title: Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β- Interface.
abstract: In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABAARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABAAR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343-19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABAAR responses. In the α1β3γ2 GABAAR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ(+)-β(-) subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β(+)-α(-) subunit interfaces. GABA inhibits S-[(3)H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2-15') in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[(3)H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ(+)-β(-) site, based upon the distance in GABAAR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABAAR function and provide a first demonstration that an intersubunit-binding site in the GABAAR transmembrane domain binds negative and positive allosteric modulators.
authors.affiliation: From the Departments of Neurobiology and.
authors.forename: Selwyn S
authors.initials: SS
authors.lastname: Jayakar
authors.affiliation: the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, and.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612.
authors.forename: Pavel Y
authors.initials: PY
authors.lastname: Savechenkov
authors.affiliation: From the Departments of Neurobiology and.
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.affiliation: the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, and.
authors.forename: Rooma
authors.initials: R
authors.lastname: Desai
authors.affiliation: the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612.
authors.forename: Karol S
authors.initials: KS
authors.lastname: Bruzik
authors.affiliation: the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Miller
authors.affiliation: From the Departments of Neurobiology and jonathan_cohen@hms.harvard.edu.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid
keywords: Cys loop receptor; GABA receptor; allosteric regulation; anesthesia; homology modeling; photoaffinity labeling