Wu_2014_Neuropharmacol_77_193

Recent genetic and pharmacological studies have implicated the alpha3, beta4 and alpha5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The alpha3beta4* nAChR subtype has been shown to co-assemble with the alpha5 or beta3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the alpha3beta4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [(125)I]-radiolabeled analog of a high affinity, selective small-molecule alpha3beta4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the alpha3beta4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat alpha3beta4 and alpha4beta2 nAChR transfected into HEK cells, as well as at the human alpha3beta4alpha5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat alpha3beta4 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 [(3)H]epibatidine. Saturation isotherms suggested that [(125)I]AT-1012 binds to a single site on the alpha3beta4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human alpha3beta4alpha5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from alpha4beta2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for alpha3beta4 over alpha4beta2 nAChR. K(i) values determined for known nAChR compounds using [(125)I]AT-1012 as radioligand were comparable to those obtained with [(3)H]epibatidine. [(125)I]AT-1012 was also used to label alpha3beta4 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 alpha3beta4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the alpha3beta4 nAChR in the presence of other nAChR subtypes.