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

Selwyn S Jayakar,Xiaojuan Zhou,Pavel Y Savechenkov,David C Chiara,Rooma Desai,Karol S Bruzik,Keith W Miller,Jonathan B Cohen

doi:10.1074/jbc.m115.672006

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.

Highlights

For some chiral barbiturates, one isomer potentiates and the other inhibits GABA responses by binding to unknown sites
In the ␣1␤3␥2 GABA type A receptors (GABAAR), S-mTFD-MPPB binds in the transmembrane domain with high affinity to the ␥؉-␤؊ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the ␤؉-␣؊ subunit interfaces
We demonstrate that the S-isomer of mTFDMPPB, an ␣1␤3␥2 GABAAR inhibitor, stabilizes the receptor in a closed channel state by binding with high affinity to a transmembrane domain (TMD) site in the ␥ϩ-␤Ϫ interface previously identified as a binding site for the anesthetic barbiturate R-mTFD-MPAB [23]

Summary

Background

One isomer potentiates and the other inhibits GABA responses by binding to unknown sites. In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of ␥-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 For mTFD-MPPB, differing from mTFD-MPAB by only two more hydrogen atoms ([1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl]barbituric acid, Fig. 1), the R-enantiomer acts as an anesthetic in vivo and potentiated GABA responses for expressed ␣1␤3␥2 GABAAR, whereas the S-enantiomer acts as a convulsant and inhibits GABA responses [26]. Based upon the direct identification of photolabeled amino acids and the pharmacological specificity of photolabeling, we find that S-[3H]mTFD-MPPB binds in the same ␥ϩ-␤Ϫ interface pocket as R-mTFD-MPAB It binds in a different orientation, and its binding is inhibited allosterically by GABA, indicative of negative energetic coupling between the sites. Our results provide a first demonstration that, similar to the benzodiazepine-binding site at the ␣ϩ-␥Ϫ interface in the ECD [27], at least one intersubunit-binding site in the GABAAR’s TMD is a target for negative as well as positive allosteric modulators

Experimental Procedures

Results

Discussion

39 Ϯ 5a 29 Ϯ 6c