This is a competing renewal of R01GM056257 for its 10th-13th years. The ultimate goal of the study is to elucidating the molecular mechanisms of general anesthesia. The research in the previous funding periods has led the investigators to realize that protein global dynamics (i.e., the slow modes of motion at tertiary and quaternary structural levels) is one of the most crucial elements in the manifestation of neurotransmitter-gated receptors'sensitivity to general anesthetics. Modulation of protein global dynamics may underlie a common molecular mechanism of anesthetic action. The approaches in this competing renewal aim at quantifying the anesthetic-induced changes in protein structure and dynamics at or near atomic resolution, using modern molecular biology and various biophysical techniques, notably the state-of-the-art high-resolution and solid- state nuclear magnetic resonance (NMR) spectroscopy. The central hypothesis is that critical amphipathic residues of ligand-gated near the membrane interface are constantly modulated by interactions with interfacial water and lipid molecules. This modulation produces a steady-state control of the channel dynamics, which in turn governs the transport properties of the channel. Anesthetics can preferentially target these pivotal residues and offset the balance of the steady-state channel dynamics by changing water or lipid association with the critical residues, thereby altering the characteristics of synaptic transmission through the channel. Substantive published as well as new preliminary results support the following three specific aims: (1) to determine and identify the structure differences between the anesthetic-supersensitive (14)2(22)3 and anesthetic-insensitive (17)5 neuronal nicotinic acetylcholine receptor (nAChR) transmembrane (TM) domains so that their different sensitivities to volatile anesthetics (halothane and isoflurane) can be understood on a structural basis;(2) to quantify the global dynamics of the (14)2(22)3 and (17)5 nAChR TM domains in membrane-mimetic environments in the absence and presence of anesthetics (halothane and isoflurane);and (3) to correlate channel global dynamics with ion transport in the presence and absence of anesthetics (a) by determining the dynamics of channel mutants having altered sensitivity to anesthetics, such as the double mutants of 14 (I268M and V274S) and 22 (V262M and D268S) with the corresponding residues in the TM2-TM3 loop of 17 subunit, and (b) by determining channel formation and ion transport rate of the wild type and mutants of the (14)2(22)3 and (17)5 nAChRs in membrane vesicles using fluorescence imaging and NMR exchange experiments. The results of this investigation will lead to a new paradigm for the analysis and interpretation of general anesthetic action on neuronal proteins.
This research will advance our understanding on how and where general anesthetics act at the molecular level. It will also solve the structure of the transmembrane domain of a class of neurotransmitter-gated receptor channels, for which no high-resolution structure is currently available. The research will eventually lead to better design of safer and more effective new anesthetic drugs.
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