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CELLULAR AND MOLECULAR

Synthesis and Characterization of ¹²⁵I--Conotoxin ArIB[V11L;V16A], a Selective 7 Nicotinic Acetylcholine Receptor Antagonist

Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (P.W.); Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado (M.J.M., A.C.C.); and Departments of Biology (S.C., C.D., J.M.M.) and Psychiatry (J.M.M.), University of Utah, Salt Lake City, Utah

The 7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, ¹²⁵I--bungarotoxin is commonly used to identify 7 nAChRs specifically. However, -bungarotoxin also interacts potently with 1^* and 910 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the 7 subtype. [³H]Methyllycaconitine is also frequently used as an 7-selective antagonist, but it has significant affinity for 6^* and 910 nAChR subtypes. In this study, we have developed a highly 7-selective -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--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 7-null mutant tissue: specific binding was abolished in all regions of 7^-/- brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. ¹²⁵I--conotoxin ArIB[V11L; V16A] should prove useful where 7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on 7 nAChRs, something that is impossible with ¹²⁵I--bungarotoxin.

Address correspondence to: Dr. J. Michael McIntosh, University of Utah, 257 S. 1400 East, Salt Lake City, UT 84112. E-mail: mcintosh.mike{at}gmail.com