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.
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