The GABA receptor is a ligand-gated chloride channel which mediates the inhibitory effects of the neurotransmitter GABA (gamma-aminobutyric acid). In addition to binding GABA, the receptor is modulated by several therapeutic agents and endogenous neurosteroids which act through distinct, allosteric binding sites. Over fifteen different receptor subunits have been cloned and sequenced and multiple receptor subtypes exist in vivo. There is evidence that the GABAa receptor is regulated by protein phosphorylation and that is this regulation is complex and involves multiple second messenger system pathways. The kinases and phosphatases which accompany this regulation are not well-characterized. The hypotheses of the present proposal are: 1) multiple kinases and phosphatases participate in the phosphorylation/dephosphorylation of the receptor, 2) the subunit composition of the receptor determines how the receptor is regulated by phosphorylation, and 3) phosphorylation alters the allosteric modulatory interactions of the receptor complex. To test these hypotheses, protein kinases and phosphatases will be microinjected into Xenopus oocytes expressing either recombinant wildtype or mutagenized human GABA receptor subunit cDNAs. The effect of these enzymes on both GABA receptor-gated chloride currents and the allosteric modulation of these currents by benzodiazepines, barbiturates and neurosteroids, will be measured using the two-electrode voltage-clamp technique. Thus, the proposed research adopts a reductionistic strategy to study GABA receptor phosphorylation/dephosphorylation under conditions where the receptor composition is defined and site-directed mutagenesis can be used to identify phosphorylation sites on the receptor. These studies will aid in the understanding of GABA receptor function in the overall scheme of neuronal signaling. The involvement of the GABA receptor in hyperexcitable states such as anxiety disorders and epilepsy, underscores the importance of understanding GABA receptor function.
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