Regulation of human D 1 , D 2(long) , D 2(short) , D 3 and D 4 dopamine receptors by amiloride and amiloride analogues

The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D 1 , D 2 , D 3 and D 4 dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D 3 subtype for acceleration of radioligand dissociation. In equilibrium binding (pseudo‐competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D 1 , D 2(short) , D 2(long) and D 3 receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). At the D 4 subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D 4 subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. MIA reduced the potency with which dopamine stimulated [ 35 S]‐GTPγS binding at the D 2 receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response. British Journal of Pharmacology (2000) 130 , 1045–1059; doi: 10.1038/sj.bjp.0703370