Artificial Brain Extracellular Fluid in Radioligand Binding Assays: Effects on Affinities of Ligands at Serotonin 5-HT7 Receptors

Abstract

Radioligands are well-established tools for measuring ligand binding affinities at receptors. Determining affinities of test ligands at many G protein-coupled receptors (GPCRs), including serotonin (5-HT) GPCRs, often involves incubating a radioligand, test ligands, and receptors expressed in cell membranes in Tris buffers, and commonly in a standard binding buffer (SBB) containing Tris HCl, MgCl2, and EDTA until ligand- receptor equilibrium binding is established. However, the composition of extracellular fluid (ECF), where ligands first encounter GPCRs in vivo, differs from that of SBB, which we hypothesized impacts ligand affinity. We conducted radioligand binding assays to compare the affinities of the agonist 5-carboxamidotryptamine (5-CT) and two antagonists/inverse agonists, lurasidone and SB-269970, at [3H]5-CT-labeled 5-HT7 GPCRs stably expressed in HEK293 cells using SBB or artificial brain ECF (abECF) as the medium at room or physiological temperatures (RT or 37{degrees}C). The rank order of ligand potencies, as well as 5-CTs affinity, was unaffected by the different experimental environments. [3H]5-CT 5-HT7R Bmax values increased in abECF and modestly at 37{degrees}C, without affecting Kd, suggesting an increase in active state conformations. In contrast to 5-CT, antagonist/inverse agonist affinities depended on both media and temperature. The affinities of lurasidone and SB-269970 at 5-HT7 receptors were substantially higher at 37{degrees}C than at RT. Also, incubation of lurasidone and SB-269970 in abECF resulted in significantly higher affinities compared to incubation in SBB (e.g., [~]10-fold higher for lurasidone), indicating that temperature and the buffer and ionic composition of abECF influence 5-HT7 antagonist/inverse agonist ligand binding. As a high concentration of NaCl in abECF is a remarkable difference from the composition of SBB, we probed the impact of removing NaCl from abECF; removal of NaCl had a minor affinity-enhancing effect on the antagonists, inferring that other ions, glucose, or sodium phosphates in abECF underlie significant changes in ligand-receptor binding interactions. Overall, the observations indicate that measuring 5-HT7 antagonist affinities at [3H]5-CT-labeled 5-HT7Rs with abECF at physiological temperature--modeling the in vivo brain environment where ligands and GPCRs interact--yields distinct affinity values that may be more physiologically accurate than values obtained from SBB. Moreover, several historical reports demonstrate that temperature, ions, and buffers have no consistent effect on the affinities of distinct ligands at various other GPCRs, and there is no consensus binding buffer used in the literature for any GPCR, which may contribute to the variability in ligand-GPCR affinities reported. These findings show that buffer and temperature impacted 5-HT7R ligand binding affinities and highlight the importance of considering such conditions when performing experiments.