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.