Cholesterol-induced Changes Research Articles

Cholesterol modulates the structural dynamics of the paddle motif loop of KvAP voltage sensor

KvAP is a prokaryotic Kv channel, which has been widely used as a model system to understand voltage- and lipid-dependent gating mechanisms. In phospholipid membranes, the KvAP-VSD adopts the activated/‘Up’ conformation, whereas the presence of non-phospholipids in membranes favours the structural transition to resting/‘Down’ state. The S3b-S4 paddle motif loop of KvAP-VSD is functionally important as this participates in protein-protein interactions and is the target for animal toxins. In this study, we have monitored the modulatory role of cholesterol – the physiologically-relevant non-phospholipid – on the organization and dynamics of the S3b-S4 loop of the isolated KvAP-VSD in membranes by site-directed fluorescence approaches using the environmental sensitivity of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-ethylenediamine (NBD) fluorescence. Our results show that cholesterol alters the dynamic nature (rotational and hydration dynamics) of S3b-S4 loop in a segmental fashion, i.e., the residues 110 to 114 and 115 to 117 behave differently in the presence of cholesterol, which is accompanied by considerable change in conformational heterogeneity. Further, quantitative depth measurements using the parallax quenching method reveal that the sensor loop is located at the shallow interfacial region of cholesterol-containing membranes, suggesting that the sensor loop organization is not directly correlated with S4 helix movement. Our results clearly show that cholesterol-induced changes in bilayer properties may not be the predominant factor for the sensor loop's altered structural dynamics, but can be attributed to the conformational change of the KvAP-VSD in cholesterol-containing membranes. Overall, these results are relevant for gating mechanisms, particularly the lipid-dependent gating, of Kv channels in membranes.

Dietary cholesterol increases body levels of oral administered vitamin D3 in mice.

Vitamin D and cholesterol share the same intestinal transporters. Thus, it was hypothesized that dietary cholesterol adversely affects vitamin D uptake. The current studies investigated the influence of cholesterol on the availability of oral vitamin D. First, 42 wild-type mice received a diet with 25 µg/kg labelled vitamin D3 (vitamin D3-d3), supplemented with either 0% (control), 0.2%, 0.4%, 0.6%, 0.8%, 1.0% or 2.0% cholesterol for four weeks to investigate vitamin D uptake. In a second study, 10 wild-type mice received diets containing 0% (control) or 1% cholesterol over four weeks to determine cholesterol-induced changes in bile acids. Finally, we investigated the impact of cholesterol versus bile acids on vitamin D uptake in Caco-2 cells. Surprisingly, dietary cholesterol intake was associated with 40% higher serum levels of vitamin D3-d3 and 2.3-fold higher vitamin D3-d3 concentrations in the liver compared to controls. The second study showed that cholesterol intake resulted in higher concentrations of faecal bile acids (control: 3.55 ± 1.71 mg/g dry matter; 1% dietary cholesterol: 8.95 ± 3.69 mg/g dry matter; P < 0.05) and changes in the bile acid profile with lower contents of muricholic acids (P < 0.1) and higher contents of taurodeoxycholic acid (P < 0.01) compared to controls. In-vitro analyses revealed that taurocholic acid (P < 0.001) but not cholesterol increased the cellular uptake of vitamin D by Caco-2 cells. To conclude, dietary cholesterol seems to improve the bioavailability of oral vitamin D by stimulating the release of bile acids and increasing the hydrophobicity of bile.

Abstract P5-18-12: Cardioprotective effects of walnuts against aromatase inhibitor: Pilot study in a zebrafish model

Abstract BACKGROUND: Breast cancer patients on aromatase inhibitor (AI) are at a significantly higher risk of heart failure and cardiovascular disease (CVD) compared with patients on tamoxifen, and trend toward increased risks of myocardial infarction and ischemic stroke. In a clinical setting, AI has been shown to induce endothelial dysfunction that is a predictor of CVD. Further, patients on extended AI therapy exhibit significantly higher number of cardiovascular events when compared with patients who took AI for no longer than 5 years. AI effects on cardiovascular health may reflect the expression of functional estrogen receptors (ERs) in vascular endothelial and smooth muscle cells and cardiomyocytes, especially the membrane bound G-protein coupled ER (GPER). Activation of nuclear ER causes enhanced vasorelaxation and re-endovascularization, whilst GPER has been identified as a mediator of estrogen-induced vasodilation, either acting through nitric oxide (NO) or independent of it. GPER activation also protects against cholesterol-induced endothelial changes and endothelial inflammation. Since walnuts and walnut oil have many cardioprotective properties, we investigated if walnuts might prevent adverse effects of AI on a zebrafish model. Walnuts are rich in plant-derived n-3 PUFA alpha-linolenic acid (ALA).METHODS: Zebrafish embryos are used in cardiotoxicity studies (reduction of heartbeat) to model various human diseases. It has been shown that at 72 hours post fertilization (hpf) the primary and sprout vessels in the zebrafish embryo heart become active, and the blood vessel structure closely resembles of that in the human body. In our study, zebrafish embryos were treated with 4 mL of corn oil, soybean oil, docosahexaenoic acid (DHA) added to soybean oil, walnut oil or walnut powder added to walnut oil when they were 24 hpf. Oils were administered to yolk sac. 10-500 mM letrozole was added to water when fish were 48 hpf, and finally heart beat was measured from 72 hpf embryos. For the heart beat measurements, zebrafish embryos were anesthetized, and recorded to calculate the heart rate. After zebrafish was euthanized, RNA and protein were collected to assess changes in Esr2a, Esr2b, Gper1, Nos1, Nos2a, and Nos2b (shares properties with Nos3/eNOS). RESULTS: Administration of 500 mM letrozole significantly (p&amp;lt;0.001) reduced the heartbeat in zebrafish, indicating that it impaired cardiac function. None of the oils significantly altered the heartbeat, compared with controls. However, both walnut oil (p&amp;lt;0.006) and walnut powder enriched walnut oil (p&amp;lt;0.001) increased heartbeat in letrozole treated zebrafish, compared with zebrafish only given letrozole. Thus, walnuts prevented letrozole induced reduction in heartbeat. RT-qPCR analysis indicated that letrozole significantly reduced Gper (p&amp;lt;0.001) levels. Walnut oil prevented this change. Walnut oil also caused a significant reduction in Nos1 (p=0.014) levels during letrozole treatment. The effect of walnut powder on gene expression has not yet been studied.CONCLUSIONS: Although preliminary, our data suggest that walnuts might be cardioprotective during letrozole treatment. The protective effect may be mediated by prevention of letrozole-induced inhibition of GPER which acts as a vasodilator in the vasculature and cardiomyocytes. Future studies are planned to investigate if feeding adult zebrafish walnut powder and/or walnut oil has cardioprotective effects against chronic letrozole administration, whether GPER is the mediator of these effects, and if GPER acts through activating pathways such as EGFR, ERK, PI3K/Akt and cAMP/PKA. Further, the possibility that walnuts might either adversely or beneficially affect AIs’ effects on breast cancer will be studied. Citation Format: Leena Hilakivi-Clarke, Joas Lucas Da Silva, Anne Blaes, Luke Hoeppner. Cardioprotective effects of walnuts against aromatase inhibitor: Pilot study in a zebrafish model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-18-12.

Interactions of Novel Phosphonium Dye with Lipid Bilayers: A Fluorescence Study

The phosphonium-based optical probes attract ever growing interest due to their excellent chemical and photophysical stability, high aqueous solubility, long wavelength absorption and emission, large extinction coefficient, high fluorescence quantum yield, low cytotoxicity, etc. The present study was focused on assessing the ability of the novel phosphonium dye TDV to monitor the changes in physicochemical properties of the model lipid membranes. To this end, the fluorescence spectral properties of TDV have been explored in lipid bilayers composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) or/and anionic phospholipid cardiolipin (CL). It was observed that in the buffer solution TDV possesses one well-defined fluorescence peak with the emission maximum at 533 nm. The dye transfer from the aqueous to lipid phase was followed by the enhancement of the fluorescence intensity coupled with a red shift of the emission maximum up to 67 nm, depending on the liposome composition. The quantitative information about the dye partitioning into lipid phase of the model membranes was obtained through approximating the experimental dependencies of the fluorescence intensity increase vs lipid concentration by the partition model. Analysis of the partition coefficients showed that TDV has a rather high lipid-associating ability and displays sensitivity to the changes in physicochemical properties of the model lipid membranes. The addition of CL, Chol or both lipids to the PC bilayer gives rise to the increase of the TDV partition coefficients compared to the neat PC membranes. The enhancement of the phosphonium dye partitioning in the CL and Chol-containing lipid bilayers has been attributed to the cardiolopin- and cholesterol-induced changes in the structure and physicochemical characteristics of the polar membrane region.

Allosteric Modulation of GPCRs of Class A by Cholesterol.

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.

Cornelian cherry extract ameliorates osteoporosis associated with hypercholesterolemia in New Zealand rabbits.

Results of animal studies show that a high-cholesterol diet increases bone resorption and decreases bone formation, thus leading to osteoporosis. Previously, we reported on the beneficial influence of Cornelian cherry (Cornus mas L.) fruit on lipid profile in an animal model of diet-induced hipercholesterolemia. To investigate the influence of Cornus mas L. extract and loganic acid (LA) on cholesterol-induced bone changes. The study was conducted on 50 New Zealand rabbits. The animals were given either standard chow (group P) or the same standard chow enriched with 1% cholesterol (other groups). Additionally, the group CHOL+EX received Cornus mas L. extract, group CHOL+LA - loganic acid, and group CHOL+SIM - simvastatin. Serum concentration of bone turnover markers, bone mineral density (BMD) and bone micro-computed tomography (microCT) were assessed. In the CHOL group, a decrease in osteocalcin (OC) and an increase in C-terminated telopeptide of type I collagen (CTX) levels were detected (CHOL vs P 0.674 ±0.159 ng/mL vs 1.003 ±0.297 ng/mL and 10.049 ±1.276 ng/mL vs 7.721 ±1.187 ng/mL, respectively). The EX and LA ameliorated cholesterol-induced changes in serum OC (0.857 ±0.160 ng/mL and 1.103 ±0.356 ng/mL, respectively) and CTX (7.735 ±1.045 ng/mL and 8.128 ±1.106 ng/mL, respectively). There was a significant decrease in femoral BMD in CHOL group (0.429 ±0.11 g/cm² vs 0.449 ±0.020 g/cm²). The EX and LA ameliorated those changes (0.458 ±0.016 g/cm² and 0.449 ±0.021 g/cm², respectively). The microCT revealed increased bone volume ratio (BV/TV) and trabecular thickness (Tb.Th.) in the CHOL+EX group. Cornus mas L. inhibited bone resorption and stimulated bone formation, thereby preventing the development of cholesterol-induced osteoporosis.

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Hydration-mediated stiffening of collective membrane dynamics by cholesterol.

The collective behaviour of individual lipid molecules determines the properties of phospholipid membranes. However, the collective molecular motions often remain challenging to characterise at the desired spatial and temporal resolution. Here we study collective vibrational motion on picosecond time scales in dioleoylphosphatidylcholine lipid bilayers with varying cholesterol content using all-atom molecular dynamics simulations. Cholesterol is found to not only laterally compact the lipid bilayer, but also to change the velocity of longitudinal density fluctuations propagating in the plane of the membrane. Cholesterol-induced reduction of the area per lipid alters the collective dynamics of the lipid headgroups, but not of the lipid tails. The introduction of cholesterol reduces the number of water molecules interacting with the lipid headgroups, leading to a decrease in the velocity of the laterally-propagating sound mode. Thus, the stiffening effect of cholesterol is found to be indirect: decreasing the area per lipid weakens the interactions between the lipid headgroups and water. The collective modes characterised in this work can enable the membrane to dissipate excess energy and thus maintain its structural integrity, e.g., under mechanical stress.

Cholesterol-Induced Conformational Changes Drive Smoothened Activation

Cholesterol-Induced Conformational Changes Drive Smoothened Activation

Cholesterol Changes the Mechanisms of Aβ Peptide Binding to the DMPC Bilayer.

Using isobaric-isothermal all-atom replica-exchange molecular dynamics (REMD) simulations, we investigated the equilibrium binding of Aβ10-40 monomers to the zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer containing cholesterol. Our previous REMD simulations, which studied binding of the same peptide to the cholesterol-free DMPC bilayer, served as a control, against which we measured the impact of cholesterol. Our findings are as follows. First, addition of cholesterol to the DMPC bilayer partially expels the Aβ peptide from the hydrophobic core and promotes its binding to bilayer polar headgroups. Using thermodynamic and energetics analyses, we argued that Aβ partial expulsion is not related to cholesterol-induced changes in lateral pressure within the bilayer but is caused by binding energetics, which favors Aβ binding to the surface of the densely packed cholesterol-rich bilayer. Second, cholesterol has a protective effect on the DMPC bilayer structure against perturbations caused by Aβ binding. More specifically, cholesterol reduces bilayer thinning and overall depletion of bilayer density beneath the Aβ binding footprint. Third, we found that the Aβ peptide contains a single cholesterol binding site, which involves hydrophobic C-terminal amino acids (Ile31-Val36), Phe19, and Phe20 from the central hydrophobic cluster, and cationic Lys28 from the turn region. This binding site accounts for about 76% of all Aβ-cholesterol interactions. Because cholesterol binding site in the Aβ10-40 peptide does not contain the GXXXG motif featured in cholesterol interactions with the transmembrane domain C99 of the β-amyloid precursor protein, we argued that the binding mechanisms for Aβ and C99 are distinct reflecting their different conformations and positions in the lipid bilayer. Fourth, cholesterol sharply reduces the helical propensity in the bound Aβ peptide. As a result, cholesterol largely eliminates the emergence of helical structure observed upon Aβ transition from a water environment to the cholesterol-free DMPC bilayer. We explain this effect by the formation of hydrogen bonds between cholesterol and the Aβ backbone, which prevent helix formation. Taken together, we expect that our simulations will advance understanding of a molecular-level mechanism behind the role of cholesterol in Alzheimer's disease.

The effect of defatted cocoa powder on cholesterol-induced changes of serum lipids in rats

Cocoa has been known for many health benefits, but its lipid-lowering activity still remains unresolved. To investigate effects of varying amounts of defatted cocoa on serum lipids in cholesterol-fed rats. Forty-eight male Sprague-Dawley rats were randomly assigned into four cholesterol-free (control) and four cholesterol-supplemented (experimental) diets containing 0, 1, 2 or 3% defatted cocoa (DC) and given ad libitumto the rats for ten weeks. Serum total cholesterol (TC), low- and very low-density lipoprotein cholesterol (LDL-C and VLDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were quantified, atherogenic index (AI) was calculated, and other biological parameters were assessed. Food intake and body weight did not respond to DC. Compared to 0% DC, 3% DC had the most prominent effect on serum lipids inducing significant fall in LDL-C and TG, and rise in TC/TG in cholesterol-deprived rats, and increase in VLDL-C and AI, and decrease in HDL-C in cholesterol-fed rats. Compared to cholesterol-deprived rats, 3% DC caused significant rise in VLDL-C, AI and TC/TG, and fall in TG in cholesterol-fed rats. This lipid-modifying effect was markedly substantiated by corresponding linear trend responses to DC. Differences in lipid variables of rats fed on DC diets were less evident. Results suggest that, in contrast to cholesterol-free situations, defatted cocoa is seemingly incapable of counteracting the atherogenic effect of cholesterol in rats, perhaps in an interaction that is likely to have clinical implications in cardiometabolic conditions.

The Role of Cholesterol in the Activation of Nicotinic Acetylcholine Receptors.

Cholesterol is a potent modulator of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Here, we review current understanding of the mechanisms underlying cholesterol-nAChR interactions in the context of increasingly available high-resolution structural and functional data. Cholesterol and other lipids influence function by conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable vs nonactivatable conformations, as well as influencing the rates of transitions between conformational states. In the absence of cholesterol and anionic lipids, the nAChR adopts an uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions-unless the nAChR is located in a relatively thick lipid bilayer, such as that found in cholesterol-rich lipid rafts. We highlight different sites of cholesterol action, including the lipid-exposed M4 transmembrane α-helix. Cholesterol and other lipids likely alter function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These same interactions have been implicated in both the folding and trafficking of nAChRs to the cell surface. We evaluate the nature of cholesterol-nAChR interactions, considering the evidence supporting the roles of both direct binding to allosteric sites and cholesterol-induced changes in bulk membrane physical properties.

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations of asymmetric lipid membranes, whose composition is largely consistent with eukaryotic plasma membranes. We show that the membrane dipole potential changes in a cholesterol-dependent manner. Remarkably, moving cholesterol from the extracellular to the cytosolic leaflet increases the dipole potential on the cytosolic side, and vice versa. Biologically this implies that by altering the dipole potential, cholesterol can provide a driving force for cholesterol molecules to favor the cytosolic leaflet, in order to compensate for the intramembrane field that arises from the resting potential.

The Water Permeability and Pore Entrance Structure of Aquaporin-4 Depend on Lipid Bilayer Thickness

Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10−13 cm3/s (mean ± SE), 3.0 ± 0.3 × 10−13 cm3/s, 2.5 ± 0.2 × 10−13 cm3/s, and 0.9 ± 0.1 × 10−13 cm3/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10−13 cm3/s and 2.5 ± 0.1 × 10−13 cm3/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and α-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon thickness, could explain the observed differences in water permeability with changes in bilayer thickness.

Investigating Influenza a M2-Cholesterol Interactions by Oriented Sample Magnetic Resonance

The influenza A M2 protein is a homotetrameric membrane protein that is vital to the viral life cycle. M2 has been shown to interact with cholesterol to induce cell membrane curvature and scission in the production of new virus particles. Previous work from our group used site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe the conformation and dynamics of M2 constructs consisting of the transmembrane and C-terminal domains, revealing multiple conformational states in a cholesterol-modulated equilibrium. The observed conformational changes were proposed to arise from both a direct M2-cholesterol interaction through an amino acid consensus region in the C-terminal domain and indirectly through cholesterol-induced changes to the bilayer environment.We present work using lipid bicelles, which spontaneously orient in a magnetic field, as a membrane alternative to detergent micelles and bilayered vesicles. Nuclear magnetic resonance (NMR) and EPR were used to obtain new information describing the specific M2-cholesterol interaction.

Type VIII Collagen Mediates Vessel Wall Remodeling after Arterial Injury and Fibrous Cap Formation in Atherosclerosis

Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions invivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.

Single-molecule observations for determining the orientation and diffusivity of dye molecules in lipid bilayers

The molecular orientation and diffusion of dye molecules in artificial lipid bilayers were observed using total internal reflection fluorescence microscopy. An artificial lipid bilayer composed of a ternary lipid mixture of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and cholesterol was used. The molecular orientation, which was obtained through defocused imaging, clarified the microscopic features, including cholesterol-induced changes in the local packing structure. Diffusion analysis gave insights into the macroscopic aspects of phase distribution in the heterogeneous bilayer system. Combining these two independent investigations, we revealed the effect of cholesterol addition on microscopic local packing and macroscopic phase structures. Our observations showed a transition from a DLPC-network-like structure to a DPPC-network-like structure upon the addition of cholesterol, which was not evident from previous domain shape observations. The present single-molecule observations yielded the actual phase structure that controls the motion of molecules in the membrane. The results imply that the orientation and diffusivity of molecules offer useful information regarding the phase distribution, which may be hindered by the apparent phase structure in a heterogeneous lipid bilayer that contains cholesterol.

Membrane Cholesterol Strongly Influences Confined Diffusion of Prestin

Prestin is the membrane motor protein that drives outer hair cell (OHC) electromotility, a process that is essential for mammalian hearing. Prestin function is sensitive to membrane cholesterol levels, and numerous studies have suggested that prestin localizes in cholesterol-rich membrane microdomains. Previously, fluorescence recovery after photobleaching experiments were performed in HEK cells expressing prestin-GFP after cholesterol manipulations, and revealed evidence of transient confinement. To further characterize this apparent confined diffusion of prestin, we conjugated prestin to a photostable fluorophore (tetramethylrhodamine) and performed single-molecule fluorescence microscopy. Using single-particle tracking, we determined the microscopic diffusion coefficient from the full time course of the mean-squared deviation. Our results indicate that prestin undergoes diffusion in confinement regions, and that depletion of membrane cholesterol increases confinement size and decreases confinement strength. By interpreting the data in terms of a mathematical model of hop-diffusion, we quantified these cholesterol-induced changes in membrane organization. A complementary analysis of the distribution of squared displacements confirmed that cholesterol depletion reduces prestin confinement. These findings support the hypothesis that prestin function is intimately linked to membrane organization, and further promote a regulatory role for cholesterol in OHC and auditory function.

Cholesterol-induced conformational changes in the oxytocin receptor

Recent studies suggest that cholesterol binding is widespread among GPCRs (G-protein-coupled receptors). In the present study, we analysed putative cholesterol-induced changes in the OTR [OT (oxytocin) receptor], a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant OTRs that are able to bind two small-sized fluorescence-labelled ligands simultaneously. An OTR antagonist was chosen as one of the ligands. To create a second ligand-binding site, a small-sized α-BTB (bungarotoxin binding) site was inserted at the N-terminus or within the third extracellular loop of the OTR. All receptor constructs were functionally active and bound both ligands with high affinity in the nanomolar range. Measurements of the quenching behaviour, fluorescence anisotropy and energy transfer of both receptor-bound ligands were performed to monitor receptor states at various cholesterol concentrations. The quenching studies suggested no major changes in the molecular environment of the fluorophores in response to cholesterol. The fluorescence anisotropy data indicated that cholesterol affects the dynamics or orientation of the antagonist. The energy transfer efficiency between both ligands clearly increased with increasing cholesterol. Overall, cholesterol induced both a changed orientation and a decreased distance of the receptor-bound ligands, suggesting a more compact receptor state in association with cholesterol.

Temperature‐ and cholesterol‐induced changes in eicosapentaenoic acid limitation of Daphnia magna determined by a promising method to estimate growth saturation thresholds

We present data on eicosapentaenoic acid (EPA)‐limited growth responses of Daphnia magna under different temperatures and different dietary cholesterol availabilities to assess how EPA growth saturation thresholds depend on changing environmental conditions. D. magna was raised on gradients of dietary EPA at 15°C and 20°C with high cholesterol supply and at 20°C with low and high cholesterol supply in laboratory experiments. A new method was applied to estimate EPA growth saturation thresholds on the basis of fitted saturation curves using bootstrapped data. The EPA threshold at which 75% and 90% of maximum growth was reached ranged from 0.7 to 1.6 μg EPA (mg dietary C)−1 and 2.0 to 4.9 μg EPA (mg dietary C)−1, respectively. Previously reported EPA concentrations in natural seston of many different lakes suggest that the thresholds measured here indicate a frequent potential for at least moderate EPA limitation in nature. Furthermore, the calculated EPA thresholds were higher in treatments of low compared with high temperature and higher in treatments of low compared with high cholesterol availability. The EPA‐dependent growth responses were more strongly affected by temperature than by cholesterol availability. Our results suggest that EPA growth saturation thresholds for a particular Daphnia species probably vary in nature under different environmental conditions.