PT  - JOURNAL ARTICLE
AU  - Arnault Massink
AU  - Hugo Gutierrez-de-Teran
AU  - Eelke B Lenselink
AU  - Natalia V Ortiz Zacarias
AU  - Lizi Xia
AU  - Laura H Heitman
AU  - Vsevolod Katritch
AU  - Raymond C Stevens
AU  - Adriaan P IJzerman
TI  - Sodium Ion Binding Pocket Mutations and Adenosine A<sub>2A</sub> Receptor Function
AID  - 10.1124/mol.114.095737
DP  - 2014 Dec 03
TA  - Molecular Pharmacology
PG  - mol.114.095737
4099  - http://molpharm.aspetjournals.org/content/early/2014/12/03/mol.114.095737.short
4100  - http://molpharm.aspetjournals.org/content/early/2014/12/03/mol.114.095737.full
AB  - Recently we identified a sodium ion binding pocket in a high resolution structure of the human adenosine A2A receptor. In the present study we explored this binding site through site-directed mutagenesis and molecular dynamics simulations. Amino acids in the pocket were mutated to alanine, and their influence on agonist and antagonist affinity, allosterism by sodium ions and amilorides, and receptor functionality was explored. Mutation of the polar residues in the Na+ pocket were shown to either abrogate (D52A2.50 and N284A7.49) or reduce (S91A3.39, W246A6.48, and N280A7.45) the negative allosteric effect of sodium ions on agonist binding. Mutations D52A2.50 and N284A7.49 completely abolished receptor signaling, while mutations S91A3.39 and N280A7.45 elevated basal activity and mutations S91A3.39, W246A6.48, and N280A7.45 decreased agonist-stimulated receptor signaling. In molecular dynamics simulations D52A2.50 directly affected the mobility of sodium ions, which readily migrated to another pocket formed by Glu131.39 and His2787.43. The D52A2.50 mutation also decreased the potency of amiloride with respect to ligand displacement, but did not change orthosteric ligand affinity. In contrast, W246A6.48 increased some of the allosteric effects of sodium ions and amiloride, while orthosteric ligand binding was decreased. These new findings suggest that the sodium ion in the allosteric binding pocket not only impacts ligand affinity, but also plays a vital role in receptor signaling. Because the sodium ion binding pocket is highly conserved in other class A GPCRs, our findings may have a general relevance for these receptors and may guide the design of novel synthetic allosteric modulators or bitopic ligands.