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 PURPOSEFluorescent 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 APPROACHThe 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 RESULTSMepyramine-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 IMPLICATIONSMepyramine-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.

The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

“…These profound differences in diffusion rates, allow the molecular composition of signalling complexes to be defined. To date, we have used fluorescent ligands in conjunction with FCS to characterize the adenosine A1 [12,44], adenosine A 3 [1,11] and histamine H 1 receptors [14], whereas other research groups have used the same approach to investigate β 2 -adrenoceptors [16], galanin [45] and somatostatin receptors [46]. In all FCS studies utilizing fluorescent ligands, ligand/receptor complexes have been found to exist as two distinct components within the autocorrelation trace (termed τ D2 and τ D3 ) with discrete rates of diffusion (typically 1–20 ms for τ D2 and 10–700 ms for τ D3 respectively) implying that multiple states or populations of receptors may exist at any one time.…”

“…Typically within a trace, the faster component termed τ D1 (usually measured in microseconds), represents the photophysics of the fluorophore used or the free diffusion of a fluorescent ligand (if used), whereas more slowly diffusing species ( τ D2 , τ D3 ) represent molecular complexes (typically measured in milliseconds). FCS has been used by researchers in our laboratory to investigate the diffusion of a range of fluorescently tagged proteins including the Class A GPCRs adenosine A 1 [12,13], adenosine A 2A [13], adenosine A 3 [1,11], histamine H 1 [14] and neuropeptide (NPY) Y receptors [15]. FCS has also been used by other research groups to investigate β-adrenoceptors [16], somatostatin receptors [17], type 2 bradykinin [18] and the biogenic amine α 1b -adrenoceptors, β 2 -adrenoceptors, muscarinic M 1 and M 3 and dopamine D 1 receptors [19].…”

Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors

“…However a significant limitation of the combined technique has been that FCS measurements are more problematic using YFP tagged proteins. Such measurements are limited by the more complex photophysics and increased photobleaching inherent to these GFP variants [28].…”

Dynamics of ligand binding to GPCR: Residence time of melanocortins and its modulation

“…The advantage of FCS is that it can be used on a single cell level and actual amounts of ligand can be measured. This approach has been used for characterization of ligand binding to different GPCRs, including adenosine [20] and histamine [21] receptors, but the complexity of the equipment complicates its implementation in high throughput screening. FCS can also be used for assessment of the localization of receptors in different membrane domains within the cell, as well as the formation of complexes or aggregates of receptors [22].…”