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 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced 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 alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.
Machold
J
J
Institut für Biochemie, Freie Universität Berlin, Germany.
Utkin
Y
Y
Kirsch
D
D
Kaufmann
R
R
Tsetlin
V
V
Hucho
F
F
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010777:Photochemistry; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D003039:Cobra Neurotoxin Proteins; D003432:Cross-Linking Reagents; D007473:Ion Channels; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; C076649:neurotoxin II, Naja naja oxiana; D008239:Lysine