Engineering of α-conotoxin MII-derived peptides with increased selectivity for native α6β2* nicotinic acetylcholine receptors.

α6β2* Nicotinic acetylcholine receptors are expressed in selected central nervous system areas, where they are involved in striatal dopamine (DA) release and its behavioral consequences, and other still uncharacterized brain activities. α6β2* receptors are selectively blocked by the α-conotoxins MII and PIA, which bear a characteristic N-terminal amino acid tail [arginine (R), aspartic acid (D), and proline (P)]. We synthesized a group of PIA-related peptides in which R1 was mutated or the RDP motif gradually removed. Binding and striatal DA release assays of native rat α6β2* receptors showed that the RDP sequence, and particularly residue R1, is essential for the activity of PIA. On the basis of molecular modeling analyses, we synthesized a hybrid peptide (RDP-MII) that had increased potency (7-fold) and affinity (13-fold) for α6β2* receptors but not for the very similar α3β2* subtype. As docking studies also suggested that E11 of MII might be a key residue engendering α6β2* vs. α3β2* selectivity, we prepared MII[E11R] and RDP-MII[E11R] peptides. Their affinity and potency for native α6β2* receptors were similar to those of their parent analogues, whereas, for the oocyte expressed rat α3β2* subtype, they showed a 31- and 14-fold lower affinity and 21- and 3.5-fold lower potency. Thus, MII[E11R] and RDP-MII[E11R] are potent antagonists showing a degree of α6β2* vs. α3β2* selectivity in vivo.