This is a competitive renewal of R01 GM56257, which has over the past four years supported in-depth investigations on the molecular nature of general anesthetic interaction with transmembrane (TM) channel proteins and model peptides. Various biophysical approaches, notably the state-of-the-art high-resolution nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD), have been combined with modem molecular biology techniques to elucidate general anesthetic effects on the structures and dynamics of TM channels. In this continuation project, a research expansion will be made from the development of conceptual framework using a simplified model channel peptide to the systematic characterization of a physiologically relevant system consisting of alpha4 (a4) and beta2 (b2) subunits of neuronal nicotinic acetylcholine receptors (nAChR). With the fulfillment of the specific aims for the previous funding period, a significant step will be made toward the long-term goal of the project, that is, to identify the molecular events responsible for the physiological effects of general anesthesia. Unlike many functional analyses that establish the protein sequence-function relationship based on inferences from anesthetic sensitivity data, the approach taken in this project aims at identifying the structure-function and dynamics-function relationships with direct binding and dynamics analysis at sub-molecular and atomic resolutions. A substantial amount of new structural and dynamical data on alpha4 and beta2 subunits of nAChR has been obtained to support the following four new specific aims: (1) to express and engineer functional TM domains of the anesthetic-sensitive alpha4 and beta2 subunits and the anesthetic-insensitive (or less-sensitive) a7 subunit of neuronal nAChR with selective and segmental isotopic labeling so that their structures and dynamics can be examined by NMR in membrane-mimetic and native lipid environments; (2) to determine the TM1+TM2+TM3 and TM4 structures of nAChR alpha4, beta2, and alpha7 subunits in homo-oligomeric states and in the 2alpha4:3beta2 mixture in membrane-mimetic micelles by NMR; (3) to differentiate anesthetic and nonimmobilizer effects on the structures of TM1+TM2+TM3 and TM4 of nAChR alpha4, beta2, and alpha7subunits in homo-oligomeric states and in the 2a4:3b2 mixture in membrane-mimetic micelles; and (4) to quantify anesthetic and nonimmobilizer effects on water exchange and the backbone and side chain dynamics of TM1+TM2+TM3 of nAChR alpha4, beta2, and alpha7 subunits in homo-oligomeric states and in 2alpha4:3beta2 mixtures in membrane-mimetic micelles, thereby elucidating the structural and dynamical requirement controlling the channel sensitivity to general anesthetics. These new studies will eventually help to close the ever-widening gulf between a plethora of sequence-based characterizations of channel function and a dearth of high-resolution structural and dynamical information.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056257-09
Application #
7057785
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Cole, Alison E
Project Start
1998-05-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2008-04-30
Support Year
9
Fiscal Year
2006
Total Cost
$290,021
Indirect Cost
Name
University of Pittsburgh
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Chen, Qiang; Xu, Yan; Tang, Pei (2018) X-Ray Crystallographic Studies for Revealing Binding Sites of General Anesthetics in Pentameric Ligand-Gated Ion Channels. Methods Enzymol 603:21-47
Bondarenko, Vasyl; Wells, Marta; Xu, Yan et al. (2018) Solution NMR Studies of Anesthetic Interactions with Ion Channels. Methods Enzymol 603:49-66
Tang, Pei; Eckenhoff, Roderic (2018) Recent progress on the molecular pharmacology of propofol. F1000Res 7:123
Chen, Qiang; Wells, Marta M; Arjunan, Palaniappa et al. (2018) Structural basis of neurosteroid anesthetic action on GABAA receptors. Nat Commun 9:3972
Wang, Yali; Yang, Elaine; Wells, Marta M et al. (2018) Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites. J Gen Physiol 150:1317-1331
Ion, Bogdan F; Wells, Marta M; Chen, Qiang et al. (2017) Ketamine Inhibition of the Pentameric Ligand-Gated Ion Channel GLIC. Biophys J 113:605-612
Chen, Qiang; Wells, Marta M; Tillman, Tommy S et al. (2017) Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC. Structure 25:180-187
Wu, Jie; Liu, Qiang; Tang, Pei et al. (2016) Heteromeric ?7?2 Nicotinic Acetylcholine Receptors in the Brain. Trends Pharmacol Sci 37:562-574
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
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

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