The Handedness of the Subunit Arrangement of the Nicotinic Acetylcholine Receptor from Torpedo californica

Abstract

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.