Membrane-mediated allosteric modulation of the κ-opioid receptor by nitrous oxide

Nitrous oxide (N₂O) is a widely used inhalational analgesic whose antinociceptive effects depend critically on the κ-opioid receptor (KOR) and its endogenous ligand dynorphin (Dyn). Although N₂O is known to promote Dyn release, whether it can also modulate KOR structure and signaling more directly remains unclear. Here, using all-atom molecular dynamics simulations together with free-energy calculations, we identify a membrane-mediated mechanism by which N₂O allosterically biases KOR toward its active state. N₂O rapidly permeates POPC/cholesterol membranes and preferentially accumulates within the hydrophobic membrane core, where it disrupts lipid packing and enhances membrane fluidity in a concentration-dependent but saturable manner. In receptor-containing systems, N₂O enrichment within the transmembrane region increases the conformational flexibility of the KOR extracellular vestibule, weakens Dyn binding, and reshapes ligand-receptor interactions. Notably, despite reducing Dyn affinity, N₂O markedly lowers the energetic barrier for KOR activation by weakening inhibitory helix-helix interactions and protein-lipid contacts, thereby stabilizing the active receptor conformation. These results support a previously unrecognized membrane-mediated allosteric effect of N₂O on KOR and suggest that N₂O can partially decouple ligand binding from receptor activation through modulation of the lipid environment. More broadly, this work provides a mechanistic link between gaseous anesthetics, membrane physical properties, and GPCR signaling, and highlights the plasma membrane as an active regulator of opioid receptor pharmacology.