Abstract

The purpose of this study was to determine whether there is a well-defined allosteric site on the human α_2A-adrenergic receptor. To explore this question, we examined the effects of amiloride analogues on the dissociation of [³H]yohimbine, [³H]rauwolscine, and [³H]RX821002. The dissociation data fitted well to an equation derived from the ternary complex allosteric model with amiloride analogue concentration and time as two independent variables. The estimated maximal increase in the [³H]yohimbine dissociation rate caused by the 5-N-alkyl amilorides varied from 2-fold for the parent amiloride to 140- and 160-fold for 5-(N,N-hexamethylene)-amiloride and 5-(N-ethyl-N-isopropyl)-amiloride, respectively. The calculated log affinities at the yohimbine-occupied receptor ranged from 1.75 for 5-(N-ethyl-N-isopropyl)-amiloride to 2.5 for 5-(N,N-hexamethylene)-amiloride. The increase in affinity found at the yohimbine-occupied receptor was not correlated with increase in size of the 5-N-alkyl side chain, in contrast to the situation found at the unoccupied receptor. The effect of competition between two amilorides on yohimbine dissociation also was explored. The data obtained were well fitted by the equation derived from the relevant model, with the off-rate increases caused by 5-(N,N-hexamethylene)-amiloride being either decreased or increased by the competing amiloride analogue in line with predictions, and the parameters derived from the fits were in good agreement with those obtained in the above dissociation assays. Thus, the data are compatible with the amilorides competing at the one allosteric site on the α_2A-adrenergic receptor and rules out the possibility that the amilorides are acting in a nonspecific fashion.