Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

The Journal of Physiology logoLink to The Journal of Physiology
. 1990 Jan;420:207–221. doi: 10.1113/jphysiol.1990.sp017908

GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones.

Q X Chen 1, A Stelzer 1, A R Kay 1, R K Wong 1
PMCID: PMC1190045  PMID: 2157838

Abstract

1. Current mediated by GABAA receptors was examined in pyramidal cells acutely dissociated from the hippocampus of mature guinea-pigs. Current responses were measured using whole-cell voltage-clamp recordings. An internal perfusion technique was used to change the intracellular contents during recording. 2. Application of GABA (100-300 microM) by short duration pressure pulses produced outward current responses at a holding potential of -10 mV. When recordings were made with intracellular solutions which did not contain Mg-ATP, GABA responses progressively decreased to less than 10% of their initial values after 10 min. This 'run-down' of the GABA response could not be accounted for by desensitization since the rate of run-down was not dependent upon agonist application. 3. The run-down of the GABAA response was reversed when Mg2+ (4 mM) and ATP (2 mM) were introduced into the intracellular perfusate. In addition to the presence of Mg-ATP, buffering of Ca2+ in the intracellular solution to low levels (approximately 10(-8) M) was also necessary to stabilize the GABAA response. 4. The role of a phosphorylation process in regulating the GABAA receptor was tested. After the GABA response stabilized, introduction of alkaline phosphatase (100 micrograms/ml) to the intracellular perfusate caused a complete run-down of the GABA response. 5. Stable GABA responses were obtained when ATP was replaced by ATP-gamma-S (adenosine 5'-O-(thiotriphosphate), an analogue of ATP that donates a thiophosphate group resulting in a product that is more resistant to hydrolysis. Following such treatment GABA responses declined more slowly after the introduction of intracellular alkaline phosphatase. 6. Run-down of GABA responses accelerated when intracellular Ca2+ concentration ([Ca2+]i) was elevated to about 5 x 10(-4) M. The run-down caused by elevated [Ca2+]i could be stopped and reversed by reducing [Ca2+]i to about 10(-8) M. 7. The introduction of ATP-gamma-S to the intracellular medium retarded the run-down of GABA responses caused by elevation of [Ca2+]i. 8. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), a calmodulin inhibitor, reduced the rate of run-down induced by elevated [Ca2+]i. 9. These results suggest that the function of the GABAA receptor is maintained by phosphorylation of the receptor or some closely associated regulatory molecule. Elevation of [Ca2+]i destabilizes the function of the GABAA receptor, probably by activating a Ca2+/calmodulin-dependent phosphatase.

Full text

PDF
207

Selected References

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

  1. ANDERSEN P., ECCLES J. C., LOYNING Y. Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapses. Nature. 1963 May 11;198:540–542. doi: 10.1038/198540a0. [DOI] [PubMed] [Google Scholar]
  2. Alger B. E., Nicoll R. A. Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro. J Physiol. 1982 Jul;328:105–123. doi: 10.1113/jphysiol.1982.sp014255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armstrong D., Eckert R. Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2518–2522. doi: 10.1073/pnas.84.8.2518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Byerly L., Yazejian B. Intracellular factors for the maintenance of calcium currents in perfused neurones from the snail, Lymnaea stagnalis. J Physiol. 1986 Jan;370:631–650. doi: 10.1113/jphysiol.1986.sp015955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chad J. E., Eckert R. An enzymatic mechanism for calcium current inactivation in dialysed Helix neurones. J Physiol. 1986 Sep;378:31–51. doi: 10.1113/jphysiol.1986.sp016206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheung W. Y. Calmodulin: its potential role in cell proliferation and heavy metal toxicity. Fed Proc. 1984 Dec;43(15):2995–2999. [PubMed] [Google Scholar]
  7. Cohen P. The coordinated control of metabolic pathways by broad-specificity protein kinases and phosphatases. Curr Top Cell Regul. 1985;27:23–37. doi: 10.1016/b978-0-12-152827-0.50010-4. [DOI] [PubMed] [Google Scholar]
  8. Doroshenko P. A., Kostyuk P. G., Martynyuk A. E. Intracellular metabolism of adenosine 3',5'-cyclic monophosphate and calcium inward current in perfused neurones of Helix pomatia. Neuroscience. 1982;7(9):2125–2134. doi: 10.1016/0306-4522(82)90124-5. [DOI] [PubMed] [Google Scholar]
  9. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  10. Goto S., Matsukado Y., Mihara Y., Inoue N., Miyamoto E. The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay. Brain Res. 1986 Nov 5;397(1):161–172. doi: 10.1016/0006-8993(86)91381-8. [DOI] [PubMed] [Google Scholar]
  11. Gyenes M., Farrant M., Farb D. H. "Run-down" of gamma-aminobutyric acidA receptor function during whole-cell recording: a possible role for phosphorylation. Mol Pharmacol. 1988 Dec;34(6):719–723. [PubMed] [Google Scholar]
  12. 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]
  13. Inoue M., Oomura Y., Yakushiji T., Akaike N. Intracellular calcium ions decrease the affinity of the GABA receptor. Nature. 1986 Nov 13;324(6093):156–158. doi: 10.1038/324156a0. [DOI] [PubMed] [Google Scholar]
  14. Kay A. R., Wong R. K. Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems. J Neurosci Methods. 1986 May;16(3):227–238. doi: 10.1016/0165-0270(86)90040-3. [DOI] [PubMed] [Google Scholar]
  15. Klee C. B., Crouch T. H., Krinks M. H. Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6270–6273. doi: 10.1073/pnas.76.12.6270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lloyd K. G., Bossi L., Morselli P. L., Munari C., Rougier M., Loiseau H. Alterations of GABA-mediated synaptic transmission in human epilepsy. Adv Neurol. 1986;44:1033–1044. [PubMed] [Google Scholar]
  17. McCarren M., Alger B. E. Use-dependent depression of IPSPs in rat hippocampal pyramidal cells in vitro. J Neurophysiol. 1985 Feb;53(2):557–571. doi: 10.1152/jn.1985.53.2.557. [DOI] [PubMed] [Google Scholar]
  18. Miles R., Wong R. K. Latent synaptic pathways revealed after tetanic stimulation in the hippocampus. Nature. 1987 Oct 22;329(6141):724–726. doi: 10.1038/329724a0. [DOI] [PubMed] [Google Scholar]
  19. Nicoll R. A. The coupling of neurotransmitter receptors to ion channels in the brain. Science. 1988 Jul 29;241(4865):545–551. doi: 10.1126/science.2456612. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Palade P. Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. I. Use of pyrophosphate to study caffeine-induced Ca2+ release. J Biol Chem. 1987 May 5;262(13):6135–6141. [PubMed] [Google Scholar]
  22. Palvimo J., Linnala-Kankkunen A., Mäenpä P. H. Thiophosphorylation and phosphorylation of chromatin proteins from calf thymus in vitro. Biochem Biophys Res Commun. 1985 Jan 16;126(1):103–108. doi: 10.1016/0006-291x(85)90577-7. [DOI] [PubMed] [Google Scholar]
  23. Schwartzkroin P. A., Prince D. A. Changes in excitatory and inhibitory synaptic potentials leading to epileptogenic activity. Brain Res. 1980 Feb 3;183(1):61–76. doi: 10.1016/0006-8993(80)90119-5. [DOI] [PubMed] [Google Scholar]
  24. Stelzer A., Kay A. R., Wong R. K. GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors. Science. 1988 Jul 15;241(4863):339–341. doi: 10.1126/science.2455347. [DOI] [PubMed] [Google Scholar]
  25. Stewart A. A., Ingebritsen T. S., Cohen P. The protein phosphatases involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B) from rabbit skeletal muscle. Eur J Biochem. 1983 May 2;132(2):289–295. doi: 10.1111/j.1432-1033.1983.tb07361.x. [DOI] [PubMed] [Google Scholar]
  26. Sweetnam P. M., Lloyd J., Gallombardo P., Malison R. T., Gallager D. W., Tallman J. F., Nestler E. J. Phosphorylation of the GABAa/benzodiazepine receptor alpha subunit by a receptor-associated protein kinase. J Neurochem. 1988 Oct;51(4):1274–1284. doi: 10.1111/j.1471-4159.1988.tb03097.x. [DOI] [PubMed] [Google Scholar]
  27. Taleb O., Trouslard J., Demeneix B. A., Feltz P., Bossu J. L., Dupont J. L., Feltz A. Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements. Pflugers Arch. 1987 Aug;409(6):620–631. doi: 10.1007/BF00584663. [DOI] [PubMed] [Google Scholar]
  28. Tanaka T., Ohmura T., Yamakado T., Hidaka H. Two types of calcium-dependent protein phosphorylations modulated by calmodulin antagonists. Naphthalenesulfonamide derivatives. Mol Pharmacol. 1982 Sep;22(2):408–412. [PubMed] [Google Scholar]
  29. Thompson S. M., Gähwiler B. H. Activity-dependent disinhibition. I. Repetitive stimulation reduces IPSP driving force and conductance in the hippocampus in vitro. J Neurophysiol. 1989 Mar;61(3):501–511. doi: 10.1152/jn.1989.61.3.501. [DOI] [PubMed] [Google Scholar]
  30. Traub R. D., Miles R., Wong R. K. Model of the origin of rhythmic population oscillations in the hippocampal slice. Science. 1989 Mar 10;243(4896):1319–1325. doi: 10.1126/science.2646715. [DOI] [PubMed] [Google Scholar]
  31. Wong R. K., Watkins D. J. Cellular factors influencing GABA response in hippocampal pyramidal cells. J Neurophysiol. 1982 Oct;48(4):938–951. doi: 10.1152/jn.1982.48.4.938. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES