Structural Mechanism of the Acetylcholine Receptor in Tubular Membrane Crystals

Scripps Research Institute, The, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant): 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.