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.

M-current plays an important role in the regulation of neuronal excitability and stabilizing the membrane potential. KCNQ2 and KCNQ3 potassium ion channels are proposed to underlie the neuronal M-current. In this paper, we studied the permeable properties and the selective properties of the KCNQ2/3 potassium channel with molecular biology and electrophysiology methods, respectively. Then, based on the first principle and the structure of the KCNQ2/3 potassium ion channel, the potential curve is calculated by the density functional theory. And forced by the potential, the dynamical properties of KCNQ2/3 channels are also studied. Our results, not only from electrophysiology study but also stochastic dynamics simulations, indicate that heteromeric KCNQ2/3 channels all showed a permeation sequence of K+ >Rb+>>Na+. The aim of this research is looking for a possible physical basis for the permeation of ion channel and opens an avenue for further research.