αConotoxin Arenatus IB[V11I,V16D] Is a Potent and Selective Antagonist at Rat and Human Native α7 Nicotinic Acetylcholine Receptors

A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (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. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha 7 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 alpha 7 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 KCI, that did not activate alpha 7 nAChRs were unaffected. Human alpha 7 nAChRs were also sensitive to alpha-CtxArIB[V11L,V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha 7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC50, 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 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 [(2)-spiro[1-azabicyclo[2.2.2]-octane3,59- oxazolidin]-29-one] 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.