BACKGROUND G protein-coupled inwardly rectifying potassium (K IR3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action. EXPERIMENTAL APPROACH The flavonoid glycoside, naringin, was evaluated on recombinant human KIR3.1-3.4 and K IR3.1-3.2 expressed in Xenopus oocytes using two-electrode voltage clamp methods. In addition, we evaluated the activity of naringin alone and in the presence of the KIR3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant KIR3.1-3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1-M2 loop of the K IR3.1F137S mutant channel important for naringin's activity. KEY RESULTS Naringin (100 μM) had minimal effect on uninjected oocytes but activated KIR3.1-3.4 and KIR3.1-3.2 channels. The activation by naringin of KIR3.1-3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the K IR3.1F137S mutant channel, replacing one by one aromatic amino acids within the M1-M2 loop, identified tyrosines 148 and 150 to be significantly contributing to the affinity of naringin as these mutations reduced the activity of naringin by 20- and 40-fold respectively. CONCLUSIONS AND IMPLICATIONS These results show that naringin is a direct activator of KIR3 channels and that tertiapin-Q shares an overlapping binding site on the KIR3.1-3.4. This is the first example of a ligand that activates KIR3 channels by binding to the extracellular M1-M2 linker of the channel.