On the Structure of the Acetylcholine Receptor from Torpedo californica Electroplaques

Abstract

Membranes highly enriched in nicotinic acetylcholine receptor from the electroplaque organ of Torpedo californica were studied using x-ray diffraction, electron microscopy, Fourier image analysis and biochemical techniques. High angle diffraction indicated that the receptor molecule contains 8 nm long alpha-helical stretches that run perpendicular to the membrane plane. The lipids are mostly fluid even at 2°C. Low angle diffraction analysis of oriented membranes under conditions where the continuous diffraction is recorded resulted in electron density profiles of the receptor-rich membranes in which the protein component has an overall length of 11 nm and is asymmetrically distributed with respect to the membrane bilayer, extending by 5.5 ± 0.5 nm on the extracellular side and 1.0 ± 0.5 nm on the other. Greater detail of the individual receptor molecule's structure normal to the membrane bilayer was attained by direct electron microscopic imaging which revealed the flaired, rivet-like shape of the extracellular part of the molecule and the extent to which the molecule extends beyond the intracellular surface (less than 1.0 nm). The receptor molecules were directly identified in negative-stain electron microscopy for the first time by immunological techniques using anti-receptor antibodies and recognized either by direct imaging, via ferritin-conjugated second antibody, or colloidal-gold labeled primary antibody (the first time the colloidal gold technique had been used in negative-stain microscopy). Small areas of receptor molecules form ordered lattices and this ordering is increased if the receptor membranes are prepared in native dimeric form, and stripped of peripheral proteins by alkali-treatment. The ordered areas have been Fourier image processed and result in a low resolution (3.8 nm) model of the surface projection in negative-stain of the receptor. A single monomeric unit packs in an a=8.8, b=8.8 nm, γ= 118° unit cell. The molecule's circular shape results in roughly centrosymmetric phases in the reflections observed, and yet the image reconstructions consistently reveal a common asymmetric structure. Combination of electron microscopic and x-ray diffraction data allows an estimate of the molecular weight of the membrane-bound monomer of 280000 and a radius of gyration of 4.2 nm. Image analysis tentatively indicates that the dimeric molecule does not contain a two-fold symmetry axis . The conventionally prepared membrane-bound receptor molecule is resistant to trypsin degradation, but removal of peripheral protein by alkali-treatment or solubilization of the membrane by detergents renders the molecule much more susceptible . All four subunits of the receptor can then be degraded and yet the receptor still binds both d-tubocurarine and α-bungarotoxin, and remains an approximately 9S structure in detergent. Proteolysis of the receptor results in a radical change in overall membrane morphology most probably the result of receptor aggregation in the plane of the membrane. Ethanol concentrations above 10% radically effect membrane morphology and may be useful in producing receptor membranes depleted in lipids. The presence of an endogenous protease activity in highly purified membrane fractions and the formation of receptor membrane sheets by osmotic shock onto clean water surfaces are described. Such sheets may hold a possibility for two-dimensional crystallization of the membrane-bound receptor.