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. 1993 Apr;64(4):1038–1050. doi: 10.1016/S0006-3495(93)81469-3

Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle.

D Naranjo 1, R Latorre 1
PMCID: PMC1262421  PMID: 8388264

Abstract

Batrachotoxin-modified Na+ channels from toad muscle were inserted into planar lipid bilayers composed of neutral phospholipids. Single-channel conductances were measured for [Na+] ranging between 0.4 mM and 3 M. When membrane preparations were made in the absence of protease inhibitors, two open conductance states were identified: a fully open state (16.6 pS in 200 mM symmetrical NaCl) and a substate that was 71% of the full conductance. The substate was predominant at [Na+] > 65 mM, whereas the presence of the fully open state was predominant at [Na+] < 15 mM. Addition of protease inhibitors during membrane preparation stabilized the fully open state over the full range of [Na+] studied. In symmetrical Na+ solutions and in biionic conditions, the ratio of amplitudes remained constant and the two open states exhibited the same permeability ratios of PLi/PNa and PCs/PNa. The current-voltage relations for both states showed inward rectification only at [Na+] < 10 mM, suggesting the presence of asymmetric negative charge densities at both channel entrances, with higher charge density in the external side. An energy barrier profile that includes double ion occupancy and asymmetric charge densities at the channel entrances was required to fit the conductance-[Na+] relations and to account for the rectification seen at low [Na+]. Energy barrier profiles differing only in the energy peaks can give account of the differences between both conductance states. Estimation of the surface charge density at the channel entrances is very dependent on the ion occupancy used and the range of [Na+] tested. Independent evidence for the existence of a charged external vestibule was obtained at low external [Na+] by identical reduction of the outward current induced by micromolar additions of Mg2+ and Ba2+.

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Selected References

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  1. Agnew W. S., Levinson S. R., Brabson J. S., Raftery M. A. Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2606–2610. doi: 10.1073/pnas.75.6.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alvarez O., Villarroel A., Eisenman G. Calculation of ion currents from energy profiles and energy profiles from ion currents in multibarrier, multisite, multioccupancy channel model. Methods Enzymol. 1992;207:816–854. doi: 10.1016/0076-6879(92)07058-v. [DOI] [PubMed] [Google Scholar]
  3. Begenisich T. B., Cahalan M. D. Sodium channel permeation in squid axons. I: Reversal potential experiments. J Physiol. 1980 Oct;307:217–242. doi: 10.1113/jphysiol.1980.sp013432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Begenisich T. B., Cahalan M. D. Sodium channel permeation in squid axons. II: Non-independence and current-voltage relations. J Physiol. 1980 Oct;307:243–257. doi: 10.1113/jphysiol.1980.sp013433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cachelin A. B., De Peyer J. E., Kokubun S., Reuter H. Sodium channels in cultured cardiac cells. J Physiol. 1983 Jul;340:389–401. doi: 10.1113/jphysiol.1983.sp014768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai M., Jordan P. C. How does vestibule surface charge affect ion conduction and toxin binding in a sodium channel? Biophys J. 1990 Apr;57(4):883–891. doi: 10.1016/S0006-3495(90)82608-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Castillo C., Villegas R., Recio-Pinto E. Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers. J Gen Physiol. 1992 Jun;99(6):897–930. doi: 10.1085/jgp.99.6.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cecchi X., Alvarez O., Wolff D. Characterization of a calcium-activated potassium channel from rabbit intestinal smooth muscle incorporated into planar bilayers. J Membr Biol. 1986;91(1):11–18. doi: 10.1007/BF01870210. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dani J. A., Eisenman G. Monovalent and divalent cation permeation in acetylcholine receptor channels. Ion transport related to structure. J Gen Physiol. 1987 Jun;89(6):959–983. doi: 10.1085/jgp.89.6.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dani J. A. Ion-channel entrances influence permeation. Net charge, size, shape, and binding considerations. Biophys J. 1986 Mar;49(3):607–618. doi: 10.1016/S0006-3495(86)83688-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fox J. A. Ion channel subconductance states. J Membr Biol. 1987;97(1):1–8. doi: 10.1007/BF01869609. [DOI] [PubMed] [Google Scholar]
  14. GRAHAME D. C. The electrical double layer and the theory of electrocapillarity. Chem Rev. 1947 Dec;41(3):441–501. doi: 10.1021/cr60130a002. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Guy H. R., Conti F. Pursuing the structure and function of voltage-gated channels. Trends Neurosci. 1990 Jun;13(6):201–206. doi: 10.1016/0166-2236(90)90160-c. [DOI] [PubMed] [Google Scholar]
  18. Hidalgo C., Parra C., Riquelme G., Jaimovich E. Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation. Biochim Biophys Acta. 1986 Feb 13;855(1):79–88. doi: 10.1016/0005-2736(86)90191-4. [DOI] [PubMed] [Google Scholar]
  19. Hille B., Schwarz W. Potassium channels as multi-ion single-file pores. J Gen Physiol. 1978 Oct;72(4):409–442. doi: 10.1085/jgp.72.4.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Krueger B. K., Worley J. F., 3rd, French R. J. Single sodium channels from rat brain incorporated into planar lipid bilayer membranes. Nature. 1983 May 12;303(5913):172–175. doi: 10.1038/303172a0. [DOI] [PubMed] [Google Scholar]
  22. Latorre R., Labarca P., Naranjo D. Surface charge effects on ion conduction in ion channels. Methods Enzymol. 1992;207:471–501. doi: 10.1016/0076-6879(92)07034-l. [DOI] [PubMed] [Google Scholar]
  23. MacKinnon R., Latorre R., Miller C. Role of surface electrostatics in the operation of a high-conductance Ca2+-activated K+ channel. Biochemistry. 1989 Oct 3;28(20):8092–8099. doi: 10.1021/bi00446a020. [DOI] [PubMed] [Google Scholar]
  24. Matsuda H., Matsuura H., Noma A. Triple-barrel structure of inwardly rectifying K+ channels revealed by Cs+ and Rb+ block in guinea-pig heart cells. J Physiol. 1989 Jun;413:139–157. doi: 10.1113/jphysiol.1989.sp017646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McLaughlin S. The electrostatic properties of membranes. Annu Rev Biophys Biophys Chem. 1989;18:113–136. doi: 10.1146/annurev.bb.18.060189.000553. [DOI] [PubMed] [Google Scholar]
  26. Meves H., Nagy K. Multiple conductance states of the sodium channel and of other ion channels. Biochim Biophys Acta. 1989 Jan 18;988(1):99–105. doi: 10.1016/0304-4157(89)90005-1. [DOI] [PubMed] [Google Scholar]
  27. Meves H., Nagy K. Multiple conductance states of the sodium channel and of other ion channels. Biochim Biophys Acta. 1989 Jan 18;988(1):99–105. doi: 10.1016/0304-4157(89)90005-1. [DOI] [PubMed] [Google Scholar]
  28. Miller C. Open-state substructure of single chloride channels from Torpedo electroplax. Philos Trans R Soc Lond B Biol Sci. 1982 Dec 1;299(1097):401–411. doi: 10.1098/rstb.1982.0140. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Nilius B., Vereecke J., Carmeliet E. Different conductance states of the bursting Na channel in guinea-pig ventricular myocytes. Pflugers Arch. 1989 Jan;413(3):242–248. doi: 10.1007/BF00583536. [DOI] [PubMed] [Google Scholar]
  31. Noda M., Suzuki H., Numa S., Stühmer W. A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. FEBS Lett. 1989 Dec 18;259(1):213–216. doi: 10.1016/0014-5793(89)81531-5. [DOI] [PubMed] [Google Scholar]
  32. Patlak J. B. Sodium channel subconductance levels measured with a new variance-mean analysis. J Gen Physiol. 1988 Oct;92(4):413–430. doi: 10.1085/jgp.92.4.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ravindran A., Kwiecinski H., Alvarez O., Eisenman G., Moczydlowski E. Modeling ion permeation through batrachotoxin-modified Na+ channels from rat skeletal muscle with a multi-ion pore. Biophys J. 1992 Feb;61(2):494–508. doi: 10.1016/S0006-3495(92)81854-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ravindran A., Schild L., Moczydlowski E. Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels. J Gen Physiol. 1991 Jan;97(1):89–115. doi: 10.1085/jgp.97.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Schild L., Ravindran A., Moczydlowski E. Zn2(+)-induced subconductance events in cardiac Na+ channels prolonged by batrachotoxin. Current-voltage behavior and single-channel kinetics. J Gen Physiol. 1991 Jan;97(1):117–142. doi: 10.1085/jgp.97.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith-Maxwell C., Begenisich T. Guanidinium analogues as probes of the squid axon sodium pore. Evidence for internal surface charges. J Gen Physiol. 1987 Sep;90(3):361–374. doi: 10.1085/jgp.90.3.361. [DOI] [PMC free article] [PubMed] [Google Scholar]

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