A novel fluorescent histamine H1 receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands

Abstract

BACKGROUND AND PURPOSE Fluorescent ligands facilitate the study of ligand–receptor interactions at the level of single cells and individual receptors. Here, we describe a novel fluorescent histamine H₁ receptor antagonist (mepyramine-BODIPY630-650) and use it to monitor the membrane diffusion of the histamine H₁ receptor. EXPERIMENTAL APPROACH The human histamine H₁ receptor fused to yellow fluorescent protein (YFP) was transiently expressed in CHO-K1 cells. The time course of binding of mepyramine-BODIPY630-650 to the H₁ receptor was determined by confocal microscopy. Additionally, fluorescence correlation spectroscopy (FCS) was used to characterize the diffusion coefficient of the H₁ receptor in cell membranes both directly (YFP fluorescence) and in its antagonist-bound state (with mepyramine-BODIPY630-650). KEY RESULTS Mepyramine-BODIPY630-650 was a high-affinity antagonist at the histamine H₁ receptor. Specific membrane binding, in addition to significant intracellular uptake of the fluorescent ligand, was detected by confocal microscopy. However, FCS was able to quantify the receptor-specific binding in the membrane, as well as the diffusion coefficient of the antagonist–H₁ receptor–YFP complexes, which was significantly slower than when determined directly using YFP. FCS also detected specific binding of mepyramine-BODIPY630-650 to the endogenous H₁ receptor in HeLa cells. CONCLUSIONS AND IMPLICATIONS Mepyramine-BODIPY630-650 is a useful tool for localizing the H₁ receptor using confocal microscopy. However, its use in conjunction with FCS allows quantification of ligand binding at the membrane, as well as determining receptor diffusion in the absence of significant bleaching effects. Finally, these methods can be successfully extended to endogenously expressed untagged receptors in HeLa cells.