Naringin directly activates inwardly rectifying potassium channels at an overlapping binding site to tertiapin-Q

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 K _IR3.1-3.4 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 K _IR3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant K _IR3.1-3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1-M2 loop of the K _IR3.1 ^F137S mutant channel important for naringin's activity. KEY RESULTS Naringin (100 μM) had minimal effect on uninjected oocytes but activated K _IR3.1-3.4 and K _IR3.1-3.2 channels. The activation by naringin of K _IR3.1-3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the K _IR3.1 ^F137S 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 K _IR3 channels and that tertiapin-Q shares an overlapping binding site on the K _IR3.1-3.4. This is the first example of a ligand that activates K _IR3 channels by binding to the extracellular M1-M2 linker of the channel.