Ethanol antagonizes human olfactory receptors through receptor-dependent competitive and noncompetitive mechanisms.

Mechanisms of ligand-receptor interactions at olfactory receptors (ORs), particularly those of antagonists, remain largely unclear. Here, we investigated how ethanol-empirically known to suppress human olfactory perception-modulates OR activity using a heterologous assay system. Ethanol did not activate OR1A1 or OR2W1 but significantly suppressed agonist-induced responses in a concentration-dependent manner. Pharmacological analyses revealed that ethanol acts as a competitive antagonist for OR1A1 and a noncompetitive antagonist for OR2W1, demonstrating receptor-dependent modes of inhibition. Washout experiments showed that ethanol-induced suppression was rapid and reversible, supporting direct receptor antagonism. Structural modeling using AlphaFold3 suggested distinct ethanol binding positions in OR1A1 and OR2W1, providing a structural basis for their different inhibition mechanisms. Furthermore, analyses of primary alcohols (C1-C6) revealed a chain-length-dependent shift in receptor pharmacology, with shorter alcohols acting as antagonists and longer alcohols exhibiting partial or full agonist activity. These findings identify ethanol as a novel OR antagonist and demonstrate receptor-dependent antagonism by a single ligand, offering new insights into olfactory receptor-ligand interactions.