Characterization of 5-HT1A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t(1/2,ass) < 1 min), reversibly (t(1/2,diss) similar to 6 min) and has high affinity(K-d = 0.30 +/- 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of alpha i subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between alpha i1 and alpha i3 subtypes, while ligand binding in the presence of alpha i2 had higher sensitivity to GDP and Mn2+. (C) 2014 Elsevier Ltd. All rights reserved.