Distant Cytosolic Residues in Kir Channels Control Channel Gating and Modulation by Cholesterol and PI(4,5)P2

Abstract

In recent years, cholesterol has been emerging as a major regulator of ion channel function. Channels regulated by cholesterol include the Kir2 channels subfamily of constitutively active, strongly inwardly rectifying K+ channels that set the resting membrane potential and modulate membrane excitability. Yet, the mechanism by which cholesterol affects channel function is unclear.We have previously shown that Kir2 channels are suppressed by the elevation of membrane cholesterol and enhanced by cholesterol depletion. We thus hypothesized that cholesterol modulates the function of Kir2 channels by stabilizing their closed state. Furthermore, we have recently identified a series of residues in the C- and N-termini of Kir2.1 that are crucial for its sensitivity to cholesterol, suggesting a critical role for the cytosolic domain in cholesterol modulation of Kir channels.Here we show that two cytosolic mutations, L222I and N251D, that are ∼24A apart from one another have similar effects on negating cholesterol and decreasing PI(4,5)P2 sensitivity. Furthermore, both residues have similar effect on the open probability of the channel. These residues are allosterically coupled as the double mutant (L222I_N251D) reverts the effects of each single mutant. This result may be the major reason underlying the differences in cholesterol sensitivity and the strength of interaction with PI(4,5)P2 of the four WT Kir2 channels, Kir2.1, Kir2.2, Kir2.3 and Kir2.4.Moreover, our simulations suggest that the two residues are connected via two β strands through a critical salt bridge between K233 and D246. In agreement with our modeling results, the D246N mutation mimics each of the L222I and N251D mutations. This relationship demonstrates how the intricate arrangement of the cytsolic β sheets connects distant regions of the channel in a manner that enables control of channel gating and modulation.