Numerous ion channels are sensitive to the quantity of cholesterol in the membrane (Fantini and Barrantes, 2013, Levitan et al., 2014, Rosenhouse-Dantsker et al., 2012a). In general, three mechanisms were proposed to underly cholesterol regulation of ion channel function: One possible mechanism is altering the biophysical properties of the membrane lipid bilayer, particularly increasing the bilayer stiffness, which increases membrane deformation energy required for the transition between different conformation states and thus the energetic cost of the gating(Andersen and Koeppe, 2007). Another possibility is the formation of cholesterol rich membrane domains that contain ion channels and may facilitate protein–protein interactions between the channels and regulatory proteins (Zakany et al., 2020). The most prevailing mechanism though of cholesterol regulation of ion channels is direct binding of the cholesterol molecule to the channel proteins (Levitan et al., 2014). Early evidence for this mechanism came from the analysis of differential effects of sterols on the channel function. Specifically, our studies indicated that cholesterol regulation of inwardly rectifying potassium channel (Kir2) is stereospecific (Romanenko et al., 2002). A similar phenomenon has been observed in other ion channels, G protein-coupled receptors, and receptor kinases (Belani, 2019). Direct binding of cholesterol was further confirmed for purified bacterial analogue of Kir channels, KirBac1.1 using biochemical approaches (Singh et al., 2011) and NMR (Borcik et al., 2022), and for purified Kir3.2 channels using cryo-electron microscopy (Mathiharan et al., 2021).

To identify cholesterol binding sites for Kir2 channels, we (Rosenhouse-Dantsker et al., 2013, Barbera et al., 2018) and others (Furst et al., 2014) used a combination of computational studies, docking and molecular dynamic (MD) simulations to predict cholesterol-binding pockets which were then validated with electrophysiology. These studies led to the identification of cholesterol binding sites in non-annular hydrophobic pockets of the transmembrane domains of the channels, where cholesterol may interact with the protein in multiple poses within the hydrophobic domain (Rosenhouse-Dantsker et al., 2013). More recently, we discovered that cholesterol binding sites in Kir2.2 channels are distinct for the two known structures of Kir2.2: the 3JYC structure that represents the closed conformation state of Kir2.2 channel (Tao et al., 2009) and 3SPI structure that is similar to the open conformation of Kir2.2 but due to the narrow bundle crossing gate is non-conductive (Hansen et al., 2011) with only partial overlap, which may lead to different modes of channel regulation (Barbera et al., 2018). The two conformation states are also termed “disengaged” and “engaged” respectively (Tao et al., 2009, Hansen et al., 2011).

A series of residues that are important for cholesterol sensitivity of Kir2.1 were also found in the cytosolic domain of Kir2.1 (Epshtein et al., 2009, Rosenhouse-Dantsker et al., 2011). Interestingly, we found an overlap between the residues that confer the sensitivity of Kir2.1 channels to cholesterol and to PI(4,5)P2 (Epshtein et al., 2009), a well-known lipid regulator of these channels (Hansen, 2005, Harraz et al., 2020). Furthermore, we found that cholesterol depletion results in strengthening of PI(4,5)P2-Kir2.1 interaction, as assessed by the kinetics of current rundown in response to sequestering PI(4,5)P2 (Epshtein et al., 2009, Rosenhouse-Dantsker et al., 2014), a well-known approach to assess the strength of PI(4,5)P2-channels interaction (Xie et al., 2005, Pattnaik and Hughes, 2009). Substitution of leucine 222 with isoleucine that renders Kir2.1 to become cholesterol insensitive, also abrogates that strengthening of PI(4,5)P2-Kir2.1 upon cholesterol depletion (Rosenhouse-Dantsker, et al., 2014).

Our recent study explored the impact of cholesterol binding on residue-residue interactions of Kir2.2 channels using MD simulations and network analysis (Barbera et al., 2022). Briefly, we identified domains of coherent motion of Kir2.2 channel, which corresponded to the known functional domains of the channels, such as the pore loop region and the helix bundle crossing region. Furthermore, calculating the net change in residue-residue contacts between these domains when the channel protein was immersed in the bilayer with low vs. high cholesterol content, revealed that cholesterol binding to Kir2.2 induces an uncoupling both, between and within the specific domains of the channel proteins, with the pattern of uncoupling distinct between the 3JYC and the 3SPI structures of the channel. Focusing on the disengaged state of the channel, which showed a stronger uncoupling within the domains than the engaged state, we identified the critical residues that are uncoupled in response to cholesterol binding and found the most pronounced effect for the amino acid residues in the cytoplasmic inter-subunit interacting domains (Barbera et al., 2022). Notably, earlier studies showed that destabilization of the inter-subunit interactions of Kir2 channels leads to the destabilization of the channel gating (Borschel et al., 2017). These predictions were confirmed by site-directed mutagenesis and electrophysiological recordings showing that mutating residues predicted to be uncoupled renders the channel to become cholesterol insensitive or even reverse its cholesterol sensitivity (Barbera et al., 2022).

In the current study, we analyze in detail the uncoupling effect induced by cholesterol binding in the engaged configuration of Kir2.2 channel and identify another critical region of the channel protein to be uncoupled by cholesterol binding. Our new findings show that in this configuration, cholesterol binding is predicted to uncouple the residues located in the vicinity of PI(4,5)P2 binding site, which suggests the mechanistic structural basis of our previous findings.