The nicotinic Acetylcholine (ACh) receptor mediates fast synaptic transmission by converting transiently to an open-channel form when activated by ACh released into the synaptic cleft. Our goal is to understand the structural transition underlying this event, i.e. to provide a definitive molecular picture of how the channel works. The ACh receptor-rich membranes for our studies come from the electric organ of the Torpedo ray, and we analyze tubular membrane crystals grown from them by cryo-electron microscopy. The proposed research builds on a long-term programmed of technical development, which recently yielded a refined atomic model of the membrane-bound receptor in the closed-channel form. To investigate the open-channel structure we will propel acetylcholine-containing spray droplets onto the tubular crystals lying within a thin aqueous film on the e.m. grid and then trap the reaction (within ~2ms) by plunge-freezing. By this means we recapitulate the brief reaction of neurotransmitter with receptors at the synapse and capture the opened channels in both a physiological ionic environment and a native membrane setting. The electron images will be recorded with an ultra- stable liquid-helium cooled stage to minimize radiation damage and to optimize electron- optical performance. Distortion correction of the tube images by alignment of successive segments will incorporate a newly developed approach to optimize the extraction of high resolution information, and the computer programs will be automated/made-more-general in collaboration with colleagues working on related specimens at Scripps. Our electron crystallographic methodology provides a unique opportunity to analyze the open ACh receptor channels because it is the only structural method devised so far that can examine this short- lived transition under essentially the same conditions as exist in vivo. It is unlikely that x-ray structures of purified ACh (or other related) receptors, if they are obtained in the future, would be able to deliver such an unambiguous result.

Public Health Relevance

The nicotinic acetylcholine receptor is the best understood member of a family of synaptic ion channels which function in the central and peripheral nervous system, and are pharmaceutical targets for numerous human diseases and psychiatric disorders, including myasthenia gravis, neuromuscular degeneration, epilepsy, depression, nicotine addiction, schizophrenia and Alzheimer's disease. Our structural studies are providing three-dimensional information about the binding sites for anesthetics and other drugs which affect the brain by modulating the function of these receptors. By understanding better the biological mechanisms of these receptors, we contribute basic insight into the nature of the disorders themselves.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Flicker, Paula F
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Scripps Research Institute
La Jolla
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Unwin, Nigel (2013) Nicotinic acetylcholine receptor and the structural basis of neuromuscular transmission: insights from Torpedo postsynaptic membranes. Q Rev Biophys 46:283-322
Zuber, BenoƮt; Unwin, Nigel (2013) Structure and superorganization of acetylcholine receptor-rapsyn complexes. Proc Natl Acad Sci U S A 110:10622-7
Unwin, Nigel; Fujiyoshi, Yoshinori (2012) Gating movement of acetylcholine receptor caught by plunge-freezing. J Mol Biol 422:617-34
Fisher, Lauren S; Ward, Andrew; Milligan, Ronald A et al. (2011) A helical processing pipeline for EM structure determination of membrane proteins. Methods 55:350-62
Wilson-Kubalek, Elizabeth M; Chappie, Joshua S; Arthur, Christopher P (2010) Helical crystallization of soluble and membrane binding proteins. Methods Enzymol 481:45-62
Fujiyoshi, Yoshinori; Unwin, Nigel (2008) Electron crystallography of proteins in membranes. Curr Opin Struct Biol 18:587-92
Unwin, Nigel (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346:967-89
Unwin, N; Miyazawa, A; Li, J et al. (2002) Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the alpha subunits. J Mol Biol 319:1165-76
Unwin, Nigel (2002) Structure of the acetylcholine-gated channel. Novartis Found Symp 245:5-15; discussion 15-21, 165-8