The study investigates the structural basis for allosteric regulation of G protein-coupled receptors (GPCRs) by sodium ions. The researchers re-engineered the human A2A adenosine receptor by replacing its third intracellular loop with apo-cytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure revealed a network of 57 ordered waters, three major clusters of water molecules, a putative sodium ion bound to the conserved Asp2-50, and two cholesterols stabilizing the conformation of helix VI. The presence of a sodium ion was confirmed through its coordination geometry and its effect on ligand binding and thermal stability. The study also explored off-target interactions of the diuretic amiloride with GPCRs, suggesting that it shares an allosteric binding site with Na+. Additionally, the structure included 23 ordered lipid chains and 3 cholesterols, forming a lipid bilayer around the protein. These findings provide insights into the role of structured waters, sodium ions, and lipids/cholesterol in GPCR stabilization and function.The study investigates the structural basis for allosteric regulation of G protein-coupled receptors (GPCRs) by sodium ions. The researchers re-engineered the human A2A adenosine receptor by replacing its third intracellular loop with apo-cytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure revealed a network of 57 ordered waters, three major clusters of water molecules, a putative sodium ion bound to the conserved Asp2-50, and two cholesterols stabilizing the conformation of helix VI. The presence of a sodium ion was confirmed through its coordination geometry and its effect on ligand binding and thermal stability. The study also explored off-target interactions of the diuretic amiloride with GPCRs, suggesting that it shares an allosteric binding site with Na+. Additionally, the structure included 23 ordered lipid chains and 3 cholesterols, forming a lipid bilayer around the protein. These findings provide insights into the role of structured waters, sodium ions, and lipids/cholesterol in GPCR stabilization and function.