Abstract

It has been demonstrated previously that amilorides can interact with a well defined allosteric site on the human α_2A-adrenergic receptor. In this study, the question was explored as to whether the human α_1A-adrenergic receptor also possesses an equivalent allosteric site. The six amilorides examined strongly increased the dissociation rate of the antagonist [³H]prazosin from the α_1A-adrenergic receptor in a concentration-dependent manner. With the parent amiloride, the dissociation data were well fitted by an equation derived from the ternary complex allosteric model, compatible with amiloride acting at a defined allosteric site on the α_1A-adrenergic receptor. In contrast, the dissociation data for [³H]prazosin in the presence of the amiloride analogs were not compatible with the equation derived from a one-allosteric-site model, but could be fitted well by an equation derived from a two-allosteric-site model. However, certain individual parameters could not be resolved. The observed dissociation rate constants increased steeply with increasing amiloride analog concentration, and in some cases the data could be fitted with a logistic equation. The slope factors calculated from such fits were 1.2 to 2.1. It is concluded that the structure-binding relationships of the amilorides at the α_1A- and α_2A-adrenergic receptors are different. The interactions of the five amiloride analogs, but not the parent amiloride, with the α_1A-adrenergic receptor are compatible with the presence of two (but not one) allosteric sites, and is thus more complex than that found for the α_2A-adrenergic receptor.