CARATTERIZZAZIONE FARMACOLOGICA E FUNZIONALE DI NUOVI LIGANDI DEI RECETTORI COLINERGICI NICOTINICI NEURONALI CHE MODULANO IL RILASCIO DI DOPAMINA NELLA VIA MESOSTRIATALE, UNA VIA IMPORTANTE PER GLI EFFETTI COMPORTAMENTALI DELLA NICOTINA.

The α6β2∗ neuronal nicotinic acetylcholine receptor (nAChR) subtype expressed in the dopaminergic mesostriatal pathway mediates many behavioural effects of nicotine, and is selectively blocked by the small disulfide-rich α-conotoxins PIA and MII. Both share a "ω-shaped" topology but PIA bears a tail in the N-terminal region containing three amino acids [arginine (R), aspartic acid (D), and proline (P)]. We synthesised a group of PIA-related peptides in which R1 was mutated or the RDP motif was gradually removed. Binding and functional studies showed that the RDP sequence is essential for the activity of PIA on the native rat α6β2* subtype, with a major role played by residue R1. Molecular modelling studies showed that recognition of PIA by α6β2* nAChRs depends on a salt bridge between the guanidine group of R1 and the highly negatively charged D166-D167 residues located on the β2 subunit. The RDP sequence was then added to the N-terminus of MII; the resulting hybrid peptide (RDP-MII) showed an increased potency (5-fold) and affinity (13-fold) for α6β2* but not for α3β2* nAChRs. Furthermore, as docking studies indicated E11 as a potential key residue engendering a α6β2* vs. α3β2* selectivity, we prepared and tested the following E11 mutated MII analogues: MII[E11R] and RDP-MII[E11R]. The binding and functional profiles of the new peptides at native rat α6β2* receptor were comparable with those of their leads while potency and affinity for native and heterologously expressed α3β2* nAChRs were reduced. Consequently, MII[E11R] and RDP-MII[E11R] are potent and α6β2* vs. α3β2* selective antagonists.