Date of Award
Doctor of Philosophy (PhD)
David A. Wagner
Robert H. Fitts, Michelle Mynlieff, Robert Peoples
The γ-aminobutyric acid type A (GABA) receptor is a member of the cys-loop family of ligand-gated ion channels that plays a crucial role in normal brain function by transducing the majority of inhibitory neurotransmission in the central nervous system. The studies documented in this dissertation were aimed at validating and refining the current best model for the interaction of GABA with the GABAA receptor via structure-function perturbation analysis. Mutational-kinetic data was used in conjunction with homology modeling knowledge to draw up architectural and functional roles of the arginines and aromatics in GABA-binding pocket. The results provide interesting new insights.
Two positively charged arginine residues, which have been implicated in ligand binding, were profiled through serial mutagenesis, to get at the specific side chain properties required for the roles they serve. The structural and functional contribution of four aromatic residues were examined through measuring the influence of their side chain on GABA binding rate with respect to changes caused by point mutations. An interaction between two aromatic residues critical for proper ligand binding was discovered through a screen for functional coupling between them and four neighboring arginines. These results were subsequently employed in an attempt to refine the current ligand-receptor interaction model, proposing specific roles for the amino acid residues studied.
In the current best model of the GABAA receptor's ligand-binding pocket, the amino moiety of GABA is coordinated by a cation-π interaction with β2Y97 and the carboxyl moiety coordinated by an interaction with either β2R207 or α1R67 (or possibly both). Here, we incorporate the results of this dissertation to modify and add significant detail to this model. The model proposed here includes the following features: a hydrophobic interaction between β2Y97 and β2F200, an inter-subunit cation-π interaction between β2Y97 and α1R132, a cation-π interaction between the amino group of GABA and β2F200, hydrogen bond(s) between the carboxyl end of GABA and the guanidinium group of α1R67, and an interaction between the side chain of β2R207 and the backbone carbonyl of β2Y97.