Some 25 million patients are given general anesthesia each year in the USA using agents with very low therapeutic indices. The molecular mechanisms of general anesthesia remain unknown, hampering the design of improved agents. General anesthetics act on a superfamily of ligand gated channels which include inhibitory anion channels gated by GABA and glycine, and excitatory cation channels gated by serotonin and acetylcholine. This PPG focuses on the ability of general anesthetics to enhance the activity of the inhibitory GABAA receptor (GABAAR) and to inhibit, and in some cases enhance, the excitatory neuronal nicotinic acetylcholine (nAcChoR) and serotonin (5HT3R) receptors. The overall hypothesis is that the various actions of general anesthetics are mediated by a number of binding sites on these receptors, that their location and affinity varies with the anesthetic's structure and the receptor's conformation. The overall aims of the PPG are to: (i) locate the anesthetic binding sites on the GABAA, nAcChoR and 5-HT3 receptors using anesthetic photolabels, and (ii) define how their occupancy allosterically modulates receptor function using rapid perfusion patch clamp techniques. Project 1 will locate sites of etomidate, propofol and barbiturate photolabels on equilibrium states of GABAAR and nAcChoRs. Project 2 focuses on the interaction of anesthetics with receptors during gating using in parallel electrophysiological and time-resolved photolabeling. Project 3 will define in detail the kinetic mechanisms of anesthetic action on GABAARS using rapid perfusion patch clamp techniques in wild type and mutated receptors, incorporating the photolabeling results to guide mutagenesis and interpretation. A Synthetic Chemistry Core develops and supplies novel photoaffinity general anesthetics. A Protein Chemistry Core locates the sites of photoincorporation and develops homology models of receptors to guide mutagenesis work. A Protein Production Core supplies large quantities of heterologously expressed, purified and reconstituted neuronal receptors for photolabeling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM058448-14
Application #
8322063
Study Section
Special Emphasis Panel (ZGM1-PPBC-0 (AN))
Program Officer
Cole, Alison E
Project Start
1998-12-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
14
Fiscal Year
2012
Total Cost
$1,843,602
Indirect Cost
$492,858
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
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