α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors

Grishin, Anton, Wang, Ching-I. A., Muttenthaler, Markus, Alewood, Paul F., Lewis, Richard J. and Adams, David J. (2010) α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors. Journal of Biological Chemistry, 285 29: 22254-22263. doi:10.1074/jbc.M110.111880

Attached Files (Some files may be inaccessible until you login with your UQ eSpace credentials)
Name Description MIMEType Size Downloads
Grishin_et_al_2010.pdf PDF of Publication application/pdf 2.40MB 0
UQ224159_OA.pdf Full text (open access) application/pdf 2.55MB 0

Author Grishin, Anton
Wang, Ching-I. A.
Muttenthaler, Markus
Alewood, Paul F.
Lewis, Richard J.
Adams, David J.
Title α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors
Formatted title
α-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of α3β4 nicotinic acetylcholine receptors
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 2010-07-16
Year available 2010
Sub-type Article (original research)
DOI 10.1074/jbc.M110.111880
Open Access Status File (Publisher version)
Volume 285
Issue 29
Start page 22254
End page 22263
Total pages 10
Editor Martha Fedor
Herbert Tabor
Place of publication Bethesda, MD, U.S.A.
Publisher American Society for Biochemistry and Molecular Biology
Language eng
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 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 EC(50) 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.
Formatted abstract
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.
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Keyword Computer modeling
Disulfide
Nicotinic acetylcholine receptors
Peptide interactions
Receptor structure-function
Conotoxin
Disulfide isomers
Electrophysiology
Subunit stoichiometry
ACh-binding protein
Subunit interfaces
Crystal-structure
Alternate stoichiometries
Subtype selectivity
Antagonists
Expression
ImII
(alpha-4)(2)(beta-2)(3)
Pharmacology
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
Queensland Brain Institute Publications
Institute for Molecular Bioscience - Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 35 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 33 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 10 Dec 2010, 21:32:51 EST by Debra McMurtrie on behalf of Queensland Brain Institute