- Project 3 This Program Project Grant aims to define the sites and mechanisms of general anesthetics on pentameric ligand-gated ion channels, specifically heteromeric GABAA and glycine receptors, and to develop novel compounds with greater selectivity for the various unique sites on these receptors. Our photolabeling and structure-function studies have established a novel and cohesive paradigm for the molecular mechanism of etomidate at its major targets, a subset of GABAA receptors. Etomidate binds in the extracellular portion of two of the five transmembrane subunit interfaces in heteromeric abg GABAARs, and functions as an allosteric co- agonist. Preliminary data indicate that other potent anesthetics that target GABAARs, including novel barbiturates (mTFD-MPAB) and propofol derivatives, act variously at the etomidate sites and/or homologs of these sites in other subunit interfaces, with different degrees of specificity. In contrast, data suggest that alphaxalone binds deeper within the transmembrane subunit interfaces. We further hypothesize that several types of subunit interfacial anesthetic sites are formed in heteromeric GlyRs, and that these are also homologs of sites in GABAA receptors. A corollary of our hypothesis is that differential anesthetic sensitivity of different GABAAR subtypes is due to natural sequence variations in the anesthetic binding sites.
The aims of Project 3 are to: 1) assess the functional roles of amino acids in the different unique anesthetic sites of several key abg and abd GABAARs as well as a1b GlyRs;2) to develop quantitative models accounting for the effects of etomidate, propofol, mTFD-MPAB, and alphaxalone at each unique interfacial site and in different types of receptors;3) identify mutations within each site that selectively ablate anesthetic effects;and 4) test novel compounds for modulatory potency and efficacy in both GABAA and Gly receptors. Based on photolabeling data from Project 1 and homology models of the transmembrane subunit interfacial cavities (Protein Chemistry Core;C), Project 3 (supported by the Protein Synthesis Core;D) will mutate single residues to tryptophan (hypothesized to mimic bound anesthetic) and electrophysiologically assess mutant effects on receptor gating, GABA sensitivity, and anesthetic sensitivity. We will quantify mutant effects using the formal mechanistic framework of two-state Monod-Wyman-Changeux allosteric co-agonism, and apply MWC models with unique binding and efficacy parameters at distinct anesthetic sites to test whether these sites equally and independently contribute to anesthetic effects. Mutations that selectively ablate anesthetic actions in single sites will be identified for drug screening in this Project and Project 2. We will also help screen novel compounds synthesized by the Synthetic Chemistry Core (B). Thus, this project synergizes with the other projects and scientific cores to help achieve the overall aims of the P01.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
2P01GM058448-16
Application #
8742134
Study Section
Special Emphasis Panel (ZGM1)
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
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