A molecular switch controls the impact of cholesterol on a Kir channel

Cholesterol decreases the activity of the majority of ion channels while increasing the activity of only a few, yet it remains unclear how. Here, we used the inwardly rectifying potassium channel Kir3.4, which is up-regulated by cholesterol, as a tool to address this question. Employing mutagenesis and electrophysiology, we discovered a molecular switch that controls the impact of cholesterol on the channel. Through a single point mutation at position 182 in the transmembrane domain of Kir3.4, we converted the cholesterol-driven up-regulation of the channel into down-regulation. Microseconds-long coarse-grained and atomistic molecular dynamics simulations revealed that the effect of the point mutation propagated toward the selectivity filter of the channel whose conformation controls the conductance of the channel. Planar lipid bilayer experiments validated these results, showing that although cholesterol up-regulated Kir3.4 by increasing its open probability, cholesterol down-regulated the mutant by decreasing its conductance. Further studies underscored the role of mutation-specific alterations of cholesterol distribution in proximity to the channel in cholesterol’s impact on channel activity, highlighting the role of subtle molecular differences in determining how cholesterol distributes around proteins and affects their function.