Differential regulation of dopamine D2S & D3 receptors by sodium ions

Claire L Newton, Martyn D Wood1 & Philip G Strange, School of Pharmacy, The University of Reading, Reading, RG6 6AJ and 1GlaxoSmithKline, New Frontiers Science Park, Harlow, CM19 5AW.

Binding of compounds to the dopamine D2 receptor is sensitive to the presence of sodium ions (Na+), due to an interaction between Na+ and Aspartate-80 of the receptor (Neve et al., 1991). This highly conserved residue is also present in the closely related dopamine D3 receptor. Therefore, the influence of Na+ on the binding of several ligands to the short isoform of the dopamine D2 receptor (D2S) and to the dopamine D3 receptor expressed in Sf9 insect cells was determined.

Membranes expressing human D2S or D3 receptors were prepared from Sf9 insect cells infected with baculoviruses (Gazi et al., 2003), and radioligand competition binding experiments performed (Armstrong & Strange, 2001). Membranes (10-25 μg) were incubated with [3H]spiperone (0.25 nM or 1 nM for D2S and D3 receptors respectively) and a range of concentrations of competing ligand in assay buffer (20 mM HEPES, 6 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, 0.1 mM DTT, pH 7.4) with or without 100 mM NaCl. Experiments were performed in triplicate, and the data are means ± SEM for 3-6 curves. Data were fitted using Prism (GraphPad, San Diego, USA), and pKi values were calculated as a measure of affinity of the compounds for the receptors. Statistical comparisons were performed using Student’s unpaired t-tests.

Binding of spiperone to both receptors was unaffected by the presence of Na+, making [3H]spiperone a suitable choice of radioligand for this study. All other compounds, with the exception of clozapine, exhibited ~5-fold increases in affinity in the presence of Na+ when binding to D2S receptors. In contrast, at the D3 receptor, only U 99194 and L-741,626 displayed increased affinity (~9-fold) in the presence of Na+. At the D2S receptor the presence of Na+ reduced the affinity of clozapine ~2-fold (Table 1).

Table 1: Competition analyses of a range of compounds versus [3H]spiperone in the presence and absence of Na+. (*p<0.05, **p<0.01, ***p<0.001 for effect of Na+)

In conclusion, Na+ differentially affects ligand binding to dopamine D2S and D3 receptors, suggesting that there may be differences in the overlap of their Na+ and ligand binding pockets, or that Na+ binding may produce different conformations of the two receptors.

Armstrong, D. & Strange, P.G., (2001). J. Biol. Chem., 276, 22621-9.
Gazi, L. et al., (2003). Br. J. Pharmacol., 138, 775-86.
Neve, K. A. et al., (1991). Mol. Pharmacol., 39, 733-739.