CHARACTERIZATION OF THE ALLOSTERIC INTERACTIONS BETWEEN ANTAGONISTS AND AMILORIDE ANALOGS AT THE HUMAN ALPHA(2A)-ADRENERGIC RECEPTOR

The purpose of this study was to determine whether there is a well-def ined allosteric site on the human alpha(2A)-adrenergic receptor. To ex plore this question, we examined the effects of amiloride analogues on the dissociation of [H-3]yohimbine, [H-3]rauwolscine, and [H-3]RX8210 02. 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 [H-3]yohimbine dissociation rate caused by the 5-N-alkyl amilo rides 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)-amilo ride, respectively. The calculated log affinities at the yohimbine-occ upied 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 f ound at the yohimbine-occupied receptor was not correlated with increa se in sire of the 5-N-alkyl side chain, in contrast to the situation f ound at the unoccupied receptor, The effect of competition between two amilorides on yohimbine dissociation also was explored. The data obta ined 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 analog ue in line with predictions, and the parameters derived from the fits were in good agreement with those obtained in the above dissociation a ssays. Thus, the data are compatible with the amilorides competing at the one allosteric site on the alpha(2A)-adrenergic receptor and rules out the possibility that the amilorides are acting in a nonspecific f ashion.