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. 2012 Mar;165(6):1789-1800.
doi: 10.1111/j.1476-5381.2011.01640.x.

A novel fluorescent histamine H(1) receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands

Rachel H Rose  1 , Stephen J Briddon  1 , Stephen J Hill  1
Affiliations

A novel fluorescent histamine H(1) receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands

Rachel H Rose et al. Br J Pharmacol. 2012 Mar.

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 H(1) receptor antagonist (mepyramine-BODIPY630-650) and use it to monitor the membrane diffusion of the histamine H(1) receptor.

Experimental approach: The human histamine H(1) 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 H(1) receptor was determined by confocal microscopy. Additionally, fluorescence correlation spectroscopy (FCS) was used to characterize the diffusion coefficient of the H(1) 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 H(1) 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-H(1) 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 H(1) receptor in HeLa cells.

Conclusions and implications: Mepyramine-BODIPY630-650 is a useful tool for localizing the H(1) 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.

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Figures

Figure 1
Figure 1
The chemical structure of mepyramine-BODIPY630-650.
Figure 2
Figure 2
Functional characterization of mepyramine-BODIPY630-650 binding to the H1 receptor. CHO-K1 cells expressing either (A) wild-type H1 receptor or (B) mycH1(F432A)-YFP were preincubated with 1 µM mepyramine or mepyramine-BODIPY630-650 and the intracellular calcium mobilization in response to histamine measured. Responses are expressed as % of the maximal calcium response to ionomycin (1 µM). Data are mean ± SEM of three to four independent experiments, each performed in duplicate.
Figure 3
Figure 3
Binding of mepyramine-BODIPY630-650 to mycH1-YFP or mycH1(F432A)-YFP-expressing cells in the absence or presence of the H1 receptor antagonist cetirizine. CHO-K1 cells were grown to 70% confluence prior to transient transfection with mycH1-YFP or mycH1(F432A)-YFP and incubated at 30°C/5%CO2 for 24 h before imaging. Cells were maintained in HBSS in the absence (left, right) or presence of 3 µM cetirizine (middle; 30 min preincubation, 37°C) prior to imaging using a Zeiss LSM 510 confocal microscope. Ligand binding was observed for 1200 s following addition of mepyramine-BODIPY630-650 (50 nM) with images taken every 10 s. The left half of each image represents mycH1-YFP (YFP channel) and the right half represents mepyramine-BODIPY630-650 (BODIPY630-650 channel). Images are from a single experiment representative of three independent experiments performed. Images of ligand binding for all transfected receptor constructs were taken using the same microscope settings for both the YFP and BODIPY630-650 channels.
Figure 4
Figure 4
Profiles of the change in fluorescence intensity over time following addition of 50 nM mepyramine-BODIPY630-650. CHO-K1 cells were transiently transfected with (A) mycH1-YFP or (B) mycH1(F432A)-YFP and incubated at 30°C for 24 h prior to imaging. Cells were imaged in HBSS over a time course of 1500 s following the addition of 50 nM mepyramine-BODIPY630-650. For both channels, the laser power, offset and gains were fixed for all experiments and tested constructs. The effect of unlabelled antagonist on mepyramine-BODIPY630-650 binding was determined by addition of 3 µM cetirizine 900 s after the addition of mepyramine-BODIPY630-650 (A; indicated by an arrow). To determine the relative amounts of membrane- and cytosolic- localized mepyramine-BODIPY630-650 at different time points, a ROI was drawn around the outside of the cell membrane (inset; ROI1) and inside the cell membrane (inset; ROI2). ROIs were drawn using fluorescence in the YFP channel. Relative fluorescence intensity was calculated as the difference between the average fluorescence intensity of ROI1 and ROI2. Data represent the mean ± SEM of data from seven cells obtained in three independent experiments.
Figure 5
Figure 5
FCS analysis of mepyramine-BODIPY630-650 binding to mycH1-YFP. Examples of FCS analysis of two data sets for the diffusion of 5 nM mepyramine-BODIPY630-650 at the surface of CHO-K1 cells transiently expressing mycH1-YFP are shown. Fluctuations in fluorescence intensity of mepyramine-BODIPY630-650 diffusion (A,C) were analysed by autocorrelation analysis, resulting in generation of autocorrelation curves (B,D). Autocorrelation curves were fit to a three component or two component diffusion models, in which the first component was fixed to the three-dimensional diffusion of the free ligand in solution. The majority of curves were best fit to a three component diffusion model (B), containing fast-moving free ligand (L) and both a fast-moving and slow-moving membrane bound components (LBF and LBS respectively). However, others (D) lacked the slower membrane-bound component (LBS) and were typically associated with a lower count rate.
Figure 6
Figure 6
Quantification of mepyramine-BODIPY630-650 binding to mycH1-YFP using FCS. The data show a comparison of the differences in particle density and diffusion coefficient of the fast (LBF) and slow (LBS) components of the mepyramine-BODIPY630-650 obtained by autocorrelation analysis. Particle density (A, C) and diffusion coefficients (B,D) of the slow membrane diffusion component (LBS; A,B) and faster membrane diffusion component (LBF, C,D) are shown for cells transiently expressing the mycH1-YFP receptor in the absence or presence of 3 µM cetirizine or the mycH1(F432A)-YFP receptor. Data represent the mean ± SEM of 39–51 cells from at least three independent experiments. *P < 0.05, significantly different from mycH1-YFP; one-way anova followed by Dunnett's multiple comparison test.
Figure 7
Figure 7
The effect of laser power on the apparent diffusion coefficient of mycH1-YFP detected by FCS using either YFP or mepyramine-BODIPY630-650 fluorescence. The diffusion coefficient (D) of the mycH1-YFP construct in the upper membrane of transiently transfected CHO-K1 cells was measured by FCS using either YFP fluorescence (open circles) or mepyramine-BODIPY630-650 fluorescence (closed circles) at different excitation laser powers. In both cases, there was a clear linear relationship between laser power and diffusion coefficient. A laser power of 0.34 kW·cm−2 was routinely used in measurements of YFP diffusion, whereas a laser power of 2.33 kW·cm−2 was normally used in measurements of mepyramine-BODIPY630-650 diffusion. Data represent the mean ± SEM of 8–46 individual cells from at least three independent experiments.
Figure 8
Figure 8
Detection of the endogenous H1 receptor on HeLa cells by (A) measuring histamine-induced changes in intracellular calcium and (B) binding of meyramine-BODIPY630-650 using FCS. (A) Changes in intracellular calcium in HeLa cells in response to histamine in the absence and presence of mepyramine (30 nM, 45 min, 37°C) were assessed, as described in the Methods section. Basal values in the absence (closed bars) and presence (open bars) of mepyramine are shown. Data are normalized to the control response to 10−5 M histamine, and are the mean ± SEM of five independent experiments performed in quadruplicate. (B) HeLa cells were stained with DiO in the absence (Ctrl) and presence of 3 µM cetirizine (+ Cet). Subsequently, cells were incubated with 3 nM mepyramine-BODIPY630-650 (5 min, 22°C) and FCS measurements performed +0.5 µm above the upper cell membrane. Left, the amount of fast-moving membrane-bound ligand in control cells and those preincubated with cetirizine was quantified by curve fitting, as described in the Methods. Right, the diffusion coefficient of the fast-moving membrane bound component was also determined from the same data. Data shown are the mean ± SEM of measurements from 12–26 cells, obtained in at least five independent experiments. *P < 0.05 and **P < 0.01, significantly different from control, unpaired Student's t-test.
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