Abstract

Allosteric regulation of rat D2 dopamine receptors by amiloride and amiloride analogs has been studied by investigating their ability to accelerate the dissociation of [3H]spiperone from the receptors expressed in Ltk- cells. The amiloride analogs were more potent and produced a greater maximal effect on the rate of [3H]spiperone dissociation than did amiloride. [3H]Spiperone dissociation was biphasic and could be resolved into contributions from fast and slow rates in the absence and presence of the modulators. Methylisobutylamiloride accelerated both the fast and slow rates of dissociation and modulated the proportions of the two rates. The association of [3H]spiperone in the absence of modulators was also biphasic. The combination of the two sets of association and dissociation rate constants gave very similar equilibrium dissociation constants, and this was confirmed in equilibrium binding experiments that were consistent with a single binding site. It is proposed that there are two binding states for [3H]spiperone that can be distinguished kinetically but not in equilibrium binding experiments. The proportions of these states are differentially modulated with the use of sodium ions and magnesium ions, whereas GTP has no significant effect. Allosteric regulation of [3H]spiperone binding by methylisobutylamiloride could also be observed in saturation and inhibition binding experiments. These effects can be accounted for in a model in which the modulator binds to the competitive site of the receptor and to an allosteric site on the receptor from which it exerts negatively cooperative effects on [3H]spiperone binding to the competitive site and positively cooperative effects on the binding of the modulator to the competitive site.