Efficient Binding of 4/7 α-Conotoxins to Nicotinic α4β2 Receptors Is Prevented by Arg185 and Pro195 in the α4 Subunit

alpha-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent alpha 3 beta 2 nAChR-selective alpha-conotoxins have been identified, currently characterized alpha-conotoxins show no or only weak affinity for alpha 4 beta 2 nAChRs, which are, besides alpha 7 receptors, the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand-binding domain of the alpha 4 subunit by the corresponding residues in the alpha 3 subunit. Two-electrode voltage clamp analysis of these mutants revealed increased affinity of alpha-conotoxins MII, TxIA, and [A10L]TxIA at the alpha 4(R185I)beta 2 receptor. Conversely, alpha-conotoxin potency was reduced at the reverse alpha 3(I186R)beta 2 mutant. Replacement of alpha 4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents alpha-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high alpha-conotoxin potency to the alpha 4 beta 2 receptor. Molecular dynamics simulations of homology models with docked alpha-conotoxin indicate that these residues control access to the alpha-conotoxin binding site.