The effect of ionic environments on the affinity of D 2 -dopamine receptor radioligands

Introduction

Interpretation of changes in PET radioligand binding to G-protein coupled receptors (GPCRs) usually assumes a single receptor population. The affinity of ligands for GPCRs such as the D2-dopamine receptor (D2-DA) depends on the coupling of the receptor to the G-protein as well as the ionic environment. In vivo, the ionic environments will vary significantly from extracellular to cytoplasmic to endosomal receptor localisation. The affinity of PET radioligands for GPCRs will therefore depend on the coupling state and the cellular location of the receptor, effectively creating several receptor populations to consider. Understanding the interaction of ionic environment and G-Protein coupling on GPCR affinity for a radioligand is essential for the accurate interpretation of PET data. We investigated the effect of extracellular and early endosomal ionic environments, and the interaction of these with the coupling states of the D2-DA receptors on the in vitro binding properties of three commonly used PET radioligands, [3H[raclopride, [3 H]FLB-457 and [3 H]spiperone in rat striatal membranes.

Methods

Rat striata (150 μg/ml protein) were incubated with either extracellular buffer (50 mM Tris HCl, 140 mM Na⁺, 5 mM K⁺, 1.5 mM Mg²⁺, 1.5 mM Ca²⁺, 110 mM Cl⁻, pH 7.4, 30 oC) or early endosomal buffer (20 mM MES, 10 mM Na⁺, 140 mM K⁺, 0.5 mM Mg²⁺, 0.003 mM Ca²⁺, 10 mM Cl⁻, pH 6.0, 30 °C), in the absence or presence of GTP (100 μM) for 30 minutes (30 °C). Subsequently the assays were incubated with one of the three radioligands, [3 H]raclopride (400 pM - 60 nM), [3 H]FLB-457 (5 pM - 1.5 nM) or [3 H]spiperone (10 pM - 3 nM) for 60 minutes (30 °C). The specific binding component was defined by addition of haloperidol (1 μM). Assays were terminated by filtration through Whatman GF/B filters, and bound radioactivity counted on a liquid scintillation counter.

Results

Equilibrium dissociation constant data (Kd) for the radioligands in the absence and presence of GTP in the two ionic environments are given (Table 1).

Table 1

Kd values (nM; mean ± sem, n=4) for each radioligand in both buffers ± GTP (100 μM)

	[3H]Raclopride	[3H]FLB-457	[3H]Spiperone
Extracellular
dH2O	1.839 ± 0.339	0.098 ± 0.021	0.322 ± 0.029
GTP	3.137 ± 1.108	0.096 ± 0.014	0.256 ± 0.013
Early endosomal
dH2O	14.047 ± 5.404	3.324 ± 1.78	0.451 ± 0.036
GTP	28.265 ± 3.013	2.006 ± 0.688	0.594 ± 0.063

Extracellular buffer

GTP altered the affinity of the butyrophenone antagonist, [3 H]spiperone, for the D2-DA receptors, but did not alter the affinity of the benzamide antagonists, [3 H]raclopride and [3 H]FLB-457 for the D2-DA receptors.

Early endosomal buffer

For both benzamide radioligands, 8 to 34 fold lower Kd values were exhibited for the D2-DA receptors. Spiperone exhibited 1.4 to 2.3 fold lower Kd values for the D2-DA receptors compared to the extracellular conditions.

Conclusion

All D2-DA radioligands examined exhibited lower Kd values for the D2-DA receptors in the early endosomal buffer compared with the extracellular buffer. As one might expect, the benzamide radioligands exhibited the greatest degree of change. In extracellular buffer, GTP altered the affinity of [3 H]spiperone for the D2-DA receptors demonstrating the sensitivity of [3 H]spiperone for the affinity state of the D2-DA receptor.