Projection structure of the nicotinic acetylcholine receptor: distinct conformations of the alpha subunits

The nicotinic acetylcholine (ACh) receptor is a neurotransmitter-gated ion channel consisting of a ring of five membrane-spanning subunits, including two (the alpha subunits) with identical amino acid sequences. To open the channel ACh has to bind at two sites, involving both alpha subunits, which have widely different affinities. An earlier three-dimensional electron microscopic study of the non-activated Torpedo receptor had suggested that these sites might be cavities with the alpha subunits, located 25 to 30 A from the membrane, and hence that the different affinities might be associated with alternative conformations of the alpha subunits. This paper compares the conformations of the alpha subunits by determining the projection structure of the non-activated receptor and correlating the projection and three-dimensional maps. The projection structure was calculated to 7.5 A resolution from images of ice-embedded "giant" tubular crystals of Torpedo membranes, which had been partially flattened to make pairs of two-dimensional sheets (rho 2 lattice: a=90.2 A, beta=162.3 A, gamma=121.7 degrees). Each subunit in projection occupied a sector of approximately 72 degrees, and had a peak of high density at a distance of 22 to 25 A from the pseudo 5-fold axis. This peak in the single subunit between the alphas was approximately 3 A further from the axis than were the other peaks, and the projected density distributions composing the two alpha subunits were different. Close matching of the projected densities with the three-dimensional densities 25 to 35 A from the membrane, showed unequivocally that corresponding (alpha-helical) rods, which encircle the cavities in the two alpha subunits, are unequally inclined. Therefore, the cavities are indeed shaped by distinct conformations of the alpha subunits before ACh has bound. A model is proposed of how both alpha subunits participate concertedly to open the channel, assuming that ACh binds to these internal sites.