A., Agnew, W.S., Levinson, S.R. 1983. Principle glycopeptide of the tetrodotoxin/saxitoxin binding protein fromElectrophorus electricus: Isolation and partial characterization.Biochemistry 22:462–470
Moczydlowski, E., Garber, S.S., Miller, C. 1984. Batrachotoxin-activated Na channels in planar lipid bilayers.J. Gen. Physiol. 84:665–686
Patlak, J.B., Ortiz, M. 1986. Two modes of gating during late Na+ channel currents in frog sartorius muscle.J. Gen. Physiol. 87:305–326
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
Rosenberg, R.L., Tomiko, S.A., Agnew, W.S. 1984b. Single-channel properties of the reconstituted voltage-regulated Na channel isolated from the electroplax ofElectroplax electricus.Proc. Natl. Acad. Sci. USA 81:5594–5598
Shoukimas, J.J., French, R.J. 1980. Incomplete inactivation of sodium currents in nonperfused squid axons.Biophys. J. 32:857–862
Talvenheimo, J.A., Tamkun, M.M., Catterall, W.A. 1982. Reconstitution of neurotoxin-stimulated sodium transport by the volgtage-sensitive sodium channel purified from rat brain.J. Biol. Chem. 257:11868–11871
Tamkun, M.M., Talvenheimo, J.A., Catterall, W.A. 1984. The sodium channel from rat brain. Reconstitution of neurotoxin-activated ion flux and scorpion-toxin binding from purified components.J. Biol. Chem. 259:1676–1688
Tanaka, J.C., Eccleston, J.F., Barchi, R.L. 1983. Cation selectivity characteristics of the reconstituted sodium channel purified from rat skeletal muscle sarcolemma.J. Biol. Chem. 258:7519–7526
Tanford, C. 1980. The Hydrophobic Effect. J. Wiley & Sons, New York
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
Weigele, J.B., Barchi, R.L. 1982. Functional reconstitution of the purified sodium channel protein from rat sarcolemma.Proc. Natl. Acad. Sci. USA 79:3651–3655
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Duch, D.S., Rock Levinson, S. Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles. J. Membrain Biol. 98, 57–68 (1987). https://doi.org/10.1007/BF01871045
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01871045