Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of 3H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT6) receptors expressed in Chinese hamster ovary (CHO)–Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT6 agonists and antagonists using intact CHO–Dukx/5-HT6 cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT6 membranes. Ki values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (Ki = 1.9 nM) > methiothepin (Ki = 6.2 nM) > mianserin (Ki = 74.3 nM) > 5-methoxytryptamine (5-MeOT, Ki = 111 nM) > 5-HT (Ki = 150 nM) > ritanserin (Ki = 207 nM) > 5-carboxamidotryptamine (5-CT, Ki = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.