The Role of a Sodium Ion Binding Site in the Allosteric Modulation of the A2A Adenosine G Protein-Coupled Receptor

Abstract

SUMMARY The function of G protein-coupled receptors (GPCRs) can be modulated by a number of endogenous allosteric molecules. In this study, we used molecular dynamics, radioligand binding and thermostability experiments to elucidate the role of the recently discovered sodium ion binding site in the allosteric modulation of the human A2A adenosine receptor, conserved among class A GPCRs. While the binding of antagonists and sodium ions to the receptor was non-competitive in nature, the binding of agonists and sodium ions appears to require mutually exclusive conformational states of the receptor. Amiloride analogs can also bind to the sodium binding pocket showing distinct patterns of agonist and antagonist modulation. These findings suggest that physiological concentrations of sodium ions affect functionally relevant conformational states of GPCRs, and can help to design novel synthetic allosteric modulators or bitopic ligands exploiting the sodium ion binding pocket.

Sodium Ion Binding Pocket Mutations and Adenosine A2AReceptor Function

“…Most importantly, we learned that all mutations in the sodium ion binding pocket impact A 2A receptor signaling significantly, including both constitutive and agonist-stimulated activity. Although all mutant data in the present study are novel, we have shown a number of similar findings on the wild-type receptor before (Lane et al, 2012;Gutiérrez-de-Terán et al, 2013b), indicative of the robustness of the assay system. We will discuss our findings in the light of available mutation data in literature by examining the mutated amino acids individually.…”

“…In the wild-type receptor, both ligands achieved an average number of four simultaneous hydrogen bonds, mainly with residues Asp52 2.50 and Trp246 6.48 [Supplemental Fig. 2;Gutiérrez-de-Terán et al (2013b)]. For amiloride, the number of hydrogen bonds dropped to approximately two in the two mutants examined, as well as for HMA with mutant W246…”

“…In a recent report we used a combination of molecular dynamics (MD) simulations and biophysical and biochemical experiments (Gutiérrez-de-Terán et al, 2013b) to conclude that sodium ions selectively stabilize the inactive conformation of the wild-type receptor and that a physiologic concentration of NaCl was sufficient to achieve this effect. This mechanism was further corroborated with radioligand binding data, indicative of a competitive interaction between the sodium ion in this allosteric pocket and an agonist in the orthosteric pocket.…”

“…This mechanism was further corroborated with radioligand binding data, indicative of a competitive interaction between the sodium ion in this allosteric pocket and an agonist in the orthosteric pocket. Furthermore, we proposed that the diuretic drug amiloride and analogs compete for the same site and exert an allosteric control on the hA 2A AR quite similar to that of sodium ions, albeit with pharmacological differences in modulation of orthosteric ligand binding (Gutiérrez-de-Terán et al, 2013b).…”

“…Molecular dynamics (MD) simulations of wild-type (WT) and mutant forms of the hA 2A AR were performed following the computational protocol published recently (Gutiérrez-de-Terán et al, 2013b). Briefly, the inactive structure of the hA 2A AR in complex with ZM241385 and a sodium ion [PDB code 4EIY (Liu et al, 2012)] was used as a basis for our simulations, after a refinement process that consisted in modeling the missing ICL3 segment and proton addition, assessing the protonation state of titratable residues (i.e., all charged) and histidine residues, which were protonated on Nd, except for His155 ECL2 (protonated on N«) and His264 ECL3 (positively charged).…”