Alpha-conotoxin Arenatus IB[V11L,V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors.

A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 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. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 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 time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 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 alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.