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

Abstract

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...

Function of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced by the α5(D398N) Variant

“…This study confirms prior reports that at least two ␣3␤4 nAChR populations may be formed and that their relative expression levels depend on the molar injection ratio of the subunit mRNAs (1:20 versus 20:1). The pharmacology observed in this study matches that reported in other recent publications (27,28) that used less-extreme injection ratios (1:9 versus 9:1 or 1:10 versus 10:1). The lack of further changes in observed pharmacology after adoption of more extreme subunit ratios indicates that, as for ␣4 and ␤2 subunits (2,40,41), relatively pure populations of two different ␣3␤4 subunit assemblies are produced at the injection ratios used in this study.…”

“…The very similar properties of nAChR arising from 1:1 or 1:20 ␣3:␤4 mRNA injection ratios indicate that the same subunit assembly pattern predominates in both cases. This confirms previous reports that Xenopus oocytes injected with 1:1 and 1:9 ␣3:␤4 mRNA ratios express similar ␣3␤4 nAChR populations, whereas high ␣3:␤4 mRNA injection ratios result in expression of a distinctly different ␣3␤4 nAChR isoform (27,28). However, none of the outcomes observed from oocytes injected with any ratio of ␣3:␤4 mRNAs closely resembled the results obtained from the concatemeric ; n ϭ 4) (B), nicotine (10 Ϫ6.5 to 10 Ϫ3.5 ; n ϭ 4) (C), or the nAChR antagonist mecamylamine (10 Ϫ7.5 to 10…”

“…2 and 3). It has recently been noted that oocytes injected with different ␣3:␤4 mRNA ratios express ␣3␤4 nAChR populations with differing pharmacological properties (27,28). By analogy to more extensively studied high and low agonist sensitivity ␣4␤2 nAChR isoforms, it has been speculated that these populations may correspond to (␣3␤4) 2 ␤4 and (␣3␤4) 2 ␣3 stoichiometries (27,28) as expressed by the concatemeric constructs produced in this study.…”

Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor

“…Mutational analyses provide evidence for PAM binding sites in the ECD at noncanonical interfaces analogous to dFBr Site III in a3b2 nAChRs at the b2/a3 interface (Seo et al, 2009) and in a4b2 nAChRs at the a4/a4 interface, which is also an ACh binding site (Mazzaferro et al, 2014;Olsen et al, 2014). Mutational analyses and computational docking also provide evidence for negative allosteric modulator binding sites equivalent to dFBr Site I at the canonical interfaces in a4b2 and a3b4 nAChRs (Grishin et al, 2010;Henderson et al, 2012). Based on our results, [ 3 H]dFBr should serve as an appropriate photoreactive PAM to identify its binding sites in a4b2 nAChRs and to identify other allosteric modulators that competitively inhibit photolabeling.…”

Conus Venom Peptide Pharmacology

“…It is noteworthy that non-native disulfide bond isomers of conopeptides can also provide useful membrane protein probes, further expanding on the pharmacology of conopeptides. For example, the two isomers of ␣-AuIB differ in their inhibitory mechanisms, with the AuIB (ribbon) form competitively inhibiting only ␣3␤4 nAChRs containing an ␣3 subunit in the fifth position, an effect distinct from the native AuIB (globular) form, which inhibited ␣3␤4 nAChRs independent of the fifth subunit, primarily through a noncompetitive mechanism (Grishin et al, 2010). Furthermore, a structural isomer of -TIA named NMB-1 was found to preferentially inhibit the sustained component of mechanically evoked current in DRG neurons, providing a novel diagnostic tool for the molecular definition of channels involved in hearing and pressureevoked pain (Drew et al, 2007).…”