J. Machold et al., PHOTOLABELING REVEALS THE PROXIMITY OF THE ALPHA-NEUROTOXIN BINDING-SITE TO THE M2 HELIX OF THE ION-CHANNEL IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR, Proceedings of the National Academy of Sciences of the United Statesof America, 92(16), 1995, pp. 7282-7286
A photoactivatable derivative of neurotoxin II from Naja naja oxiana c
ontaining a I-125-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl l
abel at Lys-25 forms a photoinduced cross-link with the delta subunit
of the membrane-bound Torpedo californica nicotinic acetylcholine rece
ptor (AChR). The cross-linked radioactive receptor peptide was isolate
d by reverse-phase HPLC after tryptic digestion of the labeled delta s
ubunit. The sequence of this peptide, delta-(260-277), and the positio
n of the label at Ala-268 were established by matrix-assisted laser-de
sorption-ionization mass spectrometry based on the molecular mass and
on post-source decay fragment analysis. With the known dimensions of t
he AChR molecule, of the photolabel, and of alpha-neurotoxin, finding
the cross-link at delta Ala-268 (located in the upper part of the chan
nel-forming transmembrane helix M2) means that the center of the a-neu
rotoxin binding site is situated at least approximate to 40 Angstrom f
rom the extracellular surface of the AChR, proximal to the channel axi
s.