Molecular Dynamics Simulations of Kir2.2 Interactions with an Ensemble of Cholesterol Molecules

Abstract

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 (μs) 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.

Defining how multiple lipid species interact with inward rectifier potassium (Kir2) channels

“…The other major regulatory lipid is Chol, which inhibits Kir2.2 via direct interactions (37)(38)(39). A number of combined computational and experimental papers have identified Chol interaction sites (27,28,(40)(41)(42)(43), with the most recent study (26) having been discussed above. Interplay between the various lipid species that are present in the plasma membrane (PM) in regulation of Kir channels remains to be fully explored.…”

“…Here, Kir2.2 channel-lipid interactions were determined from simulations in a bilayer containing multiple, functional lipid species. By applying analytical techniques to identify lipid interaction sites, introduced recently by Barbera et al (26) to look at Chol interaction sites of Kir2.2, we can identify multiple lipid interaction sites a priori. Further, by calculating the residence times of each interaction site, we identify their kinetic profiles, while free energy perturbation calculations (see, e.g., ref.…”

“…Recently, a methodology has been developed to use graph-theoretic community analysis (25) of simultaneously interacting residues, rather than the more straightforward cluster analysis referenced above. This community analysis approach was applied to examine Chol interactions with the Kir2.2 channel (26), which have previously proved difficult to definitively identify (27,28).…”

Modulation of Transient Receptor Potential C Channel Activity by Cholesterol

“…This limitation is addressed most recently, with the development of coarse-grained force fields such as the Martini force field, long (µs) time-scale simulations of membrane proteins allowing for the dynamic binding and unbinding of cholesterol to target proteins, providing deeper insight into the mechanisms of cholesterol regulation (Cang et al, 2013;Genheden et al, 2017;Rouviere et al, 2017;Barbera et al, 2018). Using these approaches, most recent studies discovered that in contrast to most other ligands, cholesterol binding is highly flexible and cholesterol dynamically explores its binding site, adopting multiple poses in a "cloud, " rather than occupying a single conformation (Gimpl, 2016;Genheden et al, 2017;Rouviere et al, 2017;Barbera et al, 2018). Recently electron cryo-microscopy structure of zebra fish TRPC4 (TRPC4 DR ) channel in its unliganded closed state, at an overall resolution of 3.6 Å was published (Vinayagam et al, 2018).…”

Electromechanics of lipid-modulated gating of Kv channels

“…POPA lipids have also been found to stabilize the tetrameric assembly of KcsA channels (50)(51)(52) and show stronger interaction with KcSA channels than POPG lipids (50,53,54). Similarly, CHOL has also been shown to inhibit the gating of Kv1.3 and Kir2.2 channels (16,(55)(56)(57). Going beyond Kv channels, POPA lipids have been shown to inhibit the gating of epithelial Na channels (58).…”