Gamma-aminobutyric acid(A) receptor alpha1 subunit: Hyperexpression, purification, structural characterization and ligand binding

The type A gamma-aminobutyric acid (GABAA) receptor plays a major inhibitory role in the central nervous system, and is the target of numerous therapeutic compounds, such as the benzodiazepine tranquilizers. Structural elucidation of the GABAA receptor has been impeded by the scarce and heterogeneous nature of the native receptor. In this study, the bovine GABAA receptor alpha1 subunit fragments of different lengths were hyperexpressed in Escherichia coli as inclusion bodies, purified to near homogeneity, and characterized by spectroscopic methods with respect to structural and ligand-binding properties.;Results showed that the fragments were rich in beta-strand structures and had ordered side chain packing. Through prediction-assisted progressive deletions and secondary structure evaluation with circular dichroism, a membrane-proximal beta-rich domain was delineated as Cyst80-Met293. Binding of the fluorescent benzodiazepine ligand, Bodipy-FL Ro-1986 (BFR) was evident from fluorescence resonance energy transfer and fluorescence polarization. The binding was attributed more to fragment Cys166-Leu296 than to Gln28-Glu165, and was inhibited by known central benzodiazepine site ligands. Alanine-substitution mutations were made for candidate ligand-binding residues. Alanine-substitution mutants of Tyr187, Thr234, and Tyr237, previously reported to be directly involved in benzodiazepine binding, were found to affect not only BFR binding but also protein secondary structures. This underlined the possibility that the functional roles previously attributed to these three residues could be secondary to their roles in the protein structure. Studies with the single-Trp mutants W198Y and W273Y demonstrated that Trp273 was more solvent accessible and closer to the binding site than Trp198. The study presents an alternative approach for GABAA receptor research, in which hyperexpressed fragments are subjected to spectroscopic analysis for the delineation of structural as well as ligand-binding properties.;Preliminary results also indicated that the bovine GABAA receptor alphaI subunit could be expressed in full length in the eukaryotic expression system of the methylotrophic yeast Pichia pastoris.