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. 1992 Jun 1;99(6):897–930. doi: 10.1085/jgp.99.6.897

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers

PMCID: PMC2216628  PMID: 1322451

Abstract

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non- Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX- modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.

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Selected References

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  1. Barnes S., Hille B. Veratridine modifies open sodium channels. J Gen Physiol. 1988 Mar;91(3):421–443. doi: 10.1085/jgp.91.3.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnola F. V., Villegas R. Sodium flux through the sodium channels of axon membrane fragments isolated from lobster nerves. J Gen Physiol. 1976 Jan;67(1):81–90. doi: 10.1085/jgp.67.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behrens M. I., Oberhauser A., Bezanilla F., Latorre R. Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties. J Gen Physiol. 1989 Jan;93(1):23–41. doi: 10.1085/jgp.93.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chabala L. D., Urban B. W., Weiss L. B., Green W. N., Andersen O. S. Steady-state gating of batrachotoxin-modified sodium channels. Variability and electrolyte-dependent modulation. J Gen Physiol. 1991 Jul;98(1):197–224. doi: 10.1085/jgp.98.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Corbett A. M., Krueger B. K. Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers. J Membr Biol. 1989 Sep;110(3):199–207. doi: 10.1007/BF01869150. [DOI] [PubMed] [Google Scholar]
  6. Correa A. M., Latorre R., Bezanilla F. Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon. J Gen Physiol. 1991 Mar;97(3):605–625. doi: 10.1085/jgp.97.3.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Correa A. M., Villegas G. M., Villegas R. Anemone toxin II receptor site of the lobster nerve sodium channel. Studies in membrane vesicles and in proteoliposomes. Biochim Biophys Acta. 1987 Mar 12;897(3):406–422. doi: 10.1016/0005-2736(87)90438-x. [DOI] [PubMed] [Google Scholar]
  8. Cukierman S., Zinkand W. C., French R. J., Krueger B. K. Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers. J Gen Physiol. 1988 Oct;92(4):431–447. doi: 10.1085/jgp.92.4.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dubois J. M., Bergman C. Late sodium current in the node of Ranvier. Pflugers Arch. 1975;357(1-2):145–148. doi: 10.1007/BF00584552. [DOI] [PubMed] [Google Scholar]
  10. Dubois J. M., Schneider M. F., Khodorov B. I. Voltage dependence of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier. J Gen Physiol. 1983 Jun;81(6):829–844. doi: 10.1085/jgp.81.6.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Duch D. S., Levinson S. R. Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles. J Membr Biol. 1987;98(1):43–55. doi: 10.1007/BF01871044. [DOI] [PubMed] [Google Scholar]
  12. Duch D. S., Levinson S. R. Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles. J Membr Biol. 1987;98(1):57–68. doi: 10.1007/BF01871045. [DOI] [PubMed] [Google Scholar]
  13. Duch D. S., Recio-Pinto E., Frenkel C., Levinson S. R., Urban B. W. Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax. J Gen Physiol. 1989 Nov;94(5):813–831. doi: 10.1085/jgp.94.5.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Duch D. S., Recio-Pinto E., Frenkel C., Urban B. W. Human brain sodium channels in bilayers. Brain Res. 1988 Nov;464(3):171–177. doi: 10.1016/0169-328x(88)90023-x. [DOI] [PubMed] [Google Scholar]
  15. Feller D. J., Talvenheimo J. A., Catterall W. A. The sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition. J Biol Chem. 1985 Sep 25;260(21):11542–11547. [PubMed] [Google Scholar]
  16. French C. R., Gage P. W. A threshold sodium current in pyramidal cells in rat hippocampus. Neurosci Lett. 1985 May 23;56(3):289–293. doi: 10.1016/0304-3940(85)90257-5. [DOI] [PubMed] [Google Scholar]
  17. French C. R., Sah P., Buckett K. J., Gage P. W. A voltage-dependent persistent sodium current in mammalian hippocampal neurons. J Gen Physiol. 1990 Jun;95(6):1139–1157. doi: 10.1085/jgp.95.6.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garber S. S., Miller C. Single Na+ channels activated by veratridine and batrachotoxin. J Gen Physiol. 1987 Mar;89(3):459–480. doi: 10.1085/jgp.89.3.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Garber S. S. Symmetry and asymmetry of permeation through toxin-modified Na+ channels. Biophys J. 1988 Nov;54(5):767–776. doi: 10.1016/S0006-3495(88)83014-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gilly W. F., Armstrong C. M. Threshold channels--a novel type of sodium channel in squid giant axon. 1984 May 31-Jun 6Nature. 309(5967):448–450. doi: 10.1038/309448a0. [DOI] [PubMed] [Google Scholar]
  21. Green W. N., Weiss L. B., Andersen O. S. Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures. J Gen Physiol. 1987 Jun;89(6):873–903. doi: 10.1085/jgp.89.6.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Green W. N., Weiss L. B., Andersen O. S. Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block. J Gen Physiol. 1987 Jun;89(6):841–872. doi: 10.1085/jgp.89.6.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hartshorne R. P., Keller B. U., Talvenheimo J. A., Catterall W. A., Montal M. Functional reconstitution of the purified brain sodium channel in planar lipid bilayers. Proc Natl Acad Sci U S A. 1985 Jan;82(1):240–244. doi: 10.1073/pnas.82.1.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Horn R., Vandenberg C. A., Lange K. Statistical analysis of single sodium channels. Effects of N-bromoacetamide. Biophys J. 1984 Jan;45(1):323–335. doi: 10.1016/S0006-3495(84)84158-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huang L. Y., Moran N., Ehrenstein G. Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2082–2085. doi: 10.1073/pnas.79.6.2082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Iwasa K., Ehrenstein G., Moran N., Jia M. Evidence for interactions between batrachotoxin-modified channels in hybrid neuroblastoma cells. Biophys J. 1986 Sep;50(3):531–537. doi: 10.1016/S0006-3495(86)83491-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Khodorov B. I. Batrachotoxin as a tool to study voltage-sensitive sodium channels of excitable membranes. Prog Biophys Mol Biol. 1985;45(2):57–148. doi: 10.1016/0079-6107(85)90005-7. [DOI] [PubMed] [Google Scholar]
  28. Khodorov B. I., Revenko S. V. Further analysis of the mechanisms of action of batrachotoxin on the membrane of myelinated nerve. Neuroscience. 1979;4(9):1315–1330. doi: 10.1016/0306-4522(79)90159-3. [DOI] [PubMed] [Google Scholar]
  29. Kirsch G. E., Brown A. M. Kinetic properties of single sodium channels in rat heart and rat brain. J Gen Physiol. 1989 Jan;93(1):85–99. doi: 10.1085/jgp.93.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Leibowitz M. D., Sutro J. B., Hille B. Voltage-dependent gating of veratridine-modified Na channels. J Gen Physiol. 1986 Jan;87(1):25–46. doi: 10.1085/jgp.87.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Levinson S. R., Duch D. S., Urban B. W., Recio-Pinto E. The sodium channel from Electrophorus electricus. Ann N Y Acad Sci. 1986;479:162–178. doi: 10.1111/j.1749-6632.1986.tb15568.x. [DOI] [PubMed] [Google Scholar]
  32. Levinson S. R., Thornhill W. B., Duch D. S., Recio-Pinto E., Urban B. W. The role of nonprotein domains in the function and synthesis of voltage-gated sodium channels. Ion Channels. 1990;2:33–64. doi: 10.1007/978-1-4615-7305-0_2. [DOI] [PubMed] [Google Scholar]
  33. Moczydlowski E., Garber S. S., Miller C. Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+. J Gen Physiol. 1984 Nov;84(5):665–686. doi: 10.1085/jgp.84.5.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Patlak J. B., Ortiz M. Kinetic diversity of Na+ channel bursts in frog skeletal muscle. J Gen Physiol. 1989 Aug;94(2):279–301. doi: 10.1085/jgp.94.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rando T. A. Rapid and slow gating of veratridine-modified sodium channels in frog myelinated nerve. J Gen Physiol. 1989 Jan;93(1):43–65. doi: 10.1085/jgp.93.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Recio-Pinto E., Duch D. S., Levinson S. R., Urban B. W. Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers. J Gen Physiol. 1987 Sep;90(3):375–395. doi: 10.1085/jgp.90.3.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Recio-Pinto E., Thornhill W. B., Duch D. S., Levinson S. R., Urban B. W. Neuraminidase treatment modifies the function of electroplax sodium channels in planar lipid bilayers. Neuron. 1990 Nov;5(5):675–684. doi: 10.1016/0896-6273(90)90221-z. [DOI] [PubMed] [Google Scholar]
  38. Rosenberg R. L., Tomiko S. A., Agnew W. S. Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1239–1243. doi: 10.1073/pnas.81.4.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sherman S. J., Catterall W. A. The developmental regulation of TTX-sensitive sodium channels in rat skeletal muscle in vivo and in vitro. Soc Gen Physiol Ser. 1985;39:237–263. [PubMed] [Google Scholar]
  40. Strichartz G., Rando T., Wang G. K. An integrated view of the molecular toxinology of sodium channel gating in excitable cells. Annu Rev Neurosci. 1987;10:237–267. doi: 10.1146/annurev.ne.10.030187.001321. [DOI] [PubMed] [Google Scholar]
  41. Stühmer W., Conti F., Suzuki H., Wang X. D., Noda M., Yahagi N., Kubo H., Numa S. Structural parts involved in activation and inactivation of the sodium channel. Nature. 1989 Jun 22;339(6226):597–603. doi: 10.1038/339597a0. [DOI] [PubMed] [Google Scholar]
  42. Sutro J. B. Kinetics of veratridine action on Na channels of skeletal muscle. J Gen Physiol. 1986 Jan;87(1):1–24. doi: 10.1085/jgp.87.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tamkun M. M., Talvenheimo J. A., Catterall W. A. The sodium channel from rat brain. Reconstitution of neurotoxin-activated ion flux and scorpion toxin binding from purified components. J Biol Chem. 1984 Feb 10;259(3):1676–1688. [PubMed] [Google Scholar]
  44. Tanguy J., Yeh J. Z. BTX modification of Na channels in squid axons. I. State dependence of BTX action. J Gen Physiol. 1991 Mar;97(3):499–519. doi: 10.1085/jgp.97.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ulbricht W. Rate of veratridine action on the nodal membrane. II. Fast and slow phase determined with periodic impulses in the voltage clamp. Pflugers Arch. 1972;336(3):201–212. doi: 10.1007/BF00590044. [DOI] [PubMed] [Google Scholar]
  46. Villegas R., Sorais-Landáez F., Rodŕiguez-Grille J. M., Villegas G. M. The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein. Biochim Biophys Acta. 1988 Jun 22;941(2):150–156. doi: 10.1016/0005-2736(88)90175-7. [DOI] [PubMed] [Google Scholar]
  47. Villegas R., Villegas G. M. Nerve sodium channel incorporation in vesicles. Annu Rev Biophys Bioeng. 1981;10:387–419. doi: 10.1146/annurev.bb.10.060181.002131. [DOI] [PubMed] [Google Scholar]
  48. WRIGHT E. B. The effect of low temperatures on single crustacean motor nerve fibers. J Cell Physiol. 1958 Feb;51(1):29–65. doi: 10.1002/jcp.1030510104. [DOI] [PubMed] [Google Scholar]

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