From in silico to in vitro: A trip to reveal flavonoid binding on the Rattus norvegicus Kir6.1 ATP-sensitive inward rectifier potassium channel

Background: ATP-sensitive inward rectifier potassium channels (Kir), are a potassium channel family involved in many physiological processes. KATPdysfunctions are observed in several diseases such as hypoglycaemia, hyperinsulinemia, Prinzmetal angina-like symptoms, cardiovascular diseases. Methods: A broader view of the KATPmechanism is needed in order to operate on their regulation, and in this work we clarify the structure of the Rattus norvegicus ATP-sensitive inward rectifier potassium channel 8 (Kir6.1), which has been obtained through a homology modelling procedure. Due to the medical use of flavonoids, a considerable increase in studies on their influence on human health has recently been observed, therefore our aim is to study, through computational methods, the three-dimensional (3D) conformation together with mechanism of action of Kir6.1 with three flavonoids. Results: Computational analysis by performing molecular dynamics (MD) and docking simulation on rat 3D modelled structure have been completed, in its closed and open conformation state and in complex with Quercetin, 5-Hydroxyflavone and Rutin flavonoids. Our study showed that only Quercetin and 5-Hydroxyflavone were responsible for a significant down-regulation of the Kir6.1 activity, stabilising it in a closed conformation. This hypothesis was supported by in vitro experiments demonstrating that Quercetin and 5-Hydroxyflavone were capable to inhibit KATPcurrents of rat tail main artery myocytes recorded by the patch-clamp technique. Conclusion: Combined methodological approaches, such as molecular modelling, docking and MD simulations of Kir6.1 channel, used to elucidate flavonoids intrinsic mechanism of action, are introduced, revealing a new potential druggable protein site.