2 SUBSITES IN THE BINDING DOMAIN OF THE ACETYLCHOLINE-RECEPTOR - AN AROMATIC SUBSITE AND A PROLINE SUBSITE

The ligand binding site of the nicotinic acetylcholine receptor (AcCho R) is localized in the alpha-subunit within a domain containing the ta ndem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have p reviously shown that four residues in this region, at positions 187, 1 89, 194, and 197, differ between animals sensitive (e.g., mouse) and r esistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fra gment of the mongoose AcChoR alpha-subunit (residues 122-205) and exch anged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mong oose fragment in which all four residues were mutated to the mouse one s exhibited alpha-BTX binding similar to that of the mouse fragment. T he mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mo use AcChoR and in all other toxin binders, bound alpha-BTX to approxim ate to 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsite s in the binding domain for alpha-BTX: the proline subsite, which incl udes Pro-194 and -197 and is critical for alpha-BTX binding, and the a romatic subsite, which includes amino acid residues 187 and 189 and de termines the extent of alpha-BTX binding.