Adenosine A_2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET

Materials and Reagents

1. Cell line (i.e. HEK-293 cells)
   
2. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich)
3. Sodium pyruvate
4. L-glutamine
5. Antibiotics: streptomycin and penicillin
6. Fetal bovine serum
   
7. TransFectin^TM Lipid Reagent (Bio-Rad Laboratories)
   
8. Hank’s balanced salt solution (HBSS) (see Recipes)
   
9. Cell culture medium (see Recipes)
   

Equipment

1. 18 mm diameter glass coverslips
   
2. Attofluor holder
   
3. Inverted Axio Observer microscope (ZEISS) equipped with a 63x oil immersion objective
   
4. Polychrome V (TILL Photonics)
   
5. Avalanche photodiodes (TILL Photonics)
   
6. Focal drug application system (ALA Scientific Instruments, OCTAFLOW^TM)
   
7. Digidata 1440A analog/digital converter (Molecular Devices)
   

Software

1. pCLAMP (Molecular Devices)
   
2. GraphPad Prism (GraphPad Software)
   

Procedure

1. Two days before the experiment, the cells (i.e. HEK-293 cells) were seeded onto 18 mm diameter glass coverslips and transiently transfected with an A_2AR construct tagged with the CFP at its N-terminal tail (A_2AR^CFP) (Figure 1).
   
   
   Figure 1. Cell surface localisation of the A_2AR^CFP construct. HEK-293 cells were transiently transfected with A_2AR^CFP, fixed and analyzed by confocal microscopy. The A_2AR^CFP was mainly targeted to the cell surface and scarcely accumulated at the intracellular level (Fernández-Dueñas et al., 2013). Scale bar: 10 µm
   
   
2. The day of the experiment the transiently transfected cells were mounted in an Attofluor holder and placed on an inverted Axio Observer microscope equipped with a 63x oil immersion objective and a dual-emission photometry system.
   
3. Then, cells were continuously superfused with a FRET-compatible A_2AR fluorescent ligand (i.e. MRS5424) (Fernández-Dueñas et al., 2012) dissolved in HBSS and applied with the aid of a focal drug application system.
   
4. A Polychrome V was used as the light source in our dual-emission photometry system. Upon excitation with the corresponding donor excitation wavelength (i.e. A_2AR^CFP) the fluorescent signals of the donor and acceptor fluorophores were detected by avalanche photodiodes and digitized using a Digidata 1440A analog/digital converter.
   
5. pCLAMP and GraphPad Prism softwares were used for data collection and analysis.
   
6. Accordingly, a FRET signal was measured upon donor (i.e. A_2AR^CFP) excitation at 430 ± 10 nm [beam splitter dichroic long-pass (DCLP) 460 nm] and an illumination time set to 10 ms at 10 Hz. Then, the emission light intensities were determined at 535 ± 15 nm (F₅₃₅; MRS5424 emission) and 480 ± 20 nm (F₄₈₀; A_2AR^CFP emission) with a beam splitter DCLP of 505 nm. No corrections for spillover between channels or direct MRS5424 excitation were made.
   
7. The increase in FRET ratio (F₅₃₅/F₄₈₀) was fitted to the equation: r(t) = A x (1 - e^-t/τ), where τ is the time constant (in seconds) and A is the magnitude of the FRET signal (Figure 2). When necessary for calculating τ, agonist-independent changes in FRET due to photobleaching were subtracted (Fernández-Dueñas et al., 2012, Fernández-Dueñas et al., 2013).
   
   
   Figure 2. Example of FRET ratio fitting. Time-resolved changes in A_2AR^CFP and MRS5424 fluorescence emission signals in single cells transfected with A_2AR^CFP (see Figure 1). The ratio (blue trace) of the emission intensities of the MRS5424 (F₅₃₅) and CFP (F₄₈₀) in response to MRS5424 application was recorded from single HEK293 cells expressing the A_2AR^CFP (see Figure 1). Shown are the changes induced by rapid superfusion with 2 μM MRS5424. The increase of the ratio F₅₃₅/F₄₈₀ was fitted by a simple monoexponential curve (r(t) = A x (1 - e^-t/τ)) using the GraphPad Prism software which gave a time constant (τ) in this experiment of 14 ± 1 sec. This assay is well suited for competitive ligand binding experiments using non-fluorescent compounds (Fernández-Dueñas et al., 2012, Fernández-Dueñas et al., 2013).
   

Recipes

1. HBSS
   137 mM NaCl
   5.4 mM KCl
   0.3 mM Na₂HPO₄
   0.4 mM KH₂PO₄
   4.2 mM NaHCO₃
   1.3 mM CaCl₂
   0.5 mM MgCl₂
   0.6 mM MgSO₄
   5.6 mM glucose
   pH 7.4
   
2. Cell culture medium
   Dulbecco’s modified Eagle’s medium (DMEM) supplemented with:
   1 mM sodium pyruvate
   2 mM L-glutamine
   100 U/ml streptomycin
   100 mg/ml penicillin
   5% (v/v) fetal bovine serum
   

Acknowledgments

This work was supported by grants SAF2011-24779, Consolider-Ingenio CSD2008-00005 and PCIN-2013-019-C03-03 from Ministerio de Economía y Competitividad and ICREA Academia-2010 from the Catalan Institution for Research and Advanced Studies (to FC), by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Intramural Research Program (to KAJ). FC belong to the “Neuropharmacology and Pain” accredited research group (Generalitat de Catalunya, 2014 SGR 1251). We thank E. Castaño and B. Torrejón from the Scientific and Technical Services (SCT) group at the Bellvitge Campus of the University of Barcelona for their technical assistance.

References

1. Fernandez-Duenas, V., Gomez-Soler, M., Jacobson, K. A., Kumar, S. T., Fuxe, K., Borroto-Escuela, D. O. and Ciruela, F. (2012). Molecular determinants of A_2AR-D₂R allosterism: role of the intracellular loop 3 of the D₂R. J Neurochem 123(3): 373-384.
   
2. Fernández-Dueñas, V., Gómez-Soler, M., Morato, X., Núñez, F., Das, A., Kumar, T. S., Jaumà, S., Jacobson, K. A. and Ciruela, F. (2013). Dopamine D₂ receptor-mediated modulation of adenosine A_2A receptor agonist binding within the A_2AR/D₂R oligomer framework. Neurochem Inter 63(1): 42-46.