Dual pattern of cholesterol-induced decoupling of residue-residue interactions of Kir2.2.

Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2P187V with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Membrane Cholesterol Interactions with Proteins in Hypercholesterolemia-Induced Endothelial Dysfunction.

PURPOSE OF REVIEW: The goal of this review is to highlight work identifying mechanisms driving hypercholesterolemia-mediated endothelial dysfunction. We specifically focus on cholesterol-protein interactions and address specific questions related to the impact of hypercholesterolemia on cellular cholesterol and vascular endothelial function. We describe key approaches used to determine the effects of cholesterol-protein interactions in mediating endothelial dysfunction under dyslipidemic conditions. RECENT FINDINGS: The benefits of removing the cholesterol surplus on endothelial function in models of hypercholesterolemia is clear. However, specific mechanisms driving cholesterol-induced endothelial dysfunction need to be determined. In this review, we detail the latest findings describing cholesterol-mediated endothelial dysfunction, highlighting our studies indicating that cholesterol suppresses endothelial Kir2.1 channels as a major underlying mechanism. The findings detailed in this review support the targeting of cholesterol-induced suppression of proteins in restoring endothelial function in dyslipidemic conditions. The identification of similar mechanisms regarding other cholesterol-endothelial protein interactions is warranted.

Impairment of endothelial glycocalyx in atherosclerosis and obesity.

Endothelial glycocalyx is a negatively charged gel-like layer located on the apical surface of endothelial cells. It serves as a selective two-way physical barrier between the flowing blood and the endothelium, which regulates the access of macromolecules and of blood cells to the endothelial surface. In addition, endothelial glycocalyx plays a major role in sensing mechanical signals generated by the blood flow and transducing these signals to maintain endothelial functions; Thus, dysfunction or disruption of endothelial glycocalyx in pathological condition leads to endothelial dysfunction and contributes to the development of vascular diseases. In this review, we discuss the impact of atherosclerosis with the following viewpoints: (i) hypercholesterolemic effects on endothelial glycocalyx degradation in animal models and human patients, (ii) disruption of endothelial glycocalyx by atherogenic lipoproteins, (iii) proatherogenic disturbed flow effects on endothelial glycocalyx degradation, (iv) pathological consequences of the loss of glycocalyx integrity in atherogenesis, and (v) therapeutic effect of glycocalyx supplementation on atherosclerosis development. Additionally, we also discuss recent studies in pathological effects of obesity on the disruption of endothelial glycocalyx.

Differential effects of obesity on visceral versus subcutaneous adipose arteries: role of shear-activated Kir2.1 and alterations to the glycocalyx.

Obesity imposes well-established deficits to endothelial function. We recently showed that obesity-induced endothelial dysfunction was mediated by disruption of the glycocalyx and a loss of Kir channel flow sensitivity. However, obesity-induced endothelial dysfunction is not observed in all vascular beds: visceral adipose arteries (VAAs), but not subcutaneous adipose arteries (SAAs), exhibit endothelial dysfunction. To determine whether differences in SAA versus VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx, mice were fed a normal rodent diet, or a high-fat Western diet to induce obesity. Flow-induced vasodilation (FIV) was measured ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant-negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAAs of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAAs, but had no effect on VAAs, from obese mice and humans. Obesity abolished Kir shear sensitivity in VAA endothelial cells and significantly altered the VAA glycocalyx. In contrast, Kir shear sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were less pronounced. We reveal distinct differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA versus VAA endothelial function with obesity.NEW & NOTEWORTHY We identified a role for endothelial Kir2.1 in the differences observed in VAA versus SAA endothelial function with obesity. The endothelial glycocalyx, a regulator of Kir activation by shear, is unequally perturbed in VAAs as compared with SAAs, which we propose results in a near complete loss of VAA endothelial Kir shear sensitivity and endothelial dysfunction. We propose that these differences underly the preserved endothelial function of SAA in obese mice and humans.

Effective Parameters Controlling Sterol Transfer: A Time-Resolved Small-Angle Neutron Scattering Study.

Though cholesterol is the most prevalent and essential sterol in mammalian cellular membranes, its precursors, post-synthesis cholesterol products, as well as its oxidized derivatives play many other important physiological roles. Using a non-invasive in situ technique, time-resolved small angle neutron scattering, we report on the rate of membrane desorption and corresponding activation energy for this process for a series of sterol precursors and post-synthesis cholesterol products that vary from cholesterol by the number and position of double bonds in B ring of cholesterol's steroid core. In addition, we report on sterols that have oxidation modifications in ring A and ring B of the steroid core. We find that sterols that differ in position or the number of double bonds in ring B have similar time and energy characteristics, while oxysterols have faster transfer rates and lower activation energies than cholesterol in a manner generally consistent with known sterol characteristics, like Log P, the n-octanol/water partitioning coefficient. We find, however, that membrane/water partitioning which is dependent on lipid-sterol interactions is a better predictor, shown by the correlation of the sterols' tilt modulus with both the desorption rates and activation energy.

Noncanonical function of long myosin light chain kinase in increasing ER-PM junctions and augmentation of SOCE.

Increased endothelial permeability leads to excessive exudation of plasma proteins and leukocytes in the interstitium, which characterizes several vascular diseases including acute lung injury. The myosin light chain kinase long (MYLK-L) isoform is canonically known to regulate the endothelial permeability by phosphorylating myosin light chain (MLC-P). Compared to the short MYLK isoform, MYLK-L contains an additional stretch of ~919 amino acid at the N-terminus of unknown function. We show that thapsigargin and thrombin-induced SOCE was markedly reduced in Mylk-L-/- endothelial cells (EC) or MYLK-L-depleted human EC. These agonists also failed to increase endothelial permeability in MYLK-L-depleted EC and Mylk-L-/- lungs, thus demonstrating the novel role of MYLK-L-induced SOCE in increasing vascular permeability. MYLK-L augmented SOCE by increasing endoplasmic reticulum (ER)-plasma membrane (PM) junctions and STIM1 translocation to these junctions. Transduction of N-MYLK domain (amino acids 1-919 devoid of catalytic activity) into Mylk-L-/- EC rescued SOCE to the level seen in control EC in a STIM1-dependent manner. N-MYLK-induced SOCE augmented endothelial permeability without MLC-P via an actin-binding motif, DVRGLL. Liposomal-mediated delivery of N-MYLK mutant but not ∆DVRGLL-N-MYLK mutant in Mylk-L-/- mice rescued vascular permeability increase in response to endotoxin, indicating that targeting of DVRGLL motif within MYLK-L may limit SOCE-induced vascular hyperpermeability.

Impairment of Flow-Sensitive Inwardly Rectifying K+ Channels via Disruption of Glycocalyx Mediates Obesity-Induced Endothelial Dysfunction.

OBJECTIVE: To determine if endothelial dysfunction in a mouse model of diet-induced obesity and in obese humans is mediated by the suppression of endothelial Kir (inwardly rectifying K+) channels. Approach and Results: Endothelial dysfunction, observed as reduced dilations to flow, occurred after feeding mice a high-fat, Western diet for 8 weeks. The functional downregulation of endothelial Kir2.1 using dominant-negative Kir2.1 construct resulted in substantial reductions in the response to flow in mesenteric arteries of lean mice, whereas no effect was observed in arteries of obese mice. Overexpressing wild-type-Kir2.1 in endothelium of arteries from obese mice resulted in full recovery of the flow response. Exposing freshly isolated endothelial cells to fluid shear during patch-clamp electrophysiology revealed that the flow-sensitivity of Kir was virtually abolished in cells from obese mice. Atomic force microscopy revealed that the endothelial glycocalyx was stiffer and the thickness of the glycocalyx layer reduced in arteries from obese mice. We also identified that the length of the glycocalyx is critical to the flow-activation of Kir. Overexpressing Kir2.1 in endothelium of arteries from obese mice restored flow- and heparanase-sensitivity, indicating an important role for heparan sulfates in the flow-activation of Kir. Furthermore, the Kir2.1-dependent component of flow-induced vasodilation was lost in the endothelium of resistance arteries of obese humans obtained from biopsies collected during bariatric surgery. CONCLUSIONS: We conclude that obesity-induced impairment of flow-induced vasodilation is attributed to the loss of flow-sensitivity of endothelial Kir channels and propose that the latter is mediated by the biophysical alterations of the glycocalyx.

Evaluating membrane structure by Laurdan imaging: Disruption of lipid packing by oxidized lipids.

Impact of different lipids on membrane structure/lipid order is critical for multiple biological processes. Laurdan microscopy provides a unique tool to assess this property in heterogeneous biological membranes. This review describes the general principles of the approach and its application in model membranes and cells. It also provides an in-depth discussion of the insights obtained using Laurdan microscopy to evaluate the differential effects of cholesterol, oxysterols and oxidized phospholipids on lipid packing of ordered and disordered domains in vascular endothelial cells.

LDL induces cholesterol loading and inhibits endothelial proliferation and angiogenesis in Matrigels: correlation with impaired angiogenesis during wound healing.

Hypercholesterolemia is a major risk factor for adverse cardiovascular outcomes, but its effect on angiogenesis and wound healing is not well understood. In this study, using a combination of mass spectrometry and laurdan two-photon imaging, we show that elevated levels of low-density lipoprotein (LDL), like those seen in hypercholesterolemic patients, lead to an increase in both free cholesterol and cholesterol esters, as well as increase in lipid order of endothelial cell membranes. Notably, these effects are distinct and opposite to the lack of cholesterol loading and the disruption of lipid order observed in our earlier studies in response to oxidized LDL (oxLDL). The same pathological level of LDL leads to a significant inhibition of endothelial proliferation and cell cycle arrest in G2/M phase, whereas oxLDL enhances endothelial proliferation in S phase of the cycle. LDL but not oxLDL suppresses the expression of vascular endothelial growth factor receptor-2 while enhancing the expression of vascular endothelial growth factor (VEGF). Furthermore, we show that aged (8-10 mo) hypercholesterolemic apolipoprotein E-deficient (ApoE-/-) mice display delayed wound closure compared with age-matched C57/BL6 wild-type controls following a skin punch biopsy. The delay in wound healing is associated with a decreased expression of cluster of differentiation 31 platelet endothelial cell adhesion molecule endothelial marker and decreased angiogenesis within the wound bed. Furthermore, decreased endothelial responsiveness to the growth factors VEGF and basic fibroblast growth factor is observed in ApoE-/- mice in Matrigel plugs and in Matrigels with high levels of LDL in wild-type mice. We propose that plasma hypercholesterolemia is antiangiogenic due to elevated levels of LDL.

Endothelial inwardly-rectifying K+ channels as a key component of shear stress-induced mechanotransduction.

It has been recognized for decades that fluid shear stress plays a major role in vascular function. Acting on the endothelium shear stress induces vasorelaxation of resistance arteries and plays a major role in the propensity of the major arteries to atherosclerosis. Many elements of shear-induced signaling have been identified yet we are just beginning to decipher the roles that mechanosensitive ion channels may play in the signaling pathways initiated by shear stress. Endothelial inwardly-rectifying K+ channels were identified as potential primary mechanosensors in the late 1980s yet until our recent works, highlighted in the forthcoming chapter, the functional effect of a shear-activated K+ current was completely unknown. In this chapter, we present the physiological effects of shear stress in arteries in health and disease and highlight the most prevalent of today's investigated mechanosensitive ion channels. Ultimately, we focus on Kir2.1 channels and discuss in detail our findings regarding the downstream signaling events that are induced by shear-activated endothelial Kir2.1 channels. Most importantly, we examine our findings regarding hypercholesterolemia-induced inhibition of Kir channel shear-sensitivity and the impact on endothelial function in the context of flow (shear)-mediated vasodilation and atherosclerosis.

Mechanisms of endothelial stiffening in dyslipidemia and aging: Oxidized lipids and shear stress.

Vascular stiffening of the arterial walls is well-known as a key factor in aging and the development of cardiovascular disease; however, the role of endothelial stiffness in vascular dysfunction is still an emerging topic. In this review, the authors discuss the impact of dyslipidemia, oxidized lipids, substrate stiffness, age and pro-atherogenic disturbed flow have on endothelial stiffness. Furthermore, we investigate several mechanistic pathways that are key contributors in endothelial stiffness and discuss their physiological effects in the onset of atherogenesis in the disturbed flow regions of the aortic vasculature. The findings in this chapter describe a novel paradigm of synergistic interaction of plasma dyslipidemia/oxidized lipids and pro-atherogenic disturbed shear stress, as well as aging has on endothelial stiffness and vascular dysfunction.

Role of matrix metalloproteinases and histone deacetylase in oxidative stress-induced degradation of the endothelial glycocalyx.

The glycocalyx is crucial for normal endothelial function. It also tethers extracellular superoxide dismutase (SOD3), which protects the endothelium against oxidative damage. Proteolytic enzymes [matrix metalloproteinases (MMPs)] are capable of disrupting endothelial cell surface proteins, such as syndecans, resulting in derangements of the endothelial glycocalyx. We sought to test the role of MMPs in oxidative stress-mediated disruption of the endothelial glycocalyx and examine the effect of pharmacological inhibition of MMPs on mitigating this detrimental effect. We also examined the role of histone deacetylase (HDAC) in the oxidative stress-mediated MMP induction and glycocalyx remodeling. Oxidative stress was experimentally induced in human adipose microvascular endothelial cells using H2O2 and buthionine sulfoximine in the presence and absence of potent MMP and HDAC inhibitors. H2O2 and buthionine sulfoximine resulted in a notable loss of the endothelial glycocalyx; they also increased the expression and proteolytic activity of MMP-2 and MMP-9 and subsequently increased the shedding of syndecan-1 and SOD3 from the endothelial cell surface. MMP upregulation was accompanied by a decline in mRNA and protein levels of their inhibitors, tissue inhibitors of metalloproteinase (TIMPs; TIMP-1 and TIMP-3). Furthermore, oxidative stress induced HDAC activity. Inhibition of MMPs and HDAC reversed syndecan-1 and SOD3 shedding and maintained endothelial glycocalyx integrity. HDAC inhibition increased TIMP expression and reduced MMP expression and activity in endothelial cells. Our findings shed light on MMPs and HDAC as therapeutically targetable mechanisms in oxidative stress-induced glycocalyx remodeling. NEW & NOTEWORTHY Oxidative stress, a hallmark of many diseases, damages the endothelial glycocalyx, resulting in vascular dysfunction. Studying the mechanistic link between oxidative stress and endothelial glycocalyx derangements might help discover new therapeutic targets to preserve vascular function. In this study, we investigated the involvement of matrix metalloproteinases and histone deacetylase in oxidative stress-induced endothelial glycocalyx degradation.

Shear-Stress Sensitive Inwardly-Rectifying K+ Channels Regulate Developmental Retinal Angiogenesis by Vessel Regression.

BACKGROUND/AIMS: Shear stress plays major roles in developmental angiogenesis, particularly in blood vessel remodeling and maturation but little is known about the shear stress sensors involved in this process. Our recent study identified endothelial Kir2.1 channels as major contributors to flow-induced vasodilation, a hallmark of the endothelial flow response. The goal of this study is to establish the role of Kir2.1 in the regulation of retinal angiogenesis. METHODS: The retina of newly born Kir2.1+/- mice were used to investigate the sprouting angiogenesis and remodeling of newly formed branched vessels. The structure, blood density and mural cell coverage have been evaluated by immunohistochemistry of the whole-mount retina. Endothelial cell alignment was assessed using CD31 staining. The experiments with flow-induced vasodilation were used to study the cerebrovascular response to flow. RESULTS: Using Kir2.1-deficient mice, we show that the retinas of Kir2.1+/- mice have higher vessel density, increased lengths and increased number of the branching points, as compared to WT littermates. In contrast, the coverage by αSMA is decreased in Kir2.1+/- mice while pericyte coverage does not change. Furthermore, to determine whether deficiency of Kir2.1 affects vessel pruning, we discriminated between intact and degraded vessels or "empty matrix sleeves" and found a significant reduction in the number of empty sleeves on the peripheral part of the retina or "angiogenic front" in Kir2.1+/- mice. We also show that Kir2.1 deficiency results in decreased endothelial alignment in retinal endothelium and impaired flow-induced vasodilation of cerebral arteries, verifying the involvement of Kir2.1 in shear-stress sensing in retina and cerebral circulation. CONCLUSION: This study shows that shear-stress sensitive Kir2.1 channels play an important role in pruning of excess vessels and vascular remodeling during retinal angiogenesis. We propose that Kir2.1 mediates the effect of shear stress on vessel maturation.

Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

Chiral Specificity of Cholesterol Orientation Within Cholesterol Binding Sites in Inwardly Rectifying K+ Channels.

Cholesterol is an integral component of cellular membranes and has been shown to be an important functional regulator for many different ion channels, including inwardly rectifying potassium (Kir) channels. Consequently, understanding the molecular mechanisms underlying this regulation represents a critical field of study. Broadly speaking, cholesterol can mediate ion channel function either directly by binding to specific sites or indirectly by altering surrounding membrane properties. Owing to the similar effects of cholesterol and its chiral isomers (epicholesterol and ent-cholesterol) on membrane properties, comparative analysis of these sterols can be an effective tool for discriminating between these direct and indirect effects. Indeed, this strategy was used to demonstrate the direct effect of cholesterol on Kir channel function. However, while this approach can discriminate between direct and indirect effects, it does not account for the promiscuity of cholesterol binding sites, which can potentially accommodate cholesterol or its chiral isomers. In this chapter, we use docking analyses to explore the idea that the specificity of cholesterol's effect on Kir channels is dependent on the specific orientation of cholesterol within its putative binding pocket which its chiral isomers cannot replicate, even when bound themselves.

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

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.

Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force.

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

Membrane Stiffening in Osmotic Swelling: Analysis of Membrane Tension and Elastic Modulus.

The effects of osmotic swelling on key cellular biomechanical properties are explored in this chapter. We present the governing equations and theoretical backgrounds of the models employed to estimate cell membrane tension and elastic moduli from experimental methods, and provide a summary of the prevailing experimental approaches used to obtain these biomechanical parameters. A detailed analysis of the current evidence of the effects of osmotic swelling on membrane tension and elastic moduli is provided. Briefly, due to the buffering effect of unfolding membrane reservoirs, mild hypotonic swelling does not change membrane tension or the adhesion of the membrane to the underlying cytoskeleton. Conversely, osmotic swelling causes the cell membrane envelope to stiffen, measured as an increase in the membrane elastic modulus.