Expression and circular dichroism studies of the extracellular domain of the α subunit of the nicotinic acetylcholine receptor

Abstract

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle α subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (α210GPI) consisting of the 210 NH2- terminal amino acids of the α subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface α-bungarotoxin binding in oocytes. Coexpression of α210GPI with an analogous construct made from the δ subunit showed no evidence of heterodimer formation. The α210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The α210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded α subunit. Circular dichroism studies of α210GPI suggest that this region of the receptor includes considerable β- sheet secondary structure, with a small proportion of α-helix.