PHOTOLABELING REVEALS THE PROXIMITY OF THE ALPHA-NEUROTOXIN BINDING-SITE TO THE M2 HELIX OF THE ION-CHANNEL IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a I-125-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photoinduced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the a-neurotoxin binding site is situated at least approximate to 40 Angstrom from the extracellular surface of the AChR, proximal to the channel axis.