NICOTINIC ACETYLCHOLINE-RECEPTOR AT 9-ANGSTROM RESOLUTION

The nicotinic acetylcholine receptor is a cation-selective, ligand-gated ion channel, involved in signal transmission at the chemical synapse. This paper reports the three-dimensional appearance of the channel in the closed conformation, at 9 Å resolution. The structure was determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was carried out by averaging data from separate images, using helical diffraction methods. The images were recorded over a wide range of defocus (7000 to 18,800 Å) so that all spacings in the object were well sampled. Tubes of only one kind ((-16,6) helical family) were processed, so that the Fourier terms could be averaged directly in reciprocal space. The three-dimensional map, obtained from 26 images, resolves some elements of secondary structure within the five protein subunits. In the synaptic part of each subunit, about 30 Å from the bilayer surface, there is a group of three rods that are oriented predominantly perpendicular to the plane of the bilayer and twist around each other as in a left-handed coil. These rods presumably are α-helices. Two of them line the entrance to the channel, and the third is on the outside. The distinctive appearance of the α subunits in this region suggests that the rods may be involved in forming the binding pocket for acetylcholine. In the bilayer-spanning part of each subunit there is only one rod clearly visible, which forms the wall lining the pore, and so is assumed to be the transmembrane helix, M2. This rod does not form a straight path through the lipid bilayer, but bends, or kinks, near its mid-point, where it is closest to the axis of the pore, and tilts radially outwards on either side. It is flanked on the lipid-facing sides by a continuous rim of density, which seems likely to be composed of β-sheet. A tentative alignment is made between the three-dimensional densities and the sequence of M2, based on correlation of the appearance of the rods with a special pattern of amino acid residues in the sequence. This alignment places the charged groups at the ends of M2 symmetrically on either side of the bilayer, and a highly conserved leucine residue (Leu251 of the α subunit) at the level of the kink. It is suggested that leucine side-chains projecting into the pore from the kinks associate and create a tight hydrophobic ring, which closes the channel by making a barrier that hydrated ions cannot cross.