Identification and characterization of a novel allosteric glutamate binding site on the GABA-A receptor

The A type γ-aminobutyric acid receptor (GABAAR) mediates major inhibition to counteract glutamate receptor-mediated excitation in the Central Nervous System (CNS). However, work in this dissertation identified a novel glutamate-binding site at the α+β- interface of the GABAAR. Activation of this glutamate binding site by glutamate and analogues can potentiate both the synaptic GABAAR-mediated phasic responses and the extrasynaptic GABAAR-mediated tonic inhibition. Using systematic mutagenesis analysis, we identified a conserved group of charged amino acid residues including α1K104, α1K155, α1E137 and β2E181, that form this glutamate binding site at the extracellular domain of the GABAAR. Spatial and electrostatic accessibility are both crucial for glutamate binding on this site. Furthermore, through in-silicon and electrophysiological screening, we identified that ampicillin, an antibiotic, and BRC640 as novel compounds that can target this newly identified glutamate-binding site, leading to an enhancement of the GABAAR function. Comparing with traditional benzodiazepine drugs, these two compounds were able to regulate both synaptic and extrasynaptic GABAARs. In the cerebellum, depolarization of the Purkinje cells induces both dendritic glutamate release and a rebound potentiation of GABA responsiveness. Application of ampicillin occluded the early phase of rebound potentiation, presumably by saturating the glutamate-binding site on the GABAARs and preventing the further potentiation induced by dendritically released glutamate. Taken together, our present study demonstrated a novel glutamate-binding site on the GABAAR that might lead to future development of novel GABAAR-based therapeutics. This type of excitation/inhibition crosstalk may play an essential role in Purkinje cell inhibitory plasticity.Medicine, Faculty ofGraduat