[125I]AT-1012, a new high affinity radioligand for the α3β4 nicotinic acetylcholine receptors

Recent genetic and pharmacological studies have implicated the alpha 3, beta 4 and alpha 5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The alpha 3 beta 4* nAChR subtype has been shown to co-assemble with the alpha 5 or beta 3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the alpha 3 beta 4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [I-125]-radiolabeled analog of a high affinity, selective small-molecule alpha 3 beta 4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the alpha 3 beta 4* nAChR. Binding of [I-125]AT-1012 was characterized at the rat alpha 3 beta 4 and alpha 4 beta 2 nAChR transfected into HEM cells, as well as at the human alpha 3 beta 4 alpha 5 nAChR in HEK cells. Binding affinity of [I-125]AT-1012 at the rat alpha 3 beta 4 nAChR was 1.4 nM, with a B-max of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [H-3]epibatidine. Saturation isotherms suggested that [I-125]AT-1012 binds to a single site on the alpha 3 beta 4 nAChR. Similar high binding affinity was also observed for [I-125]AT-1012 at the human alpha 3 beta 4 alpha 5 nAChR transfected into HEM cells. [I-125]AT-1012 did not bind with high affinity to membranes from alpha 4 beta 2 nAChR-transfected HEM cells. Binding studies with [3H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for alpha 3 beta 4 over alpha 4 beta 2 nAChR. K-i values determined for known nAChR compounds using [I-125]AT-1012 as radioligand were comparable to those obtained with [H-3]epibatidine. [I-125]AT-1012 was also used to label alpha 3 beta 4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the alpha 3 beta 4 nAChR. We demonstrate that [I-125]AT-1012 is an excellent tool for labeling the alpha 3 beta 4 nAChR in the presence of other nAChR subtypes. (C) 2013 Elsevier Ltd. All rights reserved.