Expression and renaturation of the N-terminal extracellular domain of Torpedo nicotinic acetylcholine receptor alpha-subunit

The N-terminal extracellular region (amino acids 1-209) of the alpha-subuni t of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata el ectric tissue was expressed as inclusion bodies in Escherichia coli using t he pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was o btained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[H-3]bungarotoxin in 1:1 stoichiometry with a K-D value of 0.5 nM as determined from kinetic measurements (4 nM from equilibr ium binding), The kinetics of association of toxin and fragment were of sec ond order, with a similar rate constant (8.2 x 10(5) M-1 s(-1)) as observed previously for the membrane-bound heteropentameric nAChR, Binding of small ligands was demonstrated by competition with alpha-[H-3]bungarotoxin yield ing the following K-I values: acetylcholine, 69 mu M; nicotine, 0.42 mu M; anatoxin-a, 3 mu M; tubocurarine, 400 mu M; and methyllycaconitine, 0.12 mu M. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expre sses major elements of the ligand binding sites of the receptor.