Synthesis and Characterization of 125I-α-Conotoxin ArlB[V11L;V16A], a Selective α7 Nicotinic Acetylcholine Receptor Antagonist

The a7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125 I-α-bungarotoxin is commonly used to identify a7 nAChRs specifically. However, α-bungarotoxin also interacts potently with a1* and a9a10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the a7 subtype. [ 3 H]Methyllycaconitine is also frequently used as an a7-selective antagonist, but it has significant affinity for α6* and a9a10 nAChR subtypes. In this study, we have developed a highly a7-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 K d value of 1.15 ± 0.13 nM, similar to that of 125 Ι-α-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 a7-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. 125 I-α-conotoxin ArlB[V11L; V16A] should prove useful where a7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on a7 nAChRs, something that is impossible with 125 Ι-α-bungarotoxin.