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. 1989 Oct;417:483–500. doi: 10.1113/jphysiol.1989.sp017814

Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture.

R L MacDonald 1, C J Rogers 1, R E Twyman 1
PMCID: PMC1189279  PMID: 2482885

Abstract

1. Barbiturate regulation of the kinetic properties of gamma-aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole-cell and excised outside-out patch-clamp recording techniques. 2. GABA (2 microM), GABA (2 microM) plus phenobarbitone (PhB) (500 microM) and GABA (2 microM) plus pentobarbitone (PB) (50 microM), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at -75 mV and the chloride equilibrium potential was 0 mV. GABA receptor channel currents were increased by PhB and PB. 3. Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 16.5 pS. The conductances of the two states were unchanged by the barbiturates. 4. The main conductance state kinetics were analysed. GABA alone or with the barbiturates gated the channel open singly and in groups of openings. 5. The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times. 6. Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (O1, O2, O3). The time constants for the exponential functions (0.9, 2.7 and 7.8 ms, respectively) were unchanged by the barbiturates. The increases in mean open times and the shifts of the open-time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and O2) and to an increase in the relative frequency of occurrence of the longest open state (O3). 7. Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states. None of the time constants nor areas of the exponential functions were significantly changed by the barbiturates. 8. For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms. For GABA (2 microM), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, suggesting that the channel has three burst states (B1, B2, B3). The B1 burst state was probably a single opening to the O1 open state while the B2 and B3 burst states were probably composed of multiple openings to the O2 and O3 open states.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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

  1. Barker J. L., McBurney R. N., MacDonald J. F. Fluctuation analysis of neutral amino acid responses in cultured mouse spinal neurones. J Physiol. 1982 Jan;322:365–387. doi: 10.1113/jphysiol.1982.sp014042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barker J. L., McBurney R. N. Phenobarbitone modulation of postsynaptic GABA receptor function on cultured mammalian neurons. Proc R Soc Lond B Biol Sci. 1979 Dec 31;206(1164):319–327. doi: 10.1098/rspb.1979.0108. [DOI] [PubMed] [Google Scholar]
  3. Blatz A. L., Magleby K. L. Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle. J Physiol. 1986 Sep;378:141–174. doi: 10.1113/jphysiol.1986.sp016212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bormann J., Clapham D. E. gamma-Aminobutyric acid receptor channels in adrenal chromaffin cells: a patch-clamp study. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2168–2172. doi: 10.1073/pnas.82.7.2168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bormann J., Hamill O. P., Sakmann B. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. J Physiol. 1987 Apr;385:243–286. doi: 10.1113/jphysiol.1987.sp016493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colquhoun D., Hawkes A. G. On the stochastic properties of bursts of single ion channel openings and of clusters of bursts. Philos Trans R Soc Lond B Biol Sci. 1982 Dec 24;300(1098):1–59. doi: 10.1098/rstb.1982.0156. [DOI] [PubMed] [Google Scholar]
  7. Colquhoun D., Sakmann B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol. 1985 Dec;369:501–557. doi: 10.1113/jphysiol.1985.sp015912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Curtis D. R., Hösli L., Johnston G. A., Johnston I. H. The hyperpolarization of spinal motoneurones by glycine and related amino acids. Exp Brain Res. 1968;5(3):235–258. doi: 10.1007/BF00238666. [DOI] [PubMed] [Google Scholar]
  9. Hamill O. P., Bormann J., Sakmann B. Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA. 1983 Oct 27-Nov 2Nature. 305(5937):805–808. doi: 10.1038/305805a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Jackson M. B., Lecar H., Mathers D. A., Barker J. L. Single channel currents activated by gamma-aminobutyric acid, muscimol, and (-)-pentobarbital in cultured mouse spinal neurons. J Neurosci. 1982 Jul;2(7):889–894. doi: 10.1523/JNEUROSCI.02-07-00889.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. MacDonald R. L., Barker J. L. Enhancement of GABA-mediated postsynaptic inhibition in cultured mammalian spinal cord neurons: a common mode of anticonvulsant action. Brain Res. 1979 May 11;167(2):323–336. doi: 10.1016/0006-8993(79)90826-6. [DOI] [PubMed] [Google Scholar]
  13. Macdonald R. L., Barker J. L. Different actions of anticonvulsant and anesthetic barbiturates revealed by use of cultured mammalian neurons. Science. 1978 May 19;200(4343):775–777. doi: 10.1126/science.205953. [DOI] [PubMed] [Google Scholar]
  14. Macdonald R. L., Rogers C. J., Twyman R. E. Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J Physiol. 1989 Mar;410:479–499. doi: 10.1113/jphysiol.1989.sp017545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martin R. J. gamma-Aminobutyric acid- and piperazine-activated single-channel currents from Ascaris suum body muscle. Br J Pharmacol. 1985 Feb;84(2):445–461. doi: 10.1111/j.1476-5381.1985.tb12929.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mathers D. A. Pentobarbital promotes bursts of gamma-aminobutyric acid-activated single channel currents in cultured mouse central neurons. Neurosci Lett. 1985 Sep 30;60(2):121–126. doi: 10.1016/0304-3940(85)90231-9. [DOI] [PubMed] [Google Scholar]
  17. Mathers D. A. Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons. Can J Physiol Pharmacol. 1985 Oct;63(10):1228–1233. doi: 10.1139/y85-203. [DOI] [PubMed] [Google Scholar]
  18. McManus O. B., Blatz A. L., Magleby K. L. Sampling, log binning, fitting, and plotting durations of open and shut intervals from single channels and the effects of noise. Pflugers Arch. 1987 Nov;410(4-5):530–553. doi: 10.1007/BF00586537. [DOI] [PubMed] [Google Scholar]
  19. Nicoll R. A. The effects of anaesthetics on synaptic excitation and inhibition in the olfactory bulb. J Physiol. 1972 Jun;223(3):803–814. doi: 10.1113/jphysiol.1972.sp009875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nowak L. M., Young A. B., Macdonald R. L. GABA and bicuculline actions on mouse spinal cord and cortical neurons in cell culture. Brain Res. 1982 Jul 22;244(1):155–164. doi: 10.1016/0006-8993(82)90913-1. [DOI] [PubMed] [Google Scholar]
  21. Olsen R. W. Drug interactions at the GABA receptor-ionophore complex. Annu Rev Pharmacol Toxicol. 1982;22:245–277. doi: 10.1146/annurev.pa.22.040182.001333. [DOI] [PubMed] [Google Scholar]
  22. Ransom B. R., Barker J. L. Pentobarbital selectively enhances GABA-mediated post-synaptic inhibition in tissue cultured mouse spinal neurons. Brain Res. 1976 Sep 24;114(3):530–535. doi: 10.1016/0006-8993(76)90977-x. [DOI] [PubMed] [Google Scholar]
  23. Ransom B. R., Neale E., Henkart M., Bullock P. N., Nelson P. G. Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties. J Neurophysiol. 1977 Sep;40(5):1132–1150. doi: 10.1152/jn.1977.40.5.1132. [DOI] [PubMed] [Google Scholar]
  24. SCHMIDT R. F. PHARMACOLOGICAL STUDIES ON THE PRIMARY AFFERENT DEPOLARIZATION OF THE TOAD SPINAL CORD. Pflugers Arch Gesamte Physiol Menschen Tiere. 1963 Jul 2;277:325–346. doi: 10.1007/BF00362515. [DOI] [PubMed] [Google Scholar]
  25. Sakmann B., Hamill O. P., Bormann J. Patch-clamp measurements of elementary chloride currents activated by the putative inhibitory transmitter GABA and glycine in mammalian spinal neurons. J Neural Transm Suppl. 1983;18:83–95. [PubMed] [Google Scholar]
  26. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature. 1987 Jul 16;328(6127):221–227. doi: 10.1038/328221a0. [DOI] [PubMed] [Google Scholar]
  27. Schulz D. W., Macdonald R. L. Barbiturate enhancement of GABA-mediated inhibition and activation of chloride ion conductance: correlation with anticonvulsant and anesthetic actions. Brain Res. 1981 Mar 23;209(1):177–188. doi: 10.1016/0006-8993(81)91179-3. [DOI] [PubMed] [Google Scholar]
  28. Study R. E., Barker J. L. Diazepam and (--)-pentobarbital: fluctuation analysis reveals different mechanisms for potentiation of gamma-aminobutyric acid responses in cultured central neurons. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7180–7184. doi: 10.1073/pnas.78.11.7180. [DOI] [PMC free article] [PubMed] [Google Scholar]

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