Role of histidine residues in the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific alpha-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrocarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 min period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70%; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.