Abstract
Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.
Full Text
The Full Text of this article is available as a PDF (1.4 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- 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]
- Begenisich T., Busath D. Sodium flux ratio in voltage-clamped squid giant axons. J Gen Physiol. 1981 May;77(5):489–502. doi: 10.1085/jgp.77.5.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Binstock L., Lecar H. Ammonium ion currents in the squid giant axon. J Gen Physiol. 1969 Mar;53(3):342–361. doi: 10.1085/jgp.53.3.342. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Busath D., Begenisich T. Unidirectional sodium and potassium fluxes through the sodium channel of squid giant axons. Biophys J. 1982 Oct;40(1):41–49. doi: 10.1016/S0006-3495(82)84456-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Catterall W. A. Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin. J Biol Chem. 1975 Jun 10;250(11):4053–4059. [PubMed] [Google Scholar]
- Catterall W. A. Structure and function of voltage-sensitive ion channels. Science. 1988 Oct 7;242(4875):50–61. doi: 10.1126/science.2459775. [DOI] [PubMed] [Google Scholar]
- 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]
- Cecchi X., Wolff D., Alvarez O., Latorre R. Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle. Biophys J. 1987 Nov;52(5):707–716. doi: 10.1016/S0006-3495(87)83265-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coronado R., Rosenberg R. L., Miller C. Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum. J Gen Physiol. 1980 Oct;76(4):425–446. doi: 10.1085/jgp.76.4.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Correa A. M., Bezanilla F., Agnew W. S. Voltage activation of purified eel sodium channels reconstituted into artificial liposomes. Biochemistry. 1990 Jul 3;29(26):6230–6240. doi: 10.1021/bi00478a017. [DOI] [PubMed] [Google Scholar]
- 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]
- FRANKENHAEUSER B., MOORE L. E. THE SPECIFICITY OF THE INITIAL CURRENT IN MYELINATED NERVE FIBRES OF XENOPUS LAEVIS. VOLTAGE CLAMP EXPERIMENTS. J Physiol. 1963 Nov;169:438–444. doi: 10.1113/jphysiol.1963.sp007270. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- HODGKIN A. L., HUXLEY A. F., KATZ B. Measurement of current-voltage relations in the membrane of the giant axon of Loligo. J Physiol. 1952 Apr;116(4):424–448. doi: 10.1113/jphysiol.1952.sp004716. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Hille B. Charges and potentials at the nerve surface. Divalent ions and pH. J Gen Physiol. 1968 Feb;51(2):221–236. doi: 10.1085/jgp.51.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hille B. Ionic selectivity, saturation, and block in sodium channels. A four-barrier model. J Gen Physiol. 1975 Nov;66(5):535–560. doi: 10.1085/jgp.66.5.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Llano I., Bezanilla F. Analysis of sodium current fluctuations in the cut-open squid giant axon. J Gen Physiol. 1984 Feb;83(2):133–142. doi: 10.1085/jgp.83.2.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Läuger P. Ion transport through pores: a rate-theory analysis. Biochim Biophys Acta. 1973 Jul 6;311(3):423–441. doi: 10.1016/0005-2736(73)90323-4. [DOI] [PubMed] [Google Scholar]
- 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]
- Moczydlowski E., Uehara A., Guo X., Heiny J. Isochannels and blocking modes of voltage-dependent sodium channels. Ann N Y Acad Sci. 1986;479:269–292. doi: 10.1111/j.1749-6632.1986.tb15575.x. [DOI] [PubMed] [Google Scholar]
- Noda M., Ikeda T., Kayano T., Suzuki H., Takeshima H., Kurasaki M., Takahashi H., Numa S. Existence of distinct sodium channel messenger RNAs in rat brain. Nature. 1986 Mar 13;320(6058):188–192. doi: 10.1038/320188a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Schauf C. L. Temperature dependence of the ionic current kinetics of Myxicola giant axons. J Physiol. 1973 Nov;235(1):197–205. doi: 10.1113/jphysiol.1973.sp010384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shenkel S., Cooper E. C., James W., Agnew W. S., Sigworth F. J. Purified, modified eel sodium channels are active in planar bilayers in the absence of activating neurotoxins. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9592–9596. doi: 10.1073/pnas.86.23.9592. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sigworth F. J., Spalding B. C. Chemical modification reduces the conductance of sodium channels in nerve. Nature. 1980 Jan 17;283(5744):293–295. doi: 10.1038/283293a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Worley J. F., 3rd, French R. J., Krueger B. K. Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium. J Gen Physiol. 1986 Feb;87(2):327–349. doi: 10.1085/jgp.87.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto D., Yeh J. Z., Narahashi T. Interactions of permeant cations with sodium channels of squid axon membranes. Biophys J. 1985 Sep;48(3):361–368. doi: 10.1016/S0006-3495(85)83792-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto D., Yeh J. Z., Narahashi T. Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels. Biophys J. 1984 Jan;45(1):337–344. doi: 10.1016/S0006-3495(84)84159-4. [DOI] [PMC free article] [PubMed] [Google Scholar]