Vol. 58, Issue 5, 1091-1099, November 2000

Agonist Binding and Function at the Human _2A-Adrenoceptor: Allosteric Modulation by Amilorides

Ray A. Leppik and Nigel J. M. Birdsall

National Institute for Medical Research, London, United Kingdom

It has been found previously that amilorides act via an allosteric site on the _2A-adrenergic receptor to strongly inhibit antagonist binding. In this study, allosteric modulation of agonist binding and function at the _2A-adrenergic receptor was explored. The dissociation rate of the agonist [³H]UK14304 from _2A-receptors was decreased by the amilorides in a concentration-dependent manner. This contrasts with the increases in ³H-antagonist dissociation rate found previously. The agonist-amiloride analog interaction data could be fitted to equations derived from the ternary complex allosteric model. The calculated log affinities of the amilorides at the [³H]UK14304-occupied receptor increased with the size of the 5-N-alkyl side chain and ranged from 2.4 for amiloride to 4.2 for 5-(N,N-hexamethylene)-amiloride. The calculated negative cooperativities cover a narrow range, in sharp contrast to the broad range found for antagonist-amiloride analog interactions. The effects of the amilorides on the agonist actions of UK14304, epinephrine, and norepinephrine were explored using a [³⁵S]GTPS functional assay, and the parameters calculated for the cooperativities and affinities of the UK14304-amiloride analog interactions, using the equation derived from the ternary complex allosteric model, were in good agreement with those derived from the kinetic studies. Therefore both the binding and functional data provide further support for the existence of a well defined allosteric site on the human _2A-adrenergic receptor. The binding mode of the amilorides at the agonist-occupied and antagonist-occupied receptor differs markedly but, within each group, the structure of either the agonist or the antagonist examined has only a slight effect on the allosteric interactions.