α-Conotoxin AuIB Isomers Exhibit Distinct Inhibitory Mechanisms and Differential Sensitivity to Stoichiometry of α3β4 Nicotinic Acetylcholine Receptors

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 alpha 3 beta 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 similar to 50%. The maximal inhibition was stoichiometry-dependent because altering alpha 3:beta 4 RNA injection ratios either increased AuIB(ribbon) efficacy (10 alpha:1 beta) or completely abolished blockade (1 alpha:10 beta). 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 alpha 3 beta 4 nAChRs with corresponding EC50 values of 22.6 and 176.9 mu M, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha 3 beta 4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha 3 beta 4 nAChRs by AuIB-(globular) was unaffected. Inhibition of LS and HS alpha 3 beta 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 alpha 3 beta 4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha 3 beta 4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha 3 beta 4 nAChRs competitively, whereas AuIB( globular) inhibits alpha 3 beta 4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.