Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Non-native disulfide isomers of ␣-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of ␣-conotoxin AuIB have been characterized on ␣3␤4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to ϳ50%. The maximal inhibition was stoichiometry-dependent because altering ␣3:␤4 RNA injection ratios either increased AuIB(ribbon) efficacy (10␣:1␤) or completely abolished blockade (1␣:10␤). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity ␣3␤4 nAChRs with corresponding EC 50 values of 22.6 and 176.9 M, respectively. Increasing the agonist concentration antagonized the inhibition of LS ␣3␤4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS ␣3␤4 nAChRs by AuIB-(globular) was unaffected. Inhibition of LS and HS ␣3␤4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both ␣3␤4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS ␣3␤4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS ␣3␤4 nAChRs competitively, whereas AuIB-(globular) inhibits ␣3␤4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.Conotoxins are short disulfide-rich bioactive peptides that have been originally isolated from venoms of carnivorous mollusk cone snails, belonging to the genus Conus. ␣-Conotoxins are among the largest class of conotoxins found in the venom of most cone snail species (1). This class of conotoxins targets various subtypes of nicotinic acetylcholine receptors (nAChRs) 2 and is distinguished by four cysteines arranged in a CC-C-C pattern.␣-Conotoxins have attracted considerable attention as some of them, such as Vc1.1 and RgIA, have been shown to possess analgesic activity in rodent behavioral models of neuropathic pain (2, 3). Interestingly, AuIB has recently been shown to be analgesic in vivo despite the fact that it acts on the ␣3␤4 nAChR subtype different from the ␣9␣10 nAChR targeted by Vc1.1 and RgIA.3 Vc1.1 and RgIA have been shown to suppress N-type Ca 2ϩ channel currents in dorsal root ganglion (DRG) neurons of neonatal and adult rats and wild type and ␣9 knock-out mice via activation of GABA B G protein-coupled receptors (2). Similarly, AuIB inhibits N-type Ca 2ϩ channels in rat DRG neurons analogous to Vc1.1 and RgIA and its effect can be blocked with selective GABA B receptor antagonists.4 GABA B -mediated inhibition of N-type Ca 2ϩ channels...

Background: ␣-Conotoxin AuIB interacts with ␣3␤4 nAChRs and GABA B receptors, but structural determinants of these interactions are unknown. Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB⅐␣3␤4 nAChR interaction. Conclusion:We identified the key residues that mediate AuIB⅐␣3␤4 nAChR interaction. Significance: Ability to direct ␣-conotoxin binding to nAChRs or GABA B receptors will improve analgesic conopeptides.