Delineation of a membrane-proximal β-rich domain in the GABAA receptor by progressive deletions1

Abstract The type A gamma-aminobutyric acid (GABA A ) receptor plays a major inhibitory role in the central nervous system. Structural elucidation of the GABA A receptor has been impeded by the large size of the receptor. We present here the delineation of a minimal structural domain as the first step of dissecting the receptor structure. This was achieved through prediction-assisted progressive deletions: the prediction of a candidate structural domain rich in β-strands with no close similarity to known structures was tested by deleting putative secondary structure elements from the ends of the proposed domain, as well as mutations within the terminal secondary structures. Such progressive deletions revealed the limits of an integral domain, spanning Cys180 to Met293 (numbering of human α1 subunit). Below these limits the intact domain structure, as indicated by its circular dichroism, collapses. Based on its putative position, this domain is provisionally designated the membrane-proximal β-rich domain of GABA A receptor. The inclusion of sequences from the first two out of four previously suggested transmembrane segments and one of the two conserved Cys residues in this domain defines important constraints to the receptor structure.