Nicotinic acetylcholine receptors (nAChR) play important roles in various physiological functions including pain, anxiety, fatigue, memory and learning. The α3β4 nAChR subtype has been implicated in various conditions, including lung cancer and nicotine addiction. Selective nAChR antagonists are invaluable for evaluating the functional roles of nAChR subtypes in the nervous system. α-Conotoxins that act as nAChR antagonists have been identified from the venom of predatory marine cone snails. We previously reported the discovery of a new α4/7-conotoxin, RegIIA, isolated from Conus regius. RegIIA inhibits acetylcholine (ACh)-evoked currents mediated by α3β2, α3β4 and α7 nAChR subtypes. RegIIA is the most potent α3β4 nAChR antagonist known, to date, with an IC50 of 48 nM (Franco, A., et al. 2012. Biochem. Pharmacol. 83:419-426). This study aims to understand and improve RegIIA's selectivity profile at the α3β4 nAChR subtype. It uses alanine scan mutants of non-cysteine residues within loop 2 of RegIIA, which were synthesised using solid-phase peptide synthesis. These mutants were functionally tested on nAChRs expressed in Xenopus oocytes using two-electrode voltage clamp recording. Of these mutants, [N11A] and [N12A] RegIIA exhibited a three-fold increase in selectivity for α3β4, compared with the α3β2 nAChR subtype. Molecular dynamics simulations of RegIIA bound to the ACh binding pocket of the α3β4 nAChR subtype revealed crucial molecular interactions, including residues of the “aromatic cage” at the α3(+) principal face (W174, Y215, Y222) as well as the β4(-) complementary face (W79, K81, R135). This study extends our understanding of RegIIA interactions with various nAChR subtypes and elucidated the key residues involved on both the peptide and the receptor binding site. Furthermore, we have obtained valuable information about the future design and development of α3β4-selective drugs that could target lung cancer and nicotine addiction.
