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 acetylcholine 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 fold and assemble to form a functionally relevant complex. The baculovirus insect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (α, β, γ & δ-ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folded α subunit, or a NiNTA affinity resin, specific for a polyhistidine tag engineered on the δ-ECD. Western blotting with subunit specific antibodies confirmed the co-expression of each ECD and furthermore, indicated that the α, β and γ-ECDs were being co-purified with the polyhistidine-tagged δ-ECD. Chemical cross-linking was used to show that these co-purified proteins had indeed interacted specifically to form soluble oligomeric complexes. A low-resolution, three-dimensional image of these purified complexes, composed only of ECDs, was obtained by electron microscopy. They were shown to resemble the extracellular vestibule of the native receptor, having the same pseudo-pentameric symmetry, size and shape. Expression of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes.