Identification of key determinants in α subunit for α-conotoxin [D1G, ΔQ14]LvIC selectively targeting rat α6/α3β4 nicotinic acetylcholine receptor

67 single-point mutants were generated and then were assessed for properties. • The 152nd, 157th, and 195th residues of α subnuit affect selectivity for [D1G,ΔQ14]LvIC. • Combined mutations show greater impacts on the activity of [D1G,ΔQ14]LvIC. • E152K mutation in rα6/α3 most affects inhibition via ionic interaction with peptide. • The 157th and 195th residues may alter spatial conformation, affecting peptide binding potency. nAChRs containing α6 subunit (α6* nAChRs) are identified as being involved in Parkinson's disease, dyskinesia, drug addiction, and other disorders. Among these, α6β4 nAChR is recognized as a highly promising target for the development of non-opioid analgesics. However, the research advancement on α6β4 nAChR has been hindered primarily by the overlapping tissue distribution with highly homologous α3β4 nAChR, as well as the scarcity of subtype-specific ligands. Recently, the α-conotoxin analogue [D1G, ΔQ14]LvIC was identified to exhibit remarkable selectivity for rα6/α3β4 nAChR with an IC 50 of 19 nM, while showing negligible inhibitory activity against other subtypes, including closely related rα3β4 (IC 50  > 10 μM), demonstrating exceptional subtype selectivity and potency compared to previously reported ligands for this receptor. In this study, we employed a combined approach of PCR-mediated site-directed mutagenesis and two-electrode voltage clamp electrophysiology to identify the determinants governing the [D1G, ΔQ14]LvIC selectivity. 67 single-point mutants of rα6/α3β4 nAChR were generated, systematically replacing each differing residue in rα6/α3 to rα3 subunit. The activity of [D1G, ΔQ14]LvIC was subsequently assessed on the native receptor and its mutants. As the results showed, E152, I157, and T195 in rα6/α3 subunit were identified as essential for [D1G, ΔQ14]LvIC selectivity. Inverse and multiple-point mutants were subsequently constructed and assessed for further validation. Molecular modeling further shed light on the molecular basis for the unique interaction between [D1G, ΔQ14]LvIC and rα6/α3β4 nAChR. These findings advance our understanding of α6β4 nAChR-ligand interactions and pave the way for the rational design of targeted therapeutics. Download: Download high-res image (371KB) Download: Download full-size image