Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1992 May;61(5):1332–1352. doi: 10.1016/S0006-3495(92)81941-0

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

A M Correa 1, F Bezanilla 1, R Latorre 1
PMCID: PMC1260396  PMID: 1318096

Abstract

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.

Full text

PDF
1332

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albuquerque E. X., Daly J. W., Witkop B. Batrachotoxin: chemistry and pharmacology. Science. 1971 Jun 4;172(3987):995–1002. doi: 10.1126/science.172.3987.995. [DOI] [PubMed] [Google Scholar]
  2. Albuquerque E. X., Seyama I., Narahashi T. Characterization of batrachotoxin-induced depolarization of the squid giant axons. J Pharmacol Exp Ther. 1973 Feb;184(2):308–314. [PubMed] [Google Scholar]
  3. Aldrich R. W., Corey D. P., Stevens C. F. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature. 1983 Dec 1;306(5942):436–441. doi: 10.1038/306436a0. [DOI] [PubMed] [Google Scholar]
  4. Armstrong C. M., Bezanilla F. Inactivation of the sodium channel. II. Gating current experiments. J Gen Physiol. 1977 Nov;70(5):567–590. doi: 10.1085/jgp.70.5.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Armstrong C. M., Bezanilla F., Rojas E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J Gen Physiol. 1973 Oct;62(4):375–391. doi: 10.1085/jgp.62.4.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bezanilla F. A high capacity data recording device based on a digital audio processor and a video cassette recorder. Biophys J. 1985 Mar;47(3):437–441. doi: 10.1016/S0006-3495(85)83935-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bezanilla F., Armstrong C. M. Gating currents of the sodium channels: three ways to block them. Science. 1974 Feb 22;183(4126):753–754. doi: 10.1126/science.183.4126.753. [DOI] [PubMed] [Google Scholar]
  9. Bezanilla F. Single sodium channels from the squid giant axon. Biophys J. 1987 Dec;52(6):1087–1090. doi: 10.1016/S0006-3495(87)83304-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Blatz A. L., Magleby K. L. Adjacent interval analysis distinguishes among gating mechanisms for the fast chloride channel from rat skeletal muscle. J Physiol. 1989 Mar;410:561–585. doi: 10.1113/jphysiol.1989.sp017549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Campbell D. T. Sodium channel gating currents in frog skeletal muscle. J Gen Physiol. 1983 Nov;82(5):679–701. doi: 10.1085/jgp.82.5.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conti F., Stühmer W. Quantal charge redistributions accompanying the structural transitions of sodium channels. Eur Biophys J. 1989;17(2):53–59. doi: 10.1007/BF00257102. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Cukierman S. Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes. Biophys J. 1991 Oct;60(4):845–855. doi: 10.1016/S0006-3495(91)82118-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cukierman S., Krueger B. K. Modulation of sodium channel gating by external divalent cations: differential effects on opening and closing rates. Pflugers Arch. 1990 Jun;416(4):360–367. doi: 10.1007/BF00370741. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Daly J. W., Myers C. W., Whittaker N. Further classification of skin alkaloids from neotropical poison frogs (Dendrobatidae), with a general survey of toxic/noxious substances in the amphibia. Toxicon. 1987;25(10):1023–1095. doi: 10.1016/0041-0101(87)90265-0. [DOI] [PubMed] [Google Scholar]
  18. Daly J. W., Witkop B., Bommer P., Biemann K. Batrachotoxin. The active principle of the Colombian arrow poison frog, Phyllobates bicolor. J Am Chem Soc. 1965 Jan 5;87(1):124–126. doi: 10.1021/ja01079a026. [DOI] [PubMed] [Google Scholar]
  19. Daly J., Witkop B. Batrachotoxin, an extremely active cardio- and neurotoxin from the Colombian arrow poison frog Phyllobates aurotaenia. Clin Toxicol. 1971 Sep;4(3):331–342. doi: 10.3109/15563657108990484. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. HODGKIN A. L., HUXLEY A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952 Aug;117(4):500–544. doi: 10.1113/jphysiol.1952.sp004764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  24. Horn R., Lange K. Estimating kinetic constants from single channel data. Biophys J. 1983 Aug;43(2):207–223. doi: 10.1016/S0006-3495(83)84341-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Horn R., Vandenberg C. A. Statistical properties of single sodium channels. J Gen Physiol. 1984 Oct;84(4):505–534. doi: 10.1085/jgp.84.4.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huang L. Y., Catterall W. A., Ehrenstein G. Comparison of ionic selectivity of batrachotoxin-activated channels with different tetrodotoxin dissociation constants. J Gen Physiol. 1979 Jun;73(6):839–854. doi: 10.1085/jgp.73.6.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Huang L. Y., Moran N., Ehrenstein G. Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements. Biophys J. 1984 Jan;45(1):313–322. doi: 10.1016/S0006-3495(84)84157-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Huang L. Y., Yatani A., Brown A. M. The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin. J Gen Physiol. 1987 Sep;90(3):341–360. doi: 10.1085/jgp.90.3.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jackson M. B., Wong B. S., Morris C. E., Lecar H., Christian C. N. Successive openings of the same acetylcholine receptor channel are correlated in open time. Biophys J. 1983 Apr;42(1):109–114. doi: 10.1016/S0006-3495(83)84375-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Keller B. U., Hartshorne R. P., Talvenheimo J. A., Catterall W. A., Montal M. Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin. J Gen Physiol. 1986 Jul;88(1):1–23. doi: 10.1085/jgp.88.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Keller B. U., Montal M. S., Hartshorne R. P., Montal M. Two-dimensional probability density analysis of single channel currents from reconstituted acetylcholine receptors and sodium channels. Arch Biochem Biophys. 1990 Jan;276(1):47–54. doi: 10.1016/0003-9861(90)90008-m. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Llano I., Webb C. K., Bezanilla F. Potassium conductance of the squid giant axon. Single-channel studies. J Gen Physiol. 1988 Aug;92(2):179–196. doi: 10.1085/jgp.92.2.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Magleby K. L., Weiss D. S. Estimating kinetic parameters for single channels with simulation. A general method that resolves the missed event problem and accounts for noise. Biophys J. 1990 Dec;58(6):1411–1426. doi: 10.1016/S0006-3495(90)82487-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McManus O. B., Blatz A. L., Magleby K. L. Inverse relationship of the durations of adjacent open and shut intervals for C1 and K channels. Nature. 1985 Oct 17;317(6038):625–627. doi: 10.1038/317625a0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Nagy K. Evidence for multiple open states of sodium channels in neuroblastoma cells. J Membr Biol. 1987;96(3):251–262. doi: 10.1007/BF01869307. [DOI] [PubMed] [Google Scholar]
  40. Nonner W. Relations between the inactivation of sodium channels and the immobilization of gating charge in frog myelinated nerve. J Physiol. 1980 Feb;299:573–603. doi: 10.1113/jphysiol.1980.sp013143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Patlak J. B., Ortiz M. Two modes of gating during late Na+ channel currents in frog sartorius muscle. J Gen Physiol. 1986 Feb;87(2):305–326. doi: 10.1085/jgp.87.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Quandt F. N., Narahashi T. Modification of single Na+ channels by batrachotoxin. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6732–6736. doi: 10.1073/pnas.79.21.6732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Roux B., Sauvé R. A general solution to the time interval omission problem applied to single channel analysis. Biophys J. 1985 Jul;48(1):149–158. doi: 10.1016/S0006-3495(85)83768-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rudy B. Sodium gating currents in Myxicola giant axons. Proc R Soc Lond B Biol Sci. 1976 Jun 30;193(1113):469–475. doi: 10.1098/rspb.1976.0059. [DOI] [PubMed] [Google Scholar]
  46. Scanley B. E., Hanck D. A., Chay T., Fozzard H. A. Kinetic analysis of single sodium channels from canine cardiac Purkinje cells. J Gen Physiol. 1990 Mar;95(3):411–437. doi: 10.1085/jgp.95.3.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sigworth F. J., Sine S. M. Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys J. 1987 Dec;52(6):1047–1054. doi: 10.1016/S0006-3495(87)83298-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Starkus J. G., Fellmeth B. D., Rayner M. D. Gating currents in th intact crayfish giant axon. Biophys J. 1981 Aug;35(2):521–533. doi: 10.1016/S0006-3495(81)84807-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stimers J. R., Bezanilla F., Taylor R. E. Sodium channel activation in the squid giant axon. Steady state properties. J Gen Physiol. 1985 Jan;85(1):65–82. doi: 10.1085/jgp.85.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Tanguy J., Yeh J. Z. Batrachotoxin uncouples gating charge immobilization from fast Na inactivation in squid giant axons. Biophys J. 1988 Oct;54(4):719–730. doi: 10.1016/S0006-3495(88)83007-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vandenberg C. A., Bezanilla F. A sodium channel gating model based on single channel, macroscopic ionic, and gating currents in the squid giant axon. Biophys J. 1991 Dec;60(6):1511–1533. doi: 10.1016/S0006-3495(91)82186-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES