A high-resolution structure of the human A2A adenosine receptor (A2AAR) has been determined, revealing detailed insights into the structural basis of allostery regulated by sodium ions. The structure was solved at 1.8 Å resolution by replacing the third intracellular loop (ICL3) of A2AAR with apo-cytochrome b562RIL (BRIL), resulting in the chimeric protein A2AAR-BRIL-ΔC. The structure shows 57 ordered waters, a sodium ion bound to a conserved Asp2.50, and three cholesterol molecules, along with 23 lipid acyl chains. These findings highlight the role of structured waters, sodium ions, and lipids in GPCR stabilization and function. The structure reveals a continuous water channel extending from the ligand-binding site to the G protein interaction site, containing three major water clusters. The central cluster includes a sodium ion and 10 waters, filling a cavity in the 7TM bundle. The sodium ion is coordinated by five oxygen atoms, including those from Asp52 and three water molecules, consistent with known coordination patterns for Na+ in protein structures. The presence of Na+ in the structure is supported by experimental conditions used during purification and crystallization. The structure also shows that the sodium ion is surrounded by several highly conserved residues, including Asp52, Ser91, Trp246, Asn280, Asn284, and Tyr288. Structural alignment of these residues across GPCR structures reveals a conserved site capable of binding Na+ along with several water molecules. The central water cluster likely plays an important role in receptor activation, as the pocket collapses upon activation, reducing the volume available for Na+ binding. The study also investigates the interactions of amiloride with GPCRs, showing that it non-competitively displaces a radiolabeled antagonist from the α2-adrenergic receptor. Amiloride and its derivatives share an allosteric binding site with Na+ and are docked into the central cluster cavity, where their charged guanidinium group interacts with Asp52, while other polar groups form hydrogen bonds with residues in the pocket. The structure includes 23 ordered lipid chains and three cholesterol molecules, forming an almost complete lipid bilayer around each protein molecule. These lipids and cholesterol mediate crystal contacts and stabilize the receptor's conformation. The cholesterol molecules, particularly CLR2 and CLR3, occupy hydrophobic grooves along helix VI and form extensive contacts with the aromatic ring of Phe255, which is sandwiched between them. These interactions may play a functional role in stabilizing the receptor and fixing the position of Asn253 in the ligand-binding pocket. The study highlights the importance of ions like sodium, lipids, and waterA high-resolution structure of the human A2A adenosine receptor (A2AAR) has been determined, revealing detailed insights into the structural basis of allostery regulated by sodium ions. The structure was solved at 1.8 Å resolution by replacing the third intracellular loop (ICL3) of A2AAR with apo-cytochrome b562RIL (BRIL), resulting in the chimeric protein A2AAR-BRIL-ΔC. The structure shows 57 ordered waters, a sodium ion bound to a conserved Asp2.50, and three cholesterol molecules, along with 23 lipid acyl chains. These findings highlight the role of structured waters, sodium ions, and lipids in GPCR stabilization and function. The structure reveals a continuous water channel extending from the ligand-binding site to the G protein interaction site, containing three major water clusters. The central cluster includes a sodium ion and 10 waters, filling a cavity in the 7TM bundle. The sodium ion is coordinated by five oxygen atoms, including those from Asp52 and three water molecules, consistent with known coordination patterns for Na+ in protein structures. The presence of Na+ in the structure is supported by experimental conditions used during purification and crystallization. The structure also shows that the sodium ion is surrounded by several highly conserved residues, including Asp52, Ser91, Trp246, Asn280, Asn284, and Tyr288. Structural alignment of these residues across GPCR structures reveals a conserved site capable of binding Na+ along with several water molecules. The central water cluster likely plays an important role in receptor activation, as the pocket collapses upon activation, reducing the volume available for Na+ binding. The study also investigates the interactions of amiloride with GPCRs, showing that it non-competitively displaces a radiolabeled antagonist from the α2-adrenergic receptor. Amiloride and its derivatives share an allosteric binding site with Na+ and are docked into the central cluster cavity, where their charged guanidinium group interacts with Asp52, while other polar groups form hydrogen bonds with residues in the pocket. The structure includes 23 ordered lipid chains and three cholesterol molecules, forming an almost complete lipid bilayer around each protein molecule. These lipids and cholesterol mediate crystal contacts and stabilize the receptor's conformation. The cholesterol molecules, particularly CLR2 and CLR3, occupy hydrophobic grooves along helix VI and form extensive contacts with the aromatic ring of Phe255, which is sandwiched between them. These interactions may play a functional role in stabilizing the receptor and fixing the position of Asn253 in the ligand-binding pocket. The study highlights the importance of ions like sodium, lipids, and water