Abstract
Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 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 approximately 50%. The maximal inhibition was stoichiometry-dependent because altering alpha3:beta4 RNA injection ratios either increased AuIB(ribbon) efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). 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 alpha3beta4 nAChRs with corresponding EC(50) values of 22.6 and 176.9 microM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both alpha3beta4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha3beta4 nAChRs competitively, whereas AuIB(globular) inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.
Highlights
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]
We further explore the inhibitory mechanisms of globular and ribbon isomers of AuIB on rat ␣34 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes
Concentration-Response Relationships Obtained for Inhibition of ACh-evoked Currents by Globular and Ribbon Isomers of AuIB—Concentration-response curves were obtained for the inhibition of rat ␣34 nAChRs expressed in Xenopus oocytes by globular and ribbon isomers of AuIB ␣-conotoxin (Fig. 2)
Summary
Peptide Synthesis—The peptide was assembled on a 4-methylbenzhydrylamine resin by manual tert-butoxycarbonyl solidphase peptide synthesis using 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate-mediated in situ neutralization protocol with N,NЈ-dimethylformamide as solvent [7]. Each of the top 20 lowest energy NMR structures of globular (PDB code 1MXN [5]) and ribbon (PDB code 1MXP [5]) AuIB were docked to all (␣3)3(4) and (␣3)2(4) pentamer models (assembled by both approaches with different C-loop conformations) individually using the program HEX 5.1, followed by energy minimization. We tried the ␣(ϩ)(Ϫ) dimer extracted directly from the (␣3)3(4) pentamer models, an approach used previously for docking simulations at AChBP-derived models of nAChRs. The docking solutions after energy minimization showed that ribbon AuIB docked at the same location as the global docking in the FIGURE 2. B, concentration-response curves for inhibition of rat ␣34 nAChRs expressed in Xenopus oocytes by globular and ribbon isomers of ␣-conotoxin AuIB.
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