The GABA-A/benzodiazepine receptor consists of at least four types of polypeptide subunits, alpha, beta, gamma, and delta, and the individual subunits are expressed in multiple isoforms in the vertebrate brain. The long-term goal of this project is to understand the regulation of GABA-A receptor structure and function in cortical neurons. Our studies with chick neurons in culture have led to the hypothesis is that phosphorylation of the receptor, most likely a beta subunit, by a cAMP-dependent protein kinase reduces GABA-gated chloride flux. This proposal describes experimental approaches to test the validity of this hypothesis.
The specific aims are: 1) To examine the effects of cAMP derivatives and protein kinase inhibitors on the responses of cortical neurons to GABA. The activity of GABA-A receptors will be monitored by 36Cl flux and by whole-cell patch-clamp recording. The effects of cAMP on peak currents and desensitization will be recorded following rapid changes of GABA solutions. 2) To identify cAMP-linked neurotransmitters that modulate GABA receptor inactivation. We plan to examine intracellular cAMP pools, 36Cl fluxes, and electrophysiological responses in cells exposed in ligands for adenosine A2, beta-adrenergic, and dopamine D1 receptors. These responses will be pharmacologically characterized by the use of selective agonists and antagonists. 3) To examine the cAMP-dependent incorporation of 32P into GABA receptor subunits in vitro and in intact cortical neurons. The beta subunits which are phosphorylated in neurons will be immunoprecipitated and characterized by peptide mapping. The beta peptides will be identified by comparison to fragments derived from in vitro labeling of the purified receptor with protein kinase A. It is suggested that cAMP-dependent phosphorylation of the GABA receptor provides a means for altering cellular excitability in a rapid and reversible manner. Moreover, this could be controlled by extracellular signals. Thus, the information provided by this project may prove useful in understanding the molecular alterations leading to anxiety disorders and epilepsy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK017436-18
Application #
2137087
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1977-01-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1995-03-31
Support Year
18
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Tehrani, M H; Barnes Jr, E M (1997) Sequestration of gamma-aminobutyric acidA receptors on clathrin-coated vesicles during chronic benzodiazepine administration in vivo. J Pharmacol Exp Ther 283:384-90
Tehrani, M H; Tate, C A; al-Dahan, M I (1995) Age-related levels of GABA/benzodiazepine binding sites in cerebrum of F-344 rats: effects of exercise. Neurobiol Aging 16:199-204
Tehrani, M H; Barnes Jr, E M (1995) Reduced function of gamma-aminobutyric acidA receptors in tottering mouse brain: role of cAMP-dependent protein kinase. Epilepsy Res 22:13-21
Tehrani, M H; Barnes Jr, E M (1994) GABAA receptors in mouse cortical homogenates are phosphorylated by endogenous protein kinase A. Brain Res Mol Brain Res 24:55-64
Calkin, P A; Barnes Jr, E M (1994) gamma-Aminobutyric acid-A (GABAA) agonists down-regulate GABAA/benzodiazepine receptor polypeptides from the surface of chick cortical neurons. J Biol Chem 269:1548-53
Baumgartner, B J; Harvey, R J; Darlison, M G et al. (1994) Developmental up-regulation and agonist-dependent down-regulation of GABAA receptor subunit mRNAs in chick cortical neurons. Brain Res Mol Brain Res 26:9-17
Calkin, P A; Baumgartner, B J; Barnes Jr, E M (1994) Agonist administration in ovo down-regulates cerebellar GABAA receptors in the chick embryo. Brain Res Mol Brain Res 26:18-25
Tehrani, M H; Barnes Jr, E M (1993) Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain. J Neurochem 60:1755-61
Tehrani, M H; Barnes Jr, E M (1991) Agonist-dependent internalization of gamma-aminobutyric acidA/benzodiazepine receptors in chick cortical neurons. J Neurochem 57:1307-12
Tehrani, M H; Hablitz, J J; Barnes Jr, E M (1989) cAMP increases the rate of GABAA receptor desensitization in chick cortical neurons. Synapse 4:126-31

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