Regulation of human D1, D2(long), D2(short), D3 and D4 dopamine receptors by amiloride and amiloride analogues

1 The modulatory effects of the allosteric effecters 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. 2 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. 3 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). 4 At the D-4 subtype, Hill coefficients of unity described the binding data for amiloride and benzamii, 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. 5 MIA reduced the potency with which dopamine stimulated [S-35]-GTP gamma S 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.