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NEUROPHARMACOLOGY

Conotoxin Arenatus IB[V11L,V16D] Is a Potent and Selective Antagonist at Rat and Human Native 7 Nicotinic Acetylcholine Receptors

Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom (N.I., P.D.L., A.K., T.Y., S.W.); Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (A.H., J.M.M.); and Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (P.W.)

A recently developed -conotoxin, -conotoxin Arenatus IB-[V11L,V16D] (-CtxArIB[V11L,V16D]), is a potent and selective competitive antagonist at rat recombinant 7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. 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 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. -CtxArIB[V11L,V16D] specifically inhibited 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 KCl, that did not activate 7 nAChRs were unaffected. Human 7 nAChRs were also sensitive to -CtxArIB[V11L,V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human 7 nAChRs were inhibited by -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 7 nAChRs in PC12 cells. -CtxArIB[V11L,V16D] inhibited human native 7 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 7 nAChRs contribute to dopamine release from striatal minces; -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 -CtxArIB[V11L,V16D] selectively inhibits human and rat native 7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating 7 nAChR functions.

Address correspondence to: Dr. Susan Wonnacott, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. E-mail: bsssw{at}bath.ac.uk