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.
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