Cholesterol regulation of K+ channels

Elevation of plasma cholesterol is well-known to be a major risk factor in the development of Cardiovascular disease but the mechanisms by which cholesterol regulates the function of membrane proteins are still poorly understood. Our studies focus on inwardly rectifying K+ channels, a major type of K+ channels that are expressed in multiple cell types including endothelial cells, macrophages, smooth muscle cells, cardiomyocytes, and neurons and they play key roles in the regulation of electrical properties of the cellular membranes, cellular excitability and signaling.

Cholesterol-induced suppression of endothelial Kir channels
Our earlier studies have shown that endothelial Kir channels are strongly suppressed by the elevation of membrane cholesterol in vitro (Romanenko et al , Biophys J. 2002) and by plasma hypercholesterolemia in vivo (Fang et al, Circ Res. 2006).
Kir2.1 and Kir2.2 channels were identified as the main component of endothelial Kir current in human aortic endothelial cells (HAECs) (Fang et al, Am J Physiol Cell Physiol. 2005). Our current goal is to determine how cholesterol-induced suppression of endothelial Kir channels impairs endothelial function.


Suppression of endothelial Kir by hypercholesterolemia
in vivo
and rescue of Kir by MβCD ex vivo

Cholesterol suppression of Kir channels in progenitor cells
We have also shown that plasma hypercholesterolemia suppresses Kir channels in bone-marrow derived progenitor cells (Mohler et al, Biochem Biophys Res Commun. 2007
) and that this effect correlates with an inhibition of cell proliferation. Another objective in the lab is to determine how suppression of Kir channels in these cells affects their progenitor potential.

Cholesterol impact on cardiac Kir channels
Consistent with our studies in endothelial cells and progenitor cells, elevation of membrane cholesterol in vitro and plasma hypercholesterolemia in vivo suppresses Kir2 channels in cardiac myocytes. However, we also discovered that at the same time, cholesterol has an opposite effect on G-protein- activated Kir channels, another type of cardiac Kir (Deng et al, J Biol Chem. 2012
). We believe that it is crucial to determine the impact of these opposite effects on cardiac function.

Structural determinants of cholesterol-induced suppression of Kir
Focusing on Kir2.1 channels, one of the most ubiquitous Kir channel, as a model for Kir function, we have shown that cholesterol sensitivity of the channels critically depends on a specific set of cytosolic residues that form a cholesterol sensitivity belt around the cytosolic gate of the channels (Epshtein et al, Proc Natl Acad Sci USA 2009;
Rosenhouse-Dantsker et al, Biophys J. 2011). Our further studies also identified a two-way molecular switch that regulates cholesterol sensitivity of the channels through distant cytosolic residues (Rosenhouse-Dantsker et al, J Biol Chem. 2012). 

Furthermore, using bacterial homologue of Kir channels, we provided the first evidence that cholesterol regulates Kir channels by direct lipid-protein interactions (Singh et al, J Biol Chem. 2009; Singh et al, 2011 Biochim Biophys Acta. 2011). These studies provide one of the most comprehensive insights into the mechanisms of cholesterol regulation of ion channels. Our current goal is to determine how exactly cholesterol interacts with the channel protein.

Cystolic cholesterol sensitivity belt of Kir2.1. Side view of a model of Kir2.1 that includes all four subunits. Shown in the model are the residues whose mutation affects cholesterol sensitivity: D51 and H53 (cyan), E191 and V194 (blue), N216 and K219 (pink), L222 (red), and C311 (green)

Impact of Oxidized Lipids on Endothelial Biomechanics

Multiple studies established that oxidized modifications of lipoproteins, such as oxLDL, are major risk factors for the development of atherosclerosis. However, the mechanisms of oxLDL-induced endothelial dysfunction are still poorly understood. Our studies discovered that oxLDL results in an increase in stiffness of aortic endothelial cells and we propose that endothelial stiffening is the key early step in endothelial dysfunction that leads to the disruption of the endothelial barrier and alters the sensitivity of endothelial cells to shear stress forces.

OxLDL induces endothelial stiffening
Initially, our hypothesis was that cholesterol loading of endothelial cells should increase endothelial stiffness and impair the ability of endothelial cells to respond to shear stress. Our first surprise, however, was to discover that cholesterol loading has no effect on EC (endothelial cells) stiffness whereas it is cholesterol depletion that induces EC stiffening (Byfield et al, Biophys J. 2004). Furthermore, we also found that EC stiffening is observed both in vitro and in vivo and is a result of oxLDL exposure (Byfield et al, J Lipid Res. 2006). More recently, we found that EC stiffness is regulated by specific oxysterols that are found both in the oxLDL complex and in the vascular wall of dyslipidemic ApoE-/- mice. (Shentu et al, J Lipid Res. 2012).
 
Oxidized low density lipoprotein (OxLDL), but not LDL, constrains membrane deformation of human aortic endothelial cells (HAECs). Typical images of membrane deformation for cells treated with 10 μG/ml OxLDL or 10 μG/ml LDL for 1 h. The images show the maximal deformation at -5 mm Hg. Arrows indicate the positions of the aspirated projections. Scale bar, 30 μM.

oxLDL disrupts lipid packing of cholesterol-rich membrane domains
Surprisingly, our studies discovered that that oxLDL induces fluidization of ordered membrane domains as estimated by Laurdan two-photon microscopy providing the first insights into the mechanism of how oxLDL regulates endothelial biomechanics and the reason for the similarity between oxLDL and cholesterol depletion (Shentu et al, Am J Physiol Cell Physiol. 2010; Levitan and Shentu, J Lipids. 2011). Also see editorial "Lose cholesterol, get stiff!" in Am J Physiol Cell Physiol. 2010.
Impact of oxidized low-density lipoprotein (oxLDL) on lipid packing of membrane domains in bovine aortic endothelial cells (BAECs), typical general polarization (GP) images of control cells (Ctrl) and oxLDL-treated cells (oxLDL). Scale bar is 5.6 μM.


The role of EC stiffening in the sensitivity of endothelial cells to flow
To address this question, we tested how oxLDL affects the ability of ECs to realign in the direction of the flow and flow-induced cytoskeleton reorganization. Unexpectedly, we found that both oxLDL and cholesterol depletion facilitate flow-induced endothelial realignment and re-organization of the cytoskeleton suggesting that an increase in stiffness sensitizes the cells to a mechanical stimulus generated by shear stress (Kowalsky et al, Am J Physiol Cell Physiol. 2008). Most recently, we found that cell stiffening is also associated with increased recovery from cell swelling suggesting further that it sensitizes cells to mechanical stimuli (Kowalsky et al, J Biol Chem. 2012 Sep 20).
 
Additionally, oxLDL and an increase in endothelial stiffness correlated with an increase in force generated by the cells, and an increased ability of HAECs to elongate and form networks in a 3D environment suggesting that exposure to oxLDL facilitates Angiogenesis. These studies suggested a novel link between oxLDL and neovascularization of complex atheromateous lesions (Byfield et al, J Lipid Res. 2006; Shentu et al, Am J Physiol Cell Physiol. 2010).
Our current goals are to elucidate the mechanisms responsible for oxLDL-induced endothelial stiffening and the impact of this effect on endothelial sensitivity to flow and angiogenesis.