Synthesis and characterization of 125I-alpha-conotoxin ArIB[V11L;V16A], a selective alpha7 nicotinic acetylcholine receptor antagonist.

The alpha7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-alpha-bungarotoxin is commonly used to identify alpha7 nAChRs specifically. However, alpha-bungarotoxin also interacts potently with alpha1* and alpha9alpha10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the alpha7 subtype. [3H]Methyllycaconitine is also frequently used as an alpha7-selective antagonist, but it has significant affinity for alpha6* and alpha9alpha10 nAChR subtypes. In this study, we have developed a highly alpha7-selective alpha-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 K(d) value of 1.15 +/- 0.13 nM, similar to that of 125I-alpha-bungarotoxin in the same preparations (0.52 +/- 0.16 nM). Association and dissociation kinetics were relatively rapid (k(obs) for association at 1 nM was 0.027 +/- 0.007 min(-1); k(off) = 0.020 +/- 0.001 min(-1)). Selectivity was confirmed with autoradiography using alpha7-null mutant tissue: specific binding was abolished in all regions of alpha7(-/-) brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-alpha-conotoxin ArIB[V11L; V16A] should prove useful where alpha7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on alpha7 nAChRs, something that is impossible with 125I-alpha-bungarotoxin.