Agonist binding and function at the human alpha(2A)-adrenoceptor: Allosteric modulation by amilorides

It has been found previously that amilorides act via an allosteric site on the alpha(2A)-adrenergic receptor to strongly inhibit antagonist binding. I n this study, allosteric modulation of agonist binding and function at the alpha(2A)-adrenergic receptor was explored. The dissociation rate of the ag onist [H-3]UK14304 from alpha(2A)-receptors was decreased by the amilorides in a concentration-dependent manner. This contrasts with the increases in H-3-antagonist dissociation rate found previously. The agonist-amiloride an alog interaction data could be fitted to equations derived from the ternary complex allosteric model. The calculated log affinities of the amilorides at the [H-3]UK14304-occupied receptor increased with the size of the 5-N-al kyl side chain and ranged from 2.4 for amiloride to 4.2 for 5-(N,N-hexameth ylene)-amiloride. The calculated negative cooperativities cover a narrow ra nge, in sharp contrast to the broad range found for antagonist-amiloride an alog interactions. The effects of the amilorides on the agonist actions of UK14304, epinephrine, and norepinephrine were explored using a [S-35]GTP ga mma S functional assay, and the parameters calculated for the cooperativiti es and affinities of the UK14304-amiloride analog interactions, using the e quation derived from the ternary complex allosteric model, were in good agr eement with those derived from the kinetic studies. Therefore both the bind ing and functional data provide further support for the existence of a well defined allosteric site on the human alpha(2A)-adrenergic receptor. The bi nding mode of the amilorides at the agonist-occupied and antagonist-occupie d receptor differs markedly but, within each group, the structure of either the agonist or the antagonist examined has only a slight effect on the all osteric interactions.