E224G Regulation of the PIP_2-Induced Gating Kinetics of Kir2.1 Channels

As a member of the inwardly rectifying K^+ channel (Kir) family, Kir2.1 allows K^+ to influx the cell more easily than to efflux, a biophysical phenomenon named inward rectification. The function of Kir2.1 is to set the resting membrane potential and modulate membrane excitability. It has been reported that residue E224 plays a key role in regulating inward rectification. The mutant Kir2.1 (E224G) displays weaker inward rectification than the WT channel. Gating of Kir2.1 depends on the membrane lipid, PIP_2, such that the channel gates are closed in the absence of PIP_2. Here we perform electrophysiological and computational approaches, and demonstrate that E224 also plays an important role in the PIP_2-dependent activation of Kir2.1 in addition to its influence on inward rectification. The E224G mutant takes 4.5 times longer to be activated by PIP_2. To probe the mechanism by which E224G slows the channel opening kinetics, we perform targeted molecular dynamics simulations and find that the mutant weakens the interactions between CD-loop and C-linker (H221-R189) and the adjacent G-loops (R312-E303) which are thought to stabilize the open state of the channel in our previous work. These data provide new insights into the regulation of Kir2.1 channel activity and suggest that a common mechanism may be involved in the distinct biophysical processes, such as inward rectification and PIP_2-induced gating.