Molecular determinants of the selectivity and potency of a-conotoxin Vc1.1 for human nicotinic acetylcholine receptors

The a-conotoxins (a-Ctxs) are short, disulfide-rich peptides derived from the venom of the Conus marine snails, primarily acting as antagonists of nicotinic acetylcholine receptors (nAChRs). Specifically, a-Ctx Vc1.1, a 16-amino acid peptide from Conus victoriae, competitively antagonizes non-muscle nAChRs, inhibits nicotine-induced currents in bovine chromaffin cells, and alleviates neuropathic pain in rat models. Although Vc1.1 selectively inhibits rat a9a10 nAChRs, its potency and selectivity across human nAChR subtypes remain unresolved. In this study, we assessed the activity of Vc1.1 on human (h) nAChRs heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique and simulated interactions using computational modeling. Vc1.1 selectively antagonized homomeric a9 and heteromeric a3b2 nAChRs, with half-maximal inhibitory concentrations (IC50) of 160 nM and 232 nM, respectively. At ha9[N179A]a10, Vc1.1 exhibited a 20-fold decrease in potency compared to ha9a10, due to the loss of hydrogen bonding with Vc1.1-D11. Conversely, Vc1.1 was four-fold more potent at ha3b2[E86A] compared to ha3b2, possibly influenced by the proximal residue b2-K104, as suggested by molecular dynamics (MD) simulations. Additionally, Vc1.1's potency doubled at ha9[N213K] a10, whereas it remained unchanged at ha9[N213R]a10 nAChRs. MD simulations indicate that altered interactions between the mutant ha9 N179A, N213K, and N213R side chains and Vc1.1-D5 may partly explain these changes in potency. The inhibitory action of Vc1.1 at a9-containing nAChRs is particularly relevant given their role in neuroinflammation, presenting a potential therapeutic pathway for alleviating neuropathic and inflammatory pain. This study provides valuable insights into the rational design of Vc1.1-derived a-Ctxs with enhanced nAChR subtype selectivity.