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.
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