[³H]-Spiperone Saturation Binding to Dopamine D₂, D₃ and D₄ Receptors

Materials and Reagents

1. Radioligand [³H]-spiperone (e.g. PerkinElmer, catalog number: NET565250UC )
   
2. Butaclamol (e.g. Sigma-Aldrich, catalog number: 55528-07-9)
   
3. Receptor-containing membrane suspension
   
4. Whatman GF/C filters (e.g. PerkinElmer, catalog number: 6005174 )
   
5. Poly(ethyleneimine) solution (PEI) (e.g. Sigma-Aldrich, catalog number: 9002-98-6 )
   
6. Scintillation cocktail (e.g. PerkinElmer, catalog number: 6013641 )
   
7. Tris-HCl
   
8. KCl
   
9. CaCl₂
   
10. MgCl₂
    
11. Destilled water
    
12. Assay buffer (see Recipes)
    
13. Wash buffer (see Recipes)
    
14. NSB solution (see Recipes)
    
15. Radioligand solutions (see Recipes)
    
16. Radioligand Dilutions (see Recipes)
    

Equipment

1. 96 well plates (polysterene)
   
2. Cell harvester (e.g. PerkinElmer)
   
3. Ultra-Turrax^® (IKA, model: 0001602800 ) or similar disperser
   
4. Water bath
   
5. Scintillation counter
   

Software

1. Prism (Graphpad Software, San Diego, CA, USA) or similar
   

Procedure

1. Before the assay
   1. Prepare assay and wash buffer.
      
   2. With some receptor sources PEI-pretreated filterplates may improve the TB/SB ratio: Prepare 0.1% PEI solution and pipet 100 μl/filter on the filterplate, subsequently place in refrigerator (4 °C) for at least 2 hours.
      
   3. The experiment is performed in 96 well plates (polysterene) with a total assay volume of 1,000 μl (450 μl assay buffer + 200 μl radioligand + 200 μl of assay buffer or 5 μM (+)-butaclamol + 150 μl membrane preparation).
      
   4. Prepare NSB solution.
      
   5. Prepare radioligand solutions.
      
   6. Membrane preparation.
      Prepare receptor-containing membrane suspension according to preparation protocol. Re-homogenize suspension in small volume (< 2 ml) using short burst of Ultra-Turrax. Dilute to desired protein concentration and to yield a total volume of about 4 ml per 24 data point experiment. The protein concentration of the membrane suspension should be chosen so that a robust specific binding signal is obtained but at the same time total binding should be < 10% (even better < 5%) of free radioligand concentration. Protein content can be assayed by a variety of essays, e.g. Bradford (1976). Prepare the membrane suspension initially in ice.
      
   7. Pre warm all solutions for 15 min in 25 °C water bath.
      
   8. Final preparation (Table 1), add components to wells in following order:
      
      1. 450 μl assay buffer.
         
      2. 200 μl assay buffer (TB) or 200 μl 5 μM butaclamol (NSB).
         
      3. 150 μl membrane suspension.
         
      4. Start reaction by adding 200 μl radioligand.
         
         Table 1. Pipetting scheme for sample on microtiter plate

         T1	T1	T2	T2	T3	T3	T4	T4	T5	T5	T6	T6
         TB1	TB1	TB2	TB2	TB3	TB3	TB4	TB4	TB5	TB5	TB6	TB6
         NSB1	NSB1	NSB2	NSB2	NSB3	NSB3	NSB4	NSB4	NSB5	NSB5	NSB6	NSB6

         
         
2. During the assay
   1. Incubate for 120 min at 25 °C in water bath.
      
   2. Terminate reaction by rapid vacuum filtration over Whatman GF/C filters using a cell harvester. Wash filter 10 times with ice-cold wash buffer.
      
      
3. After the experiment
   1. Add aliquots of 50 μl radioligand to wells of counting plate to determine total radioactivity.
      
   2. Dry, e.g. in oven for 2 h.
      
   3. Place sticker on bottom plate. Add scintillation cocktail (20 μl) to filters, place sticker on top of plate and count in a scintillation counter, allow adequate time (15 min) before counting samples.
      
      
4. Data analysis
   The following calculations are required to derive K_d and B_max from the data.
   
   1. Carefully inspect raw data for consistency of replicates. Do not light-heartedly eliminate apparent ‘outliers’ from the data set.
      
   2. Subtract mean non-specific binding from each replicate of total binding to obtain specific binding.
      
   3. Transform totals to molar concentration of radioligand in the assay, and measured bound values (total binding, non-specific binding and specific binding) to molar amount of ligand based upon the specific radioactivity of the radioligand and the efficiency of the scintillation counter. A representative experiment with D₂ receptors may look like this.
      
      
      
      
   4. Plot specific binding (y-axis) vs. concentration of radioligand (x-axis).
      
   5. Analyze data by non-linear iterative curve fitting using a rectangular hyperbolic function using one of many available iterative curve fitting programs, e.g. Prism.
      
   6. Molar amount of B_max can be corrected for tissue content, mostly the amount of protein per well (mostly yielding fmol/mg protein); alternative normalization e.g. for tissue wet weight, number of cells or DNA content are possible.
      
   7. As internal quality check, determine the fraction of total binding from total radioactivity in the assay. This needs to be < 10% (better < 5%) as otherwise non-equilibrium conditions may exist. In the latter case, membrane concentration in the assay can be reduced if the measured signal remains robust. Alternatively, assay volume can be increased as this will increase total amount of radioligand (concentration constant) but not amount bound (membrane protein amount constant).
      
   8. In the past when no computer assisted data analysis was available K_d and B_max were estimated with the Rosenthal plots, better known as Scatchard plots (Rosenthal invented them but Scatchard made them famous). In these plots the specifically bound radioligand is on the x-axis and is plotted against the ratio of bound/unbound radioligand on the y-axis. The x-intercept corresponds to B_max and the negative reciprocal of the slope corresponds to K_d. Rosenthal/Scatchard plots can still be useful to visualize that data points indeed fall on a linear line but the estimates derived from this are less reliable than those from iterative curve fitting (the assumptions of linear regression used in these plots don’t meet because x and y are not independent of each other), making this an outdated analysis.
      
   9. If saturation binding experiments are performed in the absence and presence of an inhibitor, the data can be used to test for a competitive nature of the inhibitor. This is the case if the presence of inhibitor changes the K_d but not the B_max of the radioligand.
      

Recipes

1. Assay buffer
   50 mM TRIS: TRIS-HCl: 6.6 g and TRIS base 970 mg (or only 6.04 g TRIS base/L)
   5 mM KCl: 373 mg/L
   2 mM CaCl₂: 220 mg/L
   2 mM MgCl₂(6 H₂O): 410 mg/L
   pH 7.4
   
2. Wash buffer
   50 mM TRIS: TRIS-HCl (33 g/5 L) and TRIS base (4.85 g/5 L) (or only 30.22 g/5 L TRIS base)
   pH 7.4
   
3. 0.1% PEI (only for D₃ receptor assay)
   Prepare 0.1% solution, PEI is delivered as a 50% solution, pipet 1 ml from this with a syringe and add to 9 ml aqua dest. To get 5%, pipet 400 μl PEI 5% + 19.6 ml destilled water to get PEI 0.1%
   
4. NSB solution
   3.98 mg butaclamol HCl/10 ml assay buffer yields 1^.10^-3 M (prepare aliquots of this)
   Dilute this 1:200 to obtain 5 μM in solution, i.e. 1 μM final concentration in assay
   
5. Radioligand solutions
   The intended radioligand concentration range in the assay should be chosen to cover both the expected K_d and about 5-10x K_d (K_d spiperone in transfected cells according to literature ≈ 0.05 nM, 0.35 nM and 0.07 nM for D₂, D₃ and D₄, respectively). The stock solution to be prepared needs to be 5x the assay concentration. Thus, the stock solution should be 25-50x K_d. The following is an example calculation based on a specific activity of 16.2 Ci/mmol radioactive of the radioactive stock solution for a D₂ receptor assay. This needs to be adapted for the other subtypes based on their K_d values and for each batch of radioligand and its specific activity.
   [³H]-spiperone concentration = 1 mCi/ml/(16.2 Ci/mmol x 1,000 mCi/Ci) = 61.7 μM
   Highest concentration needed (10 x K_d x 5) = 10 x 0.05 x 5 = 2.5 nM
   2.5 ml x 2.5 nM = 0.00625 nmol [³H]-spiperone needed
   (0.00625 nmol)/61.7 x 10³ nM = 0.1013 μl of [³H]-spiperone solution needed for 2.5 ml
   Thus 1 μl [³H]-spiperone solution in 25.0 ml assay buffer to yield a concentration of 2.5 nM.
   A free tool for such calculations can be found at www.graphpad.com/quickcalcs/chemMenu/.
   
6. Radioligand Dilutions (Over a 100-fold Range) (Table 2).
   
   Table 2. Dilution scheme to for radioligand working solutions

   Number	Relative concentration	Preparation
   1	100	2.5 ml of radioligand at the highest concentration
   2	66.67	Mix 800 μl from tube 1 + 400 μl assay buffer
   3	50	Mix 1,000 μl from tube 1 + 1,000 μl assay buffer
   4	25	Mix 1,000 μl from tube 3 + 1,000 μl assay buffer
   5	12.5	Mix 1,000 μl from tube 4 + 1,000 μl assay buffer
   6	6.25	Mix 1,000 μl from tube 5 + 1,000 μl assay buffer

   
   

Acknowledgments

This protocol is the adaptation of a protocol originally published by Levant (2007).

References

1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
   
2. Levant, B. (2007). Characterization of Dopamine Receptors. Curr Protoc Pharmacol 36:1.6.1–1.6.15.
   
3. van Wieringen, J. P., Booij, J., Shalgunov, V., Elsinga, P. and Michel, M. C. (2013). Agonist high- and low-affinity states of dopamine D₂ receptors: methods of detection and clinical implications. Naunyn Schmiedebergs Arch Pharmacol 386(2): 135-154.