Abstract
The α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-α-bungarotoxin is commonly used to identify α7 nAChRs specifically. However, α-bungarotoxin also interacts potently with α1* and α9α10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the α7 subtype. [3H]Methyllycaconitine is also frequently used as an α7-selective antagonist, but it has significant affinity for α6* and α9α10 nAChR subtypes. In this study, we have developed a highly α7-selective α-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol-1, respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a Kd value of 1.15 ± 0.13 nM, similar to that of 125I-α-bungarotoxin in the same preparations (0.52 ± 0.16 nM). Association and dissociation kinetics were relatively rapid (kobs for association at 1 nM was 0.027 ± 0.007 min-1; koff = 0.020 ± 0.001 min-1). Selectivity was confirmed with autoradiography using α7-null mutant tissue: specific binding was abolished in all regions of α7-/- brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-α-conotoxin ArIB[V11L; V16A] should prove useful where α7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on α7 nAChRs, something that is impossible with 125I-α-bungarotoxin.
Footnotes
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This work was supported by National Institutes of Health Grants DA12242 (to P.W.), MH53631 and GM48677 (to J.M.M.), and DA-15663 (to A.C.C.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136895.
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ABBREVIATIONS: α-Bgt, α-bungarotoxin; nAChR, nicotinic acetylcholine receptor; CNS, central nervous system; MLA, methyllycaconitine; α-Ctx, α-conotoxin; I1, monoiodo; I2, diiodo; TFA, trifluoroacetic acid; ANOVA, analysis of variance; HSD, honestly significant difference; A85380, [3-(2(S)-azetidinylmethoxy)pyridine].
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received January 19, 2008.
- Accepted March 4, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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