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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on July 29, 2008; DOI: 10.1124/jpet.108.142943

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Received for publication July 1, 2008.
Revised July 28, 2008.
Accepted for publication July 28, 2008.

{alpha}Conotoxin ArIB[V11L,V16D] is a potent and selective antagonist at rat and human native {alpha}7 nicotinic acetylcholine receptors

Neal Innocent 1, Phil D Livingstone 1, Arik Hone 2, Atsuko Kimura 1, Tracey Young 1, Paul Whiteaker 3, J. Michael McIntosh 2, Susan Wonnacott 1*

1 University of Bath 2 University of Utah 3 Barrow Neurological Institute

* Address correspondence to: E-mail: s.wonnacott{at}bath.ac.uk

Abstract

A recently developed {alpha}-conotoxin, {alpha}-CtxArIB[V11L,V16D] is a potent and selective competitive antagonist at rat recombinant {alpha}7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. {alpha}7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues where they are implicated in a variety of functions. Here we evaluate this toxin at rat and human native nAChRs. Functional {alpha}7 responses were evoked by choline plus the allosteric potentiator PNU-120596 in rat PC12 cells and human SHSY5Y cells loaded with calcium indicators. {alpha}-CtxArIB[V11L,V16D] specifically inhibited {alpha}7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli (5-iodo-A-85380, nicotine or KCl) that did not activate {alpha}7 nAChRs were unaffected. Human {alpha}7 nAChRs were also sensitive to {alpha}-CtxArIB[V11L,V16D]: ACh-evoked currents in X. laevis oocytes expressing human {alpha}7 nAChRs were inhibited by {alpha}-CtxArIB[V11L,V16D] (IC 50 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the timecourse of recovery from blockade of rat {alpha}7 nAChRs in PC12 cells. {alpha}-CtxArIB[V11L,V16D] inhibited human native {alpha}7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 plus PNU-120596. Rat brain {alpha}7 nAChRs contribute to dopamine release from striatal minces: {alpha}-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that {alpha}-CtxArIB[V11L,V16D] selectively inhibits human and rat native {alpha}7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating {alpha}7 nAChR functions.


Key words: PC12 cells, SH-SY5Y cells, Xenopus oocytes, calcium fluorimetry, dopamine release, two electrode voltage clamp recording




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