Three pairs of weak interactions precisely regulate the G-loop gate of Kir2.1 channel

Abstract

Kir2.1 (also known as IRK1) plays key roles in the regulation of resting membrane potential and cell excitability. To achieve its physiological roles, Kir2.1 performs a series of conformational transitions, named as gating. However, the structural basis of gating is still obscure. Here, we combined site-directed mutagenesis, two-electrode voltage clamp with molecular dynamics simulations and determined that H221 regulates the gating process of Kir2.1 by involving a weak interaction network. Our data show that the H221R mutant accelerates the rundown kinetics and decelerates the reactivation kinetics of Kir2.1. Compared with the WT channel, the H221R mutation strengthens the interaction between the CD- and G-loops (E303-R221) which stabilizes the close state of the G-loop gate and weakens the interactions between C-linker and CD-loop (R221-R189) and the adjacent G-loops (E303-R312) which in turn destabilizes the open state of G-loop gate. Our data indicate that the three pairs of interactions (E303-H221, H221-R189 and E303-R312) precisely regulate the G-loop gate by controlling the conformation of the G-loop.

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

“…The most interesting observed pocket is a conservative site, consisting in physical gates formed by cytoplasmic (G loop) region ( Hibino et al, 2010 ), necessary for the regulation of the inward potassium current that obviously depends on the state of the gate ( Lü et al, 2016 ). The G loop has a crucial role with their amino acid composition in the regulation of K ATP gating kinetics, different previous works shown how mutations of the amino acid of this region are responsible of protein inactivation ( Shimomura et al, 2009 ; Li et al, 2016 ; Nishida et al, 2007 ; Pegan et al, 2006 ; Hattersley & Ashcroft, 2005 ; Proks et al, 2005 ).…”

“…3 show that Quercetin formed hydrogen bonds with the amino-groups of Asn-258 and Asn-252 of the chain A, with the hydroxyl group of Ser-222 of the chain A and with the hydroxyl group of the Thr-306 of the chains A and C. The hydrophobic interactions took place between the hydrophobic portion of Quercetin and the hydrophobic-sensing Ile-221 and Val-299 of the chain A. All these interactions take place near the G loop residues and could stabilise the K ATP channel in a closed state ( Li et al, 2016 ). The capacity of Quercetin to inhibit Kir6.1 protein is in agreement with already published data ( Chiang et al, 2002 ; Xu et al, 2015 ; Kaufmann et al, 2013 ; Ogata et al, 1997 ; Matsushita & Puro, 2006 ; Pattnaik & Hughes, 2009 ; Ma et al, 2014 ), thus to validate the hypothesis hydrogen bonds network holds a crucial role in the complex stabilisation, we chose two analogue molecules 5-Hydroxyflavone (different from Quercetin for a reduced number of OH groups) and Rutin (characterised by a bulky glycoside group not present in the other ligands), structures reported in Fig.…”

“…3 ), in order to propose a potential consensus binding site of Kir6.1 and a flavonoid mechanism of action inside the binding pocket. Through bioinformatics approaches and previous works ( Hibino et al, 2010 ), we identified the G loop as potential binding site for ligands ( Lü et al, 2016 ; Shimomura et al, 2009 ; Li et al, 2016 ; Nishida et al, 2007 ; Pegan et al, 2006 ; Hattersley & Ashcroft, 2005 ; Proks et al, 2005 ). Docking simulation and different MD simulation protocols were accomplished for explaining how the flavonoids influenced the gating process of the channel.…”

“…The TMD and CTD are linked by about 20 amino acids and four supporting proteins, the sulfonylurea receptors, which envelop the four subunits of K ATP forming a hetero-octameric complex ( Sepúlveda et al, 2015 ). The Kir channel open-closed mechanism depends on different conformational changes which regulate its state ( Li et al, 2016 ; Lü et al, 2016 ). Small molecules regulate functions of Kir channels for instance: H + , Mg 2+ , Na + ; polyamines, phosphorylation and membrane-bound phospholipids and proteins ( Xie et al, 2007 ; Hibino et al, 2010 ; Li et al, 2016 ; Fowler et al, 2014 ).…”

“…The Kir channel open-closed mechanism depends on different conformational changes which regulate its state ( Li et al, 2016 ; Lü et al, 2016 ). Small molecules regulate functions of Kir channels for instance: H + , Mg 2+ , Na + ; polyamines, phosphorylation and membrane-bound phospholipids and proteins ( Xie et al, 2007 ; Hibino et al, 2010 ; Li et al, 2016 ; Fowler et al, 2014 ). K ATP channels act as an endogenous homeostatic transducer in response to an altered demand ( Carrasco et al, 2001 ; Zingman et al, 2002 ; Miki et al, 2002 ; Gumina et al, 2003 ).…”