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

Abstract

The N-terminal extracellular region (amino acids 1-209) of the α- subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the 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 obtained displaying approximately 15% α-helical and 45% β- structure. The fragment bound α-[3H]bungarotoxin in 1:1 stoichiometry with a K(D) value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 105 M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with α-[3H]bungarotoxin yielding the following K(I) values: acetylcholine, 69 μM; nicotine, 0.42 μM; anatoxin-a, 3 μM; tubocurarine, 400 μM; and methyllycaconitine, 0.12 μM. The results demonstrate that the N-terminal extracellular region of the nAChR α-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.