Some 40 million patients are given general anesthesia each year in the USA using agents that haven't changed for decades. General anesthetics are used as hypnotics during procedures, sedatives in the ICU, and as anticonvulsants. These drugs have low safety margins and are particularly harmful to newborns, the severely ill, and the elderly. The molecular mechanisms of general anesthesia have proved difficult to reveal, hampering the design of improved agents. This interdisciplinary PPG brings together a group of experienced scientists to focus on mechanisms underlying the ability of general anesthetics to enhance the activity of the inhibitory GABAA receptors (GABAAR) and glycine receptors (GlyR), major targets of anesthetics. The overall hypothesis is that the various actions of general anesthetics are mediated by a number of distinct binding sites on these receptors, that their location and affinity varies with the receptor's subunit composition and conformational state. The overall aims of the PPG are to: (i) locate the anesthetic binding sites on heteromeric GABAARs and GlyRs using anesthetic photolabels, (ii) define anesthetic structure-activity relationships at distinct sites, and (iii) define how their occupancy allostericlly modulates receptor function using electrophysiology and targeted mutations. Project 1 (PI: Cohen) will locate the unknown binding sites for steroids and propofol anesthetics and a convulsant barbiturate on synaptic alpha-1-beta-3 gamma-2 and extrasynaptic alpha-4-beta-3-delta GABAARs and on alpha-1-beta GlyRs. Project 2 (PI: Miller) focuses on exploring the molecular pharmacology of the different anesthetic binding sites that the PPG has discovered on alpha-1-beta-3-gamma-2 and alpha-4-beta-3-delta GABAARs, with the objective of understanding how to improve the specificity of anesthetic action on synaptic and extrasynaptic receptors. In addition, new binding sites that have high affinity for the activated states will be sought. Project 3 (PI: Forman) will use mutations and electrophysiology to define the functional roles, including drug specificity, of distinct general anesthetic binding sites, particularly the iter-subunit transmembrane sites on alpha-1-beta-3-gamma-2 and alpha-4-beta-3-delta GABAARs and alpha-1-beta GlyRs. Photolabeling results and homology models will guide mutagenesis, while allosteric co-agonist modeling will be used as a quantitative analytical framework for electrophysiological data. The Projects are supported by four Core facilities. An Administrative Core (Core A;PI: Miller) coordinates the activities of the components of the PPG, also arranging external oversight through a distinguished Advisory Board. A Synthetic Chemistry Core (Core B;PI: Bruzik) develops and supplies novel general anesthetics for photolabeling and structure activity relationships. A Protein Chemistry Core (Core C;PI: Cohen) locates anesthetic sites of photoincorporation and develops homology models of receptors to guide research. A Protein Production Core (Core D;co-PIs: Miller &Forman) supplies nanomoles of heterologously expressed, purified and functionally reconstituted human neuronal receptors for photolabeling and other studies.

Public Health Relevance

General anesthetics are essential to modern medicine, yet they have low safety margins especially in sick or elderly patients, requiring delivery by highly trained clinicians. General anesthetics interact with many receptors in the brain to cause both anesthesia and side effects. This Program Project aims to provide new knowledge so that new anesthetics can target just the anesthetic receptors and not those responsible for side effects.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
2P01GM058448-16
Application #
8742127
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Cole, Alison E
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
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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

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