Characterization of Dopamine Receptors

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Dopamine receptors are the principal targets of drugs used for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. This unit details protocols for radioligand binding assays for various subtypes of dopamine receptor. The scope is limited primarily to the most widely used, well established protocols for binding assays, which employ commercially available tritiated ligands. Protocols are given for radioligand binding assays for D1?like and D2?like receptors. A protocol for the putatively selective labeling of D3 sites is also presented. The basic protocols are used to assay dopamine receptors in membrane homogenates, and support protocols describe the preparation of these homogenates from organs and from cultured cells. The calculation of the radioligand's molar concentration is also included.

Keywords: Receptor binding; dopamine; D1; D2; D3; D4; D5; radioligand binding

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Basic Protocol 1: Binding Assay for D1/D5 Receptors
Basic Protocol 2: Binding Assay for D2/D3/D4 Receptors
Basic Protocol 3: Binding Assay for Putative D3 Receptors
Support Protocol 1: Membrane Preparation from Tissue
Support Protocol 2: Membrane Preparation from Cultured Cells
Support Protocol 3: Calculation of Radioligand Concentration
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Binding Assay for D1/D5 Receptors

Materials

Membrane preparation (see protocol 4 or protocol 52 )
[³ H]SCH 23390 assay buffer (see recipe )
[³ H]SCH 23390 (see protocol 6 ; Perkin‐Elmer, GE Healthcare)
1 mM (+)‐butaclamol in ethanol (store up to 6 months at −20°C)
Test compound(s) (optional)
Wash buffer, ice cold (see recipe )
Homogenizer (e.g., Polytron from Brinkmann or Tissumizer from Tekmar)
12 × 75–mm polystyrene culture tubes
Whatman GF/B filters
Cell harvester (Brandel)

Basic Protocol 2: Binding Assay for D2/D3/D4 Receptors

Materials

Membrane preparation (see protocol 4 or protocol 52 )
Assay buffer: [³ H]spiperone assay buffer (see recipe )
[³ H]spiperone (see protocol 6 ; Perkin‐Elmer, GE Healthcare)
1 mM (+)‐butaclamol in ethanol (store up to 6 months at −20°C)
Test compound(s) (optional)
Wash buffer (see recipe )
Homogenizer (e.g., Polytron from Brinkmann or Tissumizer from Tekmar)

12 × 75–mm polystyrene culture tubes
Whatman GF/B filters
Cell harvester (Brandel)

Basic Protocol 3: Binding Assay for Putative D3 Receptors

Materials

Membrane preparation (see protocol 4 or protocol 52 )
[³ H]PD 128907 assay buffer (see recipe )
[³ H]PD 128907 (see protocol 6 ; GE Healthcare)
1 mM spiperone in ethanol (store up to 6 months at −20°C)
Test compound(s) (optional)
Wash buffer, ice cold (see recipe )
0.5% (v/v) polyethyleneimine (PEI; store up to 1 month at 4°C)

Homogenizer (e.g., Polytron from Brinkmann or Tissumizer from Tekmar)
12 × 75–mm polystyrene culture tubes
Whatman GF/B filters
Cell harvester (Brandel)

Support Protocol 1: Membrane Preparation from Tissue

Materials

Tissue (fresh or frozen; see , discussion on general considerations)
Assay buffer (see Basic Protocols protocol 11 , protocol 22 , or protocol 33 )
Homogenizer (e.g., Polytron from Brinkmann or Tissumizer from Tekmar)

Support Protocol 2: Membrane Preparation from Cultured Cells

Materials

Cultured cells
Complete Earl's balanced salt solution (EBSS; commercially available)
Lysis buffer (see recipe )
Assay buffer (see protocol 1 , protocol 22 , or protocol 33 )

Refrigerated centrifuge and centrifuge tubes
Homogenizer (e.g., Polytron from Brinkmann or Tissumizer from Tekmar)

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Figures

Figure 1.6.1 Representative saturation analysis of [³ H]SCH 23390 binding in striatal membranes. Binding parameters for the radioligand are: K _d = 0.27 nM and B _max = 835 fmol/mg protein. B/F, bound/free ligand.

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Figure 1.6.2 Representative saturation analysis of [³ H]spiperone binding in striatal membranes. Binding parameters for the radioligand are: K _d = 0.14 nM and B _max = 440 fmol/mg protein. B/F, bound/free ligand.

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Figure 1.6.3 Representative saturation analysis of [³ H]PD 128907 binding in striatal membranes. Binding parameters for the radioligand are: K _d = 0.36 nM and B _max = 25 fmol/mg protein. B/F, bound/free ligand.

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Literature Cited

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Key References
	Bylund, D.B. and Yamamura, H.I. 1990. Methods for receptor binding. In Methods in Neurotransmitter Receptor Analysis (H.I. Yamamura, S.J. Enna, and M.J. Kuhar, eds.) pp. 1‐35. Raven Press, New York, N.Y.
	Provides detailed instructions for the analysis of a variety of receptor binding experiments.
	Seeman, 1993. See above.
	Provides encyclopedic summary of literature Kd and Ki values for dopaminergic and other receptors, as well as an extensive reference list.