α‐Conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors

α‐Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of α‐conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into α‐conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in α‐conotoxins GI and SI, or D12K substitution in α‐conotoxin SIA increased the affinity for both the high‐ and low‐affinity sites in membrane‐bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30‐ and 200‐fold enhancement for the respective sites, resulting in the most potent α‐conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in α‐conotoxin [A10L]PnIA, a blocker of neuronal α7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine‐binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica . We found that [A10L,D14K]PnIA also distinguishes two α7‐like anion‐selective nAChR subtypes present on identified neurons of L. stagnalis : [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to α‐conotoxin ImI, but gave a 50‐fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C‐terminus of α‐conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle‐type α‐conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.