The molecular mechanisms of action of general anesthetics remain an enigma. A superfamily of ligand-gated ion channels has been implicated as the primary target sites for general anesthetics. It has become increasingly clear from our own and other studies that amphiphilicity in regions near the membrane interface is a unifying property of anesthetic binding site(s). Thus, general anesthetics, but not nonimmobilizers (nonanesthetics), have been shown to target amphiphilic interfacial residues of transmembrane channel peptides, and point mutations in the transmembrane domains II and III (TM2 and TM3) of glycine and gamma- aminobutyric acidA (GABAA) receptors can completely abolish or even reverse the sensitivity of these receptors to alcohol and general anesthetics. Complete and detailed elucidation of the structure-function relationship will dramatically advance our understanding of general anesthetic action beyond what was even imaginable in the recent past. This competitive renewal will quantify specific interactions of strategically selected pairs of general anesthetics and nonimmobilizers with the TM2 and TM3 domains of the alpha1 subunit of human glycine receptors (GlyR). State-of-the-art protein expression and purification techniques will be coupled with high-resolution and solid-state nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and molecular dynamic simulations to accomplish three specific aims: (1) To express the wild-type and mutated TM2 and TM3 segments of GlyR alpha1 subunit for structural study by NMR. (2) To determine, at or near atomic resolution, the structures of the functional TM2 and TM3 segments of the human GlyR alpha1 subunit and the associated anesthetic-insensitive mutants. (3) To investigate the structural motifs contained in TM2 and TM3 for general anesthetic binding, and to quantify the effects of general anesthetic binding on channel dynamics within the determined structural frame, thereby elucidating the structural requirement that controls the channel sensitivity to general anesthetics. The long-term goal is to relate the structural events to functional changes caused by general anesthetics, paving the way for future in vivo and other studies to finally identify the sites of action of general anesthetics in the central nervous system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049202-08
Application #
6490061
Study Section
Special Emphasis Panel (ZRG1-SSS-W (21))
Program Officer
Cole, Alison E
Project Start
1995-01-01
Project End
2003-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
8
Fiscal Year
2002
Total Cost
$341,134
Indirect Cost
Name
University of Pittsburgh
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Kinde, Monica N; Bondarenko, Vasyl; Granata, Daniele et al. (2016) Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac. Proc Natl Acad Sci U S A 113:13762-13767
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Kinde, Monica N; Bu, Weiming; Chen, Qiang et al. (2016) Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels. Anesthesiology 124:664-73
Mowrey, David D; Kinde, Monica N; Xu, Yan et al. (2015) Atomistic insights into human Cys-loop receptors by solution NMR. Biochim Biophys Acta 1848:307-14
Wells, Marta M; Tillman, Tommy S; Mowrey, David D et al. (2015) Ensemble-based virtual screening for cannabinoid-like potentiators of the human glycine receptor ?1 for the treatment of pain. J Med Chem 58:2958-2966
Kinde, Monica N; Chen, Qiang; Lawless, Matthew J et al. (2015) Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC. Structure 23:995-1004

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