Effect of sodium ions on a ligand binding to the D2SHORT dopamine receptor M. Vivo and P. G. Strange. School of Animal and Microbial Sciences, University of Reading, P.O. Box 228, Whiteknights, Reading, RG6 6AJ.

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Dopamine receptor ligands have been shown to exhibit different profiles of binding depending on the assay conditions. Sodium ions lead to modifications in the binding properties of antagonists such as raclopride (Armstrong et al., 2001) or sulpiride (Freedman et al., 1982). The aim of this study was to determine the influence of Na⁺ ions on the binding properties of [³H]-nemonapride and [³H]-spiperone to the short isoform of D₂ dopamine receptor (D2S) expressed in Sf9 insect cells.

Sf9 membranes were prepared from cells grown and infected as described previously (Gazi et al., 2003). The binding assays were performed incubating 20 µg of membranes in assay buffer (20 mM HEPES, 1mM EDTA and 1 mM EGTA; pH 7.4 with KOH), with or without the presence of 100 mM NaCl, in a final volume of 1 ml at 25°C for 3h. (+)-butaclamol (3µM) was used to determine the non-specific binding. In saturation experiments radioligand concentrations were from 0.05 nM to 4 nM. In competition experiments [³H]-spiperone (0.3 nM) and [³H]-nemonapride (0.8 nM) and a range of non-radioactive ligand from 0.1 mM to 1 fM were used. Data are mean±s.e.mean for 3 or 4 independent experiments performed as triplicates and statistical comparisons were made by Student's unpaired t-test (p<0.05).

Saturation binding assays were performed under different ionic conditions. The B_max for [³H]-nemonapride increased by 25% with the addition of Na⁺ ions. K_d was not statistically different in both cases. B_max and K_d values for [³H]-spiperone were not altered by the change in ionic conditions (Table 1).

Competition experiments were also performed in the presence and absence of 100 mM NaCl. The pK_i of nemonapride (vs. [³H]-spiperone) was reduced in absence of Na⁺, while spiperone (vs. [³H]-nemonapride) displayed a decrease in pK_i in presence of Na⁺ (Table 1).

The differences observed in the B_max in the saturation experiments together with the variations of the affinity in the competition assays suggest a competitor concentration and Na⁺-dependent negative cooperativity between the two ligands that does not fit with the standard ternary complex model.

Table 1. Saturation (A) and competition (B) analyses with [³H]-nemonapride or [³H]-spiperone in the presence or absence of Na⁺. (*p<0.05;**p<0.005, for effect of Na⁺).

Armstrong, D. et al. (2001). J. Biol. Chem., 276, 2621-9.
Freedman, S.B. et al. (1982). J. Neurochem., 38, 1459-65.
Gazi, L et al. (2003). Br. J. Pharmacol., 138, 775-86.

We thank the BBSRC for financial support.