Fragment of GABA(A) receptor containing key ligand-binding residues overexpressed in Escherichia coli.

GABA(A) receptor plays a major role in inhibitory synaptic transmission in the central nervous system and is the target of drugs such as the benzodiazepine tranquilizers. The polymeric membrane protein nature of GABA(A) receptor has rendered structural elucidation of the receptor a formidable task, greatly hampering structure-based drug design. We report here the first expression in Escherichia coli of a fragment of GABA(A) receptor. This 131-residue fragment, spanning Cys166 to Leu296 of human GABAA receptor alpha1 subunit, contains residues previously suggested to be involved in benzodiazepine binding. The overexpressed non-fusion recombinant protein was purified to near homogeneity and characterized by circular dichroism (CD), which showed that the recombinant protein has well defined secondary structures where beta-strands are dominant. The stability of the secondary structures was demonstrated by CD spectra at high pH and elevated temperature. Excluding part of the sequences from the carboxyl terminal of the fragment resulted in dramatic changes in the secondary structures comparable to the effects caused by SDS denaturation. Our results therefore suggest that the 131-residue fragment harbors an integral structural domain of the receptor. The overexpression of the recombinant protein fragment thus opens the way to the biochemical and structural studies of a functionally important region of the receptor, and exemplifies an effective approach of expression and characterization that potentially may be extended to other members of the ligand gated channel receptor superfamily, to which the GABA(A) receptor belongs.