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Originally published In Press as doi:10.1074/jbc.M209281200 on October 17, 2002

J. Biol. Chem., Vol. 277, Issue 51, 50036-50045, December 20, 2002
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Identification of the Bovine gamma -Aminobutyric Acid Type A Receptor alpha  Subunit Residues Photolabeled by the Imidazobenzodiazepine [3H]Ro15-4513*

Gregory W. SawyerDagger §, David C. Chiara||, Richard W. OlsenDagger **, and Jonathan B. Cohen||Dagger Dagger

From the Dagger  Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California 90095 and the || Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

Ligands binding to the benzodiazepine-binding site in gamma -aminobutyric acid type A (GABAA) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABAA receptor alpha  and gamma  subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [3H]flunitrazepam identified a primary site of incorporation at alpha 1His-102, whereas studies with [3H]Ro15-4513 suggested incorporation into the alpha 1 subunit at unidentified amino acids C-terminal to alpha 1His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified (~200-fold) GABAA receptor from detergent extracts of bovine cortex, photolabeled it with [3H]Ro15-4513, and identified 3H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of 3H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different alpha  subunits. Based upon this radiochemical sequence analysis, [3H]Ro15-4513 was found to selectively label the homologous tyrosines alpha 1Tyr-210, alpha 2Tyr-209, and alpha 3Tyr-234, in GABAA receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure.

* This work was supported in part by United States Public Health Service Grant GM 58448 and by an award to the Harvard Medical School from the Howard Hughes Medical Institute Biomedical Research Support Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health Training Grant F32NS11015. Present address: Dept. of Pharmacology and Physiology, College of Osteopathic Medicine, Oklahoma State University, Tulsa, OK 74107.

Both authors contributed equally to this work.

** To whom correspondence may be addressed: Dept. of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095. Tel.: 310-825-5093; E-mail: rolsen@mednet.ucla.edu.

Dagger Dagger To whom correspondence may be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen@hms.harvard.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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