Cholesterol is a major regulator of multiple types of ion channels, but the specific
mechanisms and the dynamics of its interactions with the channels are not well understood.
Kir2 channels were shown to be sensitive to cholesterol through direct interactions
with "cholesterol-sensitive" regions on the channel protein. In this work, we used
Martini coarse-grained simulations to analyze the long (us) timescale dynamics of
cholesterol with Kir2.2 channels embedded into a model membrane containing POPC phospholipid
with 30 mol% cholesterol. This approach allows us to simulate the dynamic, unbiased
migration of cholesterol molecules from the lipid membrane environment to the protein
surface of Kir2.2 and explore the favorability of cholesterol interactions at both
surface sites and recessed pockets of the channel. We found that the cholesterol environment
surrounding Kir channels forms a complex milieu of different short- and long-term
interactions, with multiple cholesterol molecules concurrently interacting with the
channel. Furthermore, utilizing principles from network theory, we identified four
discrete cholesterol-binding sites within the previously identified cholesterol-sensitive
region that exist depending on the conformational state of the channel-open or closed.
We also discovered that a twofold decrease in the cholesterol level of the membrane,
which we found earlier to increase Kir2 activity, results in a site-specific decrease
of cholesterol occupancy at these sites in both the open and closed states: cholesterol
molecules at the deepest of these discrete sites shows no change in occupancy at different
cholesterol levels, whereas the remaining sites showed a marked decrease in occupancy.