| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2200602 | Neurochemistry International | 2014 | 7 Pages |
•Fluorescence anisotropy of Bodipy-FL-NAN-190 for characterization 5-HT1A receptors.•New homogenous assay for characterization of binding of serotonergic ligands.•Bodipy-FL-NAN-190 binding to 5-HT1A receptors was rapid and reversible.•αi subunits increased nucleotide sensitive high affinity agonist binding.•αi2 had higher sensitivity to GDP and Mn2+ in regulation of agonist binding.
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 (t1/2,ass < 1 min), reversibly (t1/2,diss ∼ 6 min) and has high affinity (Kd = 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 αi 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 αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn2+.
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