α-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors
Non-native disulfide isomers of α-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of α-conotoxin AuIB have been characterized on α3β4 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 ∼50%. The maximal inhibition was stoichiometry-dependent because altering α3:β4 RNA injection ratios either increased AuIB(ribbon) efficacy (10α:1β) or completely abolished blockade (1α:10β). 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 α3β4 nAChRs with corresponding EC50 values of 22.6 and 176.9 μm, respectively. Increasing the agonist concentration antagonized the inhibition of LS α3β4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS α3β4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS α3β4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both α3β4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS α3β4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS α3β4 nAChRs competitively, whereas AuIB(globular) inhibits α3β4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.