title: How do acetylcholine receptor ligands reach their binding sites?
abstract: The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with alpha-neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor's binding sites. Together, our data suggest (a) that alpha-neurotoxins approach their binding sites from the membrane-facing periphery of the receptor's extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.
authors.affiliation: AG Neurochemie, Institut für Biochemie, Freie Universität Berlin, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Sáez-Briones
authors.forename: M
authors.initials: M
authors.lastname: Krauss
authors.forename: M
authors.initials: M
authors.lastname: Dreger
authors.forename: A
authors.initials: A
authors.lastname: Herrmann
authors.forename: V I
authors.initials: VI
authors.lastname: Tsetlin
authors.forename: F
authors.initials: F
authors.lastname: Hucho
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D003039:Cobra Neurotoxin Proteins / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008560:Membrane Fluidity; D008958:Models, Molecular; D018733:Nicotinic Antagonists / Q000378:metabolism; D019920:Photoaffinity Labels; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014101:Torpedo / Q000378:metabolism
chemical_list: D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D008024:Ligands; D018733:Nicotinic Antagonists; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; C076649:neurotoxin II, Naja naja oxiana