How do acetylcholine receptor ligands reach their binding sites?

The access pathway to the binding sites for large competitive antagonists o f the nicotinic acetylcholine receptor from Torpedo californica electric ti ssue was analyzed by binding and photolabeling experiments with alpha-neuro toxins. Binding assays with [I-125]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 fl uidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed memb rane state. Using a novel series of large biotinylated photoactivatable der ivatives of neurotoxin II, we observed that the accessibility to the alpha/ gamma- but not to the alpha/delta-binding site was considerably decreased f or some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. T hese observations support the nonequivalence of the receptor's binding site s. Together, our data suggest (a) that alpha-neurotoxins approach their bin ding sites from the membrane-facing periphery of the receptor's extramembra ne domain rather than through the channel mouth and (b) that different entr ance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.