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 regulation of resting membrane potential and cell excitability. To achieve its physiological roles, Kir2.1 performs a series of conformational transition, named as gating. However, the structural basis of gating is still obscure. Here, we combined site-directed mutation, 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 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 G-loop. Proteins 2016; 84:1929-1937. © 2016 Wiley Periodicals, Inc.

Keywords: Kir channel; gating kinetics; homology model; molecular dynamics; targeted molecular dynamics; weak interaction.

Figure 1

RMSD variations for four systems (a, closed and open states of wild type. b, closed and open states of H221R.) throughout the simulation. RMSDs were calculated based on all Cα atoms of the channel.

Figure 2

(a) The schematic diagram of a targeted structure, which is in the open state. (b) The schematic diagram of the initial structure, which is in the closed state. The target residues, to which the targeted force is applied (residues Lys185-Thr192 and PIP₂ binding sites), are colored blue. The G-loop is highlighted in black. (c) The schematic diagram of the final conformation is one which is achieved by our last Targeted MD simulation.

Figure 3

(a) The time course of the interaction between G-loop (blue) and CD-loop (yellow) through an intra-subunit E303-H221 interaction; (b) Variation of the strengthened interactions between the CD-loop (yellow) and C-linker (red) through H221-R189 and between adjacent G-loops through E303-R312 (c) as a function of simulation time. (a-c) are taken the average of H-bond number every 40ps.

Figure 4. H221R mutation impacts the gating kinetics of Kir2.1

(a) represents the time course of reactivation (PIP₂ re-phosphorylation, a 4-fold effect) and inhibition (PIP₂ de-phosphorylation induced by C_i-VSP in a 4-fold effect) of WT-Kir2.1 and H221R; (b) Bars in b are the time constants (mean ± SEM of at least six experiments) corresponding to a.

Figure 5. The three interactions that controls the G-loop gate

(a) The time course of the interaction between G-loop and CD-loop through intra-subunit E303-R221. (b) and (c) show the interaction between CD-loop and C-linker through R221-R189 (b) and between adjacent G-loops through E303-R312 (c). The red and blue lines are shown the average of the H-bond number every 40 ps of H221R (red) and WT-Kir2.1 (blue) as a function of simulation time, respectively.

Figure 6

RMSF (root mean square fluctuation) analysis of the CD-loop (residues 216 to 223) (a) and G-loop (residues 301 to 308) (b) of Kir2.1 and H221R. (c) The time course of the interactions between CD-loop and G-loop of H221R (black) and WT-Kir2.1 (green). (d) Correlation of movements among CD-loop and G-loop of WT and H221R.