Modulation of agonist binding to recombinant human α2-adrenoceptors by sodium ions

AbstractAgonist binding to α2-adrenoceptors is modulated by a number of factors such as Mg2+ and Na+ ions and by experimental manipulations which interfere with receptor–G-protein-coupling such as pertussis toxin pre-treatment or the presence of guanine nucleotides. Agonist binding assays may therefore offer an opportunity to make inferences, albeit indirect, about receptor states or conformations and about the molecular nature of the processes involved in receptor activation. We have investigated possible differences in the effects of Na+ ions on the binding of agonists to the three human α2-adrenoceptor subtypes, α2A, α2B and α2C, recombinantly expressed in S115 mouse mammary tumour cells. NaCl (40 mM) influenced the apparent affinity of a panel of α2-adrenoceptor ligands in a complex compound- and subtype-dependent manner. Sodium ions affected both high- and low-affinity conformations of the receptors, as defined by co-incubation with 10 μM 5′-guanylylimidodiphosphate (Gpp(NH)p). The effects of NaCl and Gpp(NH)p on agonist binding were additive indicating different modes of action for the two allosteric modulators. Thus, quite marked differences between closely related receptor subtypes were noted in the molecular details of agonist–receptor interactions and in the integration of allosteric modulation by Na+ ions. Possible explanations for the experimental findings are discussed within the theoretical framework of multi-state models, and a proposal is presented for a potential physiological role of the modulatory effect of Na+ ions, where intracellular Na+ concentrations would direct the activating influence of receptors to different G-proteins