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

Abstract

BACKGROUNDG protein-coupled inwardly rectifying potassium (KIR3) 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 APPROACHThe flavonoid glycoside, naringin, was evaluated on recombinant human KIR3.1-3.4 and KIR3.1-3.2 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 KIR3.1 F137S mutant channel important for naringin's activity. KEY RESULTSNaringin (100 mM) 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 KIR3.1 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 IMPLICATIONSThese 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. AbbreviationsCGP36742 or SGS742, 3-aminopropyl-n-butylphosphinic acid; GPCRs, G protein-coupled receptors; KIR3, G proteincoupled inwardly rectifying potassium channels; LPA, lysophosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; r KIR1.1, rat renal outer medullary potassium; TPN-Q, tertiapin-Q

ML297 (VU0456810), the First Potent and Selective Activator of the GIRK Potassium Channel, Displays Antiepileptic Properties in Mice

“…Two recent reports have disclosed additional compounds that activate GIRK. 23,24 In the case of the natural product, naringin, a potency of ∼100 μM limits the utility of the compound to in vitro assays. In, 24 the authors report the identification of both GIRK inhibitors and activators discovered by screening a library of compounds that are structurally related to fluoxetine.…”

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K+(GIRK) channels by the novel anxiolytic drug, ML297

“…Gβγ binding strengthens channel affinity for phosphatidylinositol-4,5-bisphosphate (PIP 2 ), a necessary cofactor for channel gating (4,5). GIRK channels are also activated in a G-protein-independent manner by ethanol (6,7), volatile anesthetics (8,9), and naringin (10). Many psychoactive and clinically relevant compounds with other primary molecular targets inhibit GIRK channels, albeit at relatively high doses (1,11).…”

Structural Insights into GIRK Channel Function

“…In addition to ethanol and halothane, Naringin, a bioflavonoid found in grapefruit, has been identified as a GIRK channel agonist (Yow et al, 2011). Studies suggest that Naringin might exert its agonist effects extracellularly, within a site containing aromatic tyrosine residues on the GIRK1 extracellular vestibule (GIRK1 Y148 and Y150), which are known to be important in tertiapin binding.…”

“…Studies suggest that Naringin might exert its agonist effects extracellularly, within a site containing aromatic tyrosine residues on the GIRK1 extracellular vestibule (GIRK1 Y148 and Y150), which are known to be important in tertiapin binding. Naringin and tertiapin may share a binding site (Yow et al, 2011), although there is no proposed mechanism as to how this might lead to channel activation.…”