Abstract
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, whereas 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, whereas 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 G protein–coupled receptors, our findings may have a general relevance for these receptors and may guide the design of novel synthetic allosteric modulators or bitopic ligands.
Footnotes
- Received September 8, 2014.
- Accepted December 3, 2014.
A.M. and H.G.T. contributed equally to this study as shared first authors.
This work was supported by The Netherlands Organization for Scientific Research—Chemical Sciences [Grant 714.011.001], the Swedish Research Council [Grant 521-2014-2118], the Swedish strategic research program eSSENCE, and the Swedish National Infrastructure for Computing.
↵ This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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