Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor

Exploitation of soluble extracellular domains (ECDs) of the nicotinic acety lcholine receptor may provide a route to crystallographic studies aimed at exploring the structure and function of the intact receptor. The first step towards this goal is to manufacture and isolate soluble fragments that fol d and assemble to form a functionally relevant complex. The baculovirus ins ect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (alpha, beta, gamma & delta -ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folde d alpha subunit, or a NiNTA affinity resin, specific for a polyhistidine ta g engineered on the delta -ECD. Western blotting with subunit specific anti bodies confirmed the co-expression of each ECD and furthermore, indicated t hat the alpha, beta and gamma -ECDs were being co-purified with the polyhis tidine-tagged delta -ECD. Chemical cross-linking was used to show that thes e co-purified proteins had indeed interacted specifically to form soluble o ligomeric complexes. A low-resolution, three-dimensional image of these pur ified complexes, composed only of ECDs, was obtained by electron microscopy . They were shown to resemble the extracellular vestibule of the native rec eptor, having the same pseudo-pentameric symmetry, size and shape. Expressi on of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes. (C) 2000 Academic Press.