. The overall hypothesis of Project 2 is that general anesthetics bind to several distinct sites on the GABAAR and that each distinct site has its own pharmacology. ,We aim to devise agents that bind selectively to each distinct site. Furthermore, the affinity for each site depends on: (1) the receptor's subunit composition.
We aim to develop anesthetics that target specific subpopulations of GABAARs, such as ?-subunit containing extrasynaptic vs. ?-subunit containing synaptic receptors;(2) its conformational state. The best general anesthetics will bind with higher affinity to the open than to the resting state, the ratio of affinities defining the anesthetic's efficacy. In extreme cases, affinity for the resting state may be so low that a binding site may only be detectable in the open state of a GABAAR. The project is supported by: (1) the Synthetic Chemistry Core (Core B), with whom we will devise new anesthetics and anesthetic photolabels to support all aims;(2) the Protein Chemistry Core (Core C) who provide HPLC assays required in Aim 1 and the sequencing required in Aims 2 &3, and (3) the Protein Production Core (Core D) who provide the large amounts of GABAARs of various subunit compositions required in all aims.
Aim 1 : The PPG has discovered that [3H]R-azi-etomidate and a barbiturate, [3H]R-mTFD-MPAB, bind at different sites between subunits in the transmembrane domain. These sites are homologous in secondary structure, but differ subtly in sequence. These two agents provide us with tools to study the structure activity relationships that govern selective binding to their separate sites. In addition, the PPG has determined that there is a separate nonhomologous site in the extracellular domain that a convulsant barbiturate interacts with. Because general anesthetics often cause excitation and close analogs are often convulsant or pro-convulsant, it is important to discover the structural rules governing binding to this convulsant site so that undesirable excitatory side effects can be eliminated when designing new general anesthetics, thus lowering toxicity and improving patient care.
Aim 2 seeks new sites on the GABAAR. (1) Inhalational anesthetics can fit into smaller binding pockets than those the PPG has detected to date for intravenous anesthetics, so we will devise smaller photolabels to test this hypothesis. (2) What are the structure activity relationships of the transmembrane domain sites neighboring ?-subunits in GABAARs representative of extrasynaptic receptors? Aim 3 uses time resolved photolabeling to test the hypotheses that: (1) There are intrasubunit sites within the bundle of four helices in the transmembrane domain that only become occupied upon activation, and (2) the prediction of the allosteric model that the relative affinity of an anesthetic for the open vs. the resting state is proportional to efficacy for enhancing GABA-induced currents. This interdisciplinary project aims to improve patient safety by providing new information and concepts that will guide the development of more selective general anesthetics.

National Institute of Health (NIH)
Research Program Projects (P01)
Project #
Application #
Study Section
Special Emphasis Panel (ZGM1)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts General Hospital
United States
Zip Code
Eaton, Megan M; Germann, Allison L; Arora, Ruby et al. (2016) Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol. Curr Neuropharmacol 14:772-80
Zhang, Xi (2016) Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias. Mol Cell Proteomics 15:3665-3684
Forman, Stuart A; Miller, Keith W (2016) Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes. Anesth Analg 123:1263-1273
Chiara, David C; Jounaidi, Youssef; Zhou, Xiaojuan et al. (2016) General Anesthetic Binding Sites in Human α4β3δ γ-Aminobutyric Acid Type A Receptors (GABAARs). J Biol Chem 291:26529-26539
Amlong, Corey A; Perkins, Mark G; Houle, Timothy T et al. (2016) Contrasting Effects of the γ-Aminobutyric Acid Type A Receptor β3 Subunit N265M Mutation on Loss of Righting Reflexes Induced by Etomidate and the Novel Anesthetic Barbiturate R-mTFD-MPAB. Anesth Analg 123:1241-1246
Ziemba, Alexis M; Forman, Stuart A (2016) Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of α1β3γ2L GABAA Receptors. PLoS One 11:e0154031
Nourmahnad, Anahita; Stern, Alex T; Hotta, Mayo et al. (2016) Tryptophan and Cysteine Mutations in M1 Helices of α1β3γ2L γ-Aminobutyric Acid Type A Receptors Indicate Distinct Intersubunit Sites for Four Intravenous Anesthetics and One Orphan Site. Anesthesiology 125:1144-1158
Forman, Stuart A; Chiara, David C; Miller, Keith W (2015) Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors. Neuropharmacology 96:169-77
Hamouda, Ayman K; Wang, Ze-Jun; Stewart, Deirdre S et al. (2015) Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor. Mol Pharmacol 88:1-11
Liu, K; Jounaidi, Y; Forman, S A et al. (2015) Etomidate uniquely modulates the desensitization of recombinant α1β3δ GABA(A) receptors. Neuroscience 300:307-13

Showing the most recent 10 out of 104 publications