( Key words: ...)Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System

Key words: α_2c receptor • GPCR • receptor binding assay • LEADseeker • SPA Imaging Beads

The α₂-adrenergic receptors are G protein-coupled receptors that play a key role in the control of numerous physiological functions such as renal Na⁺ re-absorption, insulin secretion, platelet aggregation, and neurotransmitter release at sympathetic nerve endings (1). They consist of three highly homologous subtypes encoded by distinct genes: α_2a, α_2b, and α_2c. Both α_2a and α_2c subtypes are required for normal presynaptic control of neurotransmitter release from sympathetic nerves from the heart and from central noradrenergic neurons. α_2a adrenergic receptors inhibit transmitter release at high stimulation frequencies whilst the α_2c subtype modulates neurotransmission at lower levels of nerve activity (2). The α_2c receptor is distinguishable from α_2a and α_2b receptors by its sensitivity to prazosin (3).

This application note describes a 384-well adrenergic α_2c receptor binding assay developed using the LEADseeker™ Multimodality Imaging System.

Materials
Products used
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) YOx RPNQ0270
SPA Imaging Beads

[0-methyl-³H]Rauwolscine TRK685

Other materials required
Human recombinant adrenergic α_2c receptor membrane preparation (Euroscreen, ES-032-M)

Prazosin hydrochloride (Sigma, P7791)

Rauwolscine hydrochloride (Sigma, R104)

Costar™ solid white 384-well microplate (Corning, 3705)

Buffer: 50-mM Tris pH 7.4
5-mM MgCl₂
50-mM NaCl

GraphPad Prism™ software v4.0 (GraphPad Software)

Protocol
Human recombinant α_2c receptor membrane preparation was used in conjunction with [0-methyl-³H]Rauwolscine ligand and WGA YOx SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 6-µM prazosin. The standard assay format was as follows:

1. Reagents were added in the following order: buffer, unlabeled ligand (NSB wells), labeled ligand, and precoupled bead and membrane. Total assay volume was 50 µl.

2. Diluted membrane and bead were precoupled for 30 min on a roller mixer at 2–8 ºC.

3. Wells contained 10 µl of 6.5-nM [0-methyl³H]Rauwolscine (final concentration 1.3 nM) unless otherwise stated. Wells also contained 5 µg of membrane and 250 µg of WGA YOx SPA Imaging Beads. Precoupled bead and membrane was added in a 20-µl volume.

4. NSB wells contained 10 µl of 30-µM prazosin (final concentration 6 µM) in addition to the above.

5. Plates were sealed and incubated in darkness for 3 h at room temperature (20–25 ºC) unless otherwise stated.

6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min using quasi-coincident averaging and 3 ×3 binning.

Results
K_d determination
Saturation binding was performed with dilutions of [0-methyl-³H]Rauwolscine to give a range of concentrations from 0.07 to 8.8 nM in the wells. Plates were incubated for 5 h prior to imaging. Figure 1 shows the saturation curve, which was fitted using nonlinear regression with the data analysis software package GraphPad Prism v4.0. Over two experiments the mean K_d value was determined to be 1.3 nM (95% confidence intervals 0.8 to 1.9 nM), estimated directly from the curve. Figure 1 is representative of the data obtained.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.25% (v/v) to 5% (v/v) in the well. The results shown in Figure 2 indicate the assay was tolerant to DMSO up to a final concentration of 1% (v/v).

IC₅₀ determination
Competitive binding of 1.3-nM [0-methyl-³H]Rauwolscine with rauwolscine (Fig 3A) and prazosin (Fig 3B) was assessed and the IC₅₀ value of each ligand calculated. Final concentrations in the wells were from 0.0004 to 4000 nM and 0.25 to 20000 nM respectively. DMSO was present at a final concentration of 1% v/v in each well.

Over two experiments, the mean rauwolscine IC₅₀ value was determined to be 3.1 nM (95% confidence interval range 2.7–3.7 nM) and the mean K_i value was 1.6 nM (95% confidence intervals 1.3–1.8 nM). The mean prazosin IC₅₀ value was determined to be 430 nM (95% confidence interval range 336–535 nM) and the mean K_i value was 215 nM (95% confidence intervals 168–267 nM).

Time course
A time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 17 h. The assay appeared to be stable for at least 17 h as demonstrated by the curve in Figure 4.

Z’ analysis
Finally, two independent Z’ analysis (4) evaluations were performed, each using 48 replicate values for "total" and NSB wells. DMSO was present at a final concentration of 1% (v/v) in each well. A mean Z’ value of 0.80 was generated. Figure 5 is representative of the data obtained. This is well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.

Conclusion
The adrenergic α_2c receptor binding assay has been successfully miniaturized to a 384-well format using the LEADseeker Multimodality Imaging System and is suitable for adaptation to automated screening formats. The assay is tolerant to DMSO up to a concentration of 1% (v/v), robust; achieving a mean Z’ value of 0.80, and has a stability window of at least 17 h.

References
1. Ruffolo, R. R. Jnr et al. Annual Review Pharmacological Toxicology 32, 243–279 (1993).

2. Hein, L. et al. Nature 402, 181–184 (1999).

3. Bylund, D.B. et al. Pharmacological Reviews 46, 121–136 (1994).

4. Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).

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