Deduction of amino acid residues in the GABAA receptor α subunits photoaffinity labeled with the benzodiazepine flunitrazepam

Peptide mapping and microsequencing were used to infer the site of photoaffinity labeling by the gamma-aminobutyric acid, receptor modulator [H-3]flunitrazepam. Peptide mapping with and without N-deglycosylation was used to restrict the domain for photoaffinity labeling to residues 74-123 of the bovine al subunit, in agreement with a previously predicted labeling domain between residues 59-148 based on cyanogen bromide fragmentation. Edman degradation of partially purified photolabeled peptides gave release of H-3 counts in the ninth cycle of a tryptic peptide sequence. A second V8/chymotryptic peptide produced an impure sequence with release of H-3 counts in the seventh through ninth cycle of sequence. The combined data support those previously reported, i.e., that the primary site for photoaffinity labeling by [H-3]flunitrazepam is His102 of the bovine al subunit. In addition we also detected possible secondary labeling of Pro97. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.