Abstract
Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.
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Introduction
The most fundamental function of biological membranes is to enclose the cell and cellular organelles. That way, the plasma membrane maintains the essential differences between the cytosol and the extracellular environment, and the other intracellular membranes maintain the characteristic differences between the contents of each organelle and the cytosol. Membranes also provide the environment in which integral membrane proteins are located, specifically oriented, and specifically assembled. The strict separation of environments by biological membranes is largely due to the hydrophobicity of their interior. However, biological membranes enable communication and transport between the interior and exterior of the cell or the organelle. Water-soluble components such as ions and sugars can cross membranes by channels and transporters [1–3]. Nonelectrolytes can directly permeate the lipid bilayer membrane portion by passive diffusion [4]. Specific assemblies of membrane proteins allow membranes to perform the most complex functions such as signal transduction [5–7] and energy transformation [8]. Nevertheless, the lipid-bilayer portion of the biological membrane determines bulk membrane properties (including diffusion barriers [9, 10]) and can affect the functioning of membrane proteins [11–14].
In this review, we assert that the diversity in the lipid composition determines the membrane properties that allow the membranes to perform certain cellular functions. Cholesterol (Chol) plays a major role in this determination. Thus, we will concentrate mainly on the role of Chol in modifying the main properties of the lipid bilayer of biological membranes such as fluidity, permeability, and hydrophobicity (dielectric properties), and also on its role in the formation of separated phases and domains in the bilayer. The major hypothesis, which we will support thorough this review, states that during biological evolution, membranes with a particular Chol content were selected to perform certain functions in the cells of eukaryotic organisms. Parallel with membrane evolution, the structure of the Chol molecule evolved over a very long time to obtain the ability to optimize certain physical properties of cell membranes with regard to biological functions [15, 16]. In effect, the Chol structure is optimal for its interactions with other lipid species [17, 18]. Like other membrane lipids, Chol is amphiphilic; however, its structure—a rigid fragment that has a smooth side and a rough side, and a short hydrocarbon chain—differs significantly from that of other lipid species of eukaryotic cell membranes.
The long-standing opinion is that the Chol content in the eukaryotic cell membranes increases along the secretory pathway, being very low in the endoplasmic reticulum, higher in the Golgi apparatus, and highest in the plasma membrane [19, 20]. Indeed, this is confirmed when the Chol contents in these biological membranes are compared. The first group comprises the endoplasmic reticulum, Golgi apparatus, and mitochondria membranes, all of which have a low Chol content, with a Chol-to-phospholipid (PL) molar ratio between 0 and 0.1 (<10 mol% Chol). Data for these membranes are scattered, but they indicate that, for the endoplasmic reticulum and Golgi apparatus, the Chol/PL molar ratios are as low as 0.06 and 0.1, respectively [21] (see, however, ref. [20] for Chol content in Golgi apparatus). The second group comprises biological membranes with a normal Chol content (this is our terminology), which are typical plasma membranes with a Chol/PL molar ratio between 0.1 and 0.5 (from 10 to 30 mol% Chol) [22]. Even though most of the cellular plasma membranes show such Chol content, the content in certain cells is significantly higher. The third group, which comprises membranes with a high Chol content, includes the plasma membrane of red blood cells and the myelin membranes of Schwann cells isolating nerve axons; the Chol/PL molar ratio in these membranes is close to 1 (~ 50 mol% Chol) [23, 24]. At a Chol/PL molar ratio of 1, Chol saturates the PL bilayer [25]. In membranes with a greater Chol content, Chol starts to form immiscible pure Chol domains, called Chol bilayer domains (CBDs), within the PL bilayer saturated with Chol [26]. We refer to membranes in which CBDs are present as having very high Chol content. This fourth group comprises fiber cell plasma membranes of the eye lens, especially those of human eyes, for which the Chol/PL molar ratio is greater than 1 (>50 mol% Chol) [27–29]. When the Chol content exceeds the Chol solubility threshold (CST), usually at a Chol/PL molar ratio of 2 (66 mol% Chol), Chol crystals are formed, presumably outside the lipid bilayer [25, 30]. This situation can be observed in the clear human eye lenses of old individuals as well as in the membranes of atherosclerotic arteries [30–32]. We call this situation Chol content beyond the CST.
PLs are the most basic molecular components of a biological membrane. They form the lipid bilayer in which integral membrane proteins are immersed. At physiological temperatures, all membrane components undergo thermal diffusion. If the membrane were to follow the fluid-mosaic model [33], the diffusional motion would be laterally unrestricted, and all membrane components would be uniformly mixed; in effect, the membrane would constitute a homogeneous and featureless bilayer structure. But the membrane is not such [34], which strongly suggests that the fluid-mosaic model is not entirely correct, even though it can describe some membrane functions [35–37]. In the membrane, lipid-lipid, lipid-protein, and protein-protein interactions lead to the formation of domains that play an essential role in the functioning of biological membranes. The relationship between the different types of domains and membrane functions is one of the key issues of membrane biology today.
As previously mentioned, Chol plays a key role in the lateral organization of the lipid bilayer and the determination of basic membrane properties. The Chol molecule contains three well-distinguished regions: a small polar hydroxyl group, a rigid plate-like steroid ring, and a flexible isooctyl chain. Chol intercalates into the membrane with a polar hydroxyl group positioned between the PL polar headgroups, and its rigid, plate-like ring structure reaches the depth of the C9–C10 carbon atoms of the PL acyl chains. The Chol isooctyl chain, the cross section of which is much smaller than that of the rigid ring, is located in the membrane center. Chol affects membrane properties differently at different depths [38–40].
In this review, the effect of Chol on the PL bilayer is illustrated with bilayers made of dimyristoylphosphatidylcholine (DMPC) and Chol. We will also demonstrate that the electron paramagnetic resonance (EPR) spin-labeling approaches can provide unique information about the lateral organization of the lipid bilayer membranes, allowing discrimination of membrane phases and domains in situ, without their physical separation. EPR spin-labeling methods also provide a number of unique approaches for determining several important membrane properties as a function of the bilayer depth. Because this issue of Cell Biochemistry and Biophysics is dedicated to the applications of EPR in biomedical research, we will also provide a short explanation of how this type of information about membrane properties and organization can be obtained from EPR signals.
Phases and Domains
The phase diagram for the DMPC/Chol mixture together with the schematic drawings shown in Figure 1 are used to illustrate how membrane organization changes with Chol content, in order to help guide readers through this review. The DMPC/Chol bilayer forms one of the simplest paradigms for the study of formation, coexistence, and separation of phases and domains (see [41] for an extended terminological discussion regarding “phases” and “domains”). The phase diagram in Fig. 1 indicates single-phase regions of liquid-disordered (l d) and solid-ordered (s o) phases (separated by the main phase transition at 23.6 °C) between 0 and ~ 10 mol% Chol and that of the liquid-ordered (l o) phase between ~ 30 and 50 mol% Chol. In the region of Chol content between ~ 10 and ~ 30 mol%, two phases coexist, l d and l o (above the phase transition temperature) and s o and l o (below the phase transition temperature). At a Chol content above 50 mol%, the l o phase becomes a structured l o phase (see [42] for terminology) because the excess Chol forms CBDs within the l o phase [25]. In the terminology of Simons and Vaz [43], such a region can be called a dispersed phase. Chol crystals do form at 66 mol% Chol (CST for DMPC [44]) and, even if the Chol exchange between the crystal and the bilayer pool is too slow to be observed, Chol crystals represent an equilibrium state of Chol [41]. The two-phase region above the 66 mol% Chol contains both Chol crystals and a structured l o phase.
The phase diagrams per se do not tell us much about the structure and properties of the single or coexisting phases. However, we can get significant information about Chol distribution between these phases at the particular Chol mixing ratio at which membranes were prepared (at a certain Chol content). In any phase, the Chol concentration has an upper limit that can be accommodated within the phase. For a DMPC/Chol mixture at 27 °C, the limit for the l d phase is 6 mol% Chol, above which the excess Chol forms an l o phase (with a Chol concentration of 27 mol%). The upper limit of the Chol concentration in the l o phase of the DMPC membrane is 50 mol%, above which the excess Chol forms CBDs. The next limit is defined by the CST; above this limit, Chol crystals are formed. The phase diagrams also do not indicate if each of the two coexisting phases is dispersed into one larger domain or into a number of smaller domains.
EPR Approach
In EPR studies of lipid bilayers, both PL-analog (1-Palmitoyl-2-(n-doxylstearoyl) phosphatidylcholine (n-PC) and n-doxylstearic acid spin labels (n-SASL)) and Chol-analog (androstane spin label (ASL) and cholestane spin label (CSL)) spin labels were used. The structures of the most commonly used spin labels are presented in Fig. 2. These spin labels, when introduced to the investigated membranes, give rise to the EPR signal at specific membrane depths and in specific membrane domains. Applying both conventional and time-domain techniques, the dynamic state of the lipid molecules as well as the physicochemical properties of the microenvironment in the immediate vicinity of the nitroxide can be characterized. The spin labels, due to their respective overall similarity to PLs and Chol, are believed to be distributed between different membrane phases and domains, similar to PLs and Chol (see Fig. 1 for membrane domains and phases). The locations of spin labels in phases and domains consisting of molecules of corresponding structures allow the phases and domains to be discriminated and, in some cases, certain transmembrane profiles for coexisting membrane domains or coexisting membrane phases to be obtained without the need for their physical separation.
Below, we provide a detailed description of how the transmembrane profiles of some EPR spectral parameters describing certain membrane properties, such as acyl chain order, fluidity, hydrophobicity, and oxygen diffusion-concentration product (oxygen transport parameter [OTP]), can be obtained. Conventional EPR spectroscopy provides the order parameter and hydrophobicity profiles, whereas the time-domain saturation-recovery (SR) EPR method, which is highly sensitive to the rotational motion of the nitroxide moiety and to collisions between nitroxide and molecular oxygen, provides profiles of the membrane fluidity and OTP. Below, the profiles are grouped into those illustrating the order and the rotational motion of the PL acyl chains and those illustrating hydrophobicity and the diffusion-concentration products of oxygen. We will show how these profiles change as a function of the Chol content in the membrane.
Order of PL Acyl Chains
In lipid bilayers, an acyl chain of a PL-analog spin label (n-PC or n-SASL, Fig. 2) undergoes rapid anisotropic rotational motion due to its elongated shape and the axial symmetry of the lipid bilayer. It rotates about its long axis, and its long axis wobbles within the confines of a cone imposed by the membrane environment. However, because the acyl chain is flexible, each of its segments has its own rotational freedom, which gives rise to the unique features of the EPR spectrum [45]. From this spectrum, the order parameter for the spin labeled acyl chain segment can be calculated (Fig. 3a).
The order parameter is a measure of the amplitude of the wobbling motion of the given chain segment averaged over time and ensemble; thus, it can be considered a nondynamic parameter. However, profiles of the order parameter (Fig. 3b) have been routinely used as a measure of the membrane fluidity [46, 47]. As shown in Fig. 3b, when the spin label moiety is moved down along the acyl chain, the effect of the wobbling (a decrease of the chain order parameter) accumulates. The effect of accumulation is clearly demonstrated by the shape of the order parameter profiles for bilayers containing Chol (Fig. 3b). The Chol ring induces ordering not only of the acyl chain segments in direct contact with the ring (close to the membrane surface) but also of the distal segments (close to the membrane center). Thus, as shown in Fig. 3b, the ordering of acyl chains by Chol proceeds from the membrane surface to the membrane center. This effect has also been observed in computer simulation studies of the DMPC/Chol bilayers, e.g., ref. [48].
As indicated by the phase diagram and schematic drawings presented in Fig. 1, at increased Chol content, the coexisting phases and domains are present in the DMPC bilayer. However, using the conventional EPR spectra of spin labels, one cannot discriminate these phases and domains. Order parameters derived from these spectra are averaged over spin labels located in the coexisting phases and domains. However, as PL-analog spin labels are located only in PL/Chol phases, the profiles obtained with these spin labels are not contaminated by the CBD. As follows from the phase diagram in Fig. 1, this is true for Chol content in the DMPC bilayer greater than 50 mol%.
The values of the order parameter of acyl chains close to the membrane surface (C5 position, data obtained with 5-PC) and in the membrane center (C14 position, data obtained with 14-PC) are presented in Fig. 3c as a function of Chol content in the DMPC bilayer. As shown in Fig. 3c, at both locations, the order parameter continuously increases with Chol content, up to the Chol/DMPC molar ratio of 1, and then becomes constant upon further increases in Chol. This makes sense because, above the Chol/DMPC molar ratio of 1 (50 mol% Chol), CBDs start to form within the DMPC/Chol bilayer, which remains saturated with Chol even though Chol content increases above 50 mol%. This agrees with the phase diagram presented in Fig. 1. Further increases in Chol beyond the CST (66 mol% Chol) cause formation of Chol crystals. Their formation does not affect the values of the order parameter for the same reasons as discussed with regard to CBD formation. These results may lead to the conclusions that (1) CBDs can be supported by the surrounding DMPC bilayer saturated with Chol when Chol content is between 50 and 66 mol% (Chol/PC molar ratio between 1 and 2), and (2) as shown in ref. [25], formation of CBDs precedes formation of Chol crystals.
Fluidity of The Bilayer
In our previous papers, we proposed use of the spin-lattice relaxation rate (T 1 −1, the inverse of the spin-lattice relaxation time T 1) as a convenient quantitative measure of local membrane dynamics commonly termed fluidity [49, 50]. In a deoxygenated spin labeled lipid bilayer, T 1 −1 depends primarily on the rate of rotational motion of the nitroxide moiety [51, 52] and thus describes the dynamics of the acyl chain fragment to which the nitroxide moiety is rigidly attached. As shown in Fig. 4a, T 1 can be obtained from SR EPR measurements, allowing studies of the effects of membrane modifiers, like Chol, on membrane fluidity. T 1 −1 measured for n-SASL or n-PC spin labels provides a fluidity profile across the lipid bilayer. The profiles across DMPC bilayers without and with 50 mol% Chol are shown in Fig. 4b. As expected, for both systems, moving the spin label position progressively deeper into the bilayer increases the value of T 1 −1, which corresponds to an increase in membrane fluidity (membrane dynamics). However, comparison of the profiles for DMPC and DMPC with 50 mol% Chol bilayers indicates that in the presence of Chol the membrane fluidity decreases close to the bilayer surface and increases near the bilayer center. The transition between the rigidifying and fluidizing effects of Chol lies approximately at the position of the C9 carbon atom, the depth to which the rigid Chol ring is immersed into the bilayer. These results are in accordance with atomistic computer simulation studies that show the exact positions of the Chol ring atoms and the effect of the Chol ring on packing of the atoms in the DMPC/Chol bilayer, refs [18, 53, 54]. Here, we would like to stress that profiles of the order parameter do not differentiate the effects of Chol at different depths (cf. Fig. 3b) as they show that the Chol ordering effect extends to all bilayer depths.
Effects of Chol on membrane fluidity close to the membrane surface (at the C5 position) and in the membrane center (at the C14 position) are summarized in Fig. 4c. Close to the surface, Chol decreases membrane fluidity up to the Chol/DMPC molar ratio of 1. Similarly, in the membrane center, Chol increases membrane fluidity up to the Chol/DMPC molar ratio of 1. A further increase in Chol content does not affect membrane fluidity at any bilayer depth. As indicated above, n-PC and n-SASL spin labels are located only in the DMPC/Chol bilayer phases and, thus, report only on their fluidity, which is not affected by the presence of CBDs or by Chol crystals, formed presumably outside the membrane. T 1s obtained from SR EPR signals of T-PC (Fig. 2) with the nitroxide moiety located in the bilayer polar headgroup region of the DMPC and DMPC with 50 mol% Chol bilayers indicate that Chol increases membrane fluidity in this region [49, 50]. Because the structure of the nitroxide moiety of T-PC is different from that of n-PC and n-SASL, we cannot include the data obtained with T-PC in Fig. 4; however, the effect of Chol on the fluidity of the bilayer headgroup region is unequivocal. Increased fluidity in the headgroup region of the DMPC/Chol bilayer relative to that of the DMPC bilayer can be explained by increased motional freedom of the DMPC phosphorylcholine group resulting from increased spacing among DMPC molecules induced by the presence of Chol molecules in the bilayer, as was shown in a computer simulation study [55].
Results obtained for the order parameters (Fig. 3b and c) and for the membrane fluidity, T 1 −1, (Fig. 4b and c) might at first seem at variance. However, as noted above, the rigid sterol ring of Chol extends only to a depth of approximately the position of the C9 carbon atom in the acyl chains. Below the sterol ring, the Chol isooctyl chain has a cross section much smaller than that of the sterol ring; in effect, the central region of the bilayer is less dense than in the bilayer without Chol. This smaller density in the center of the DMPC/Chol bilayer can be seen from mass density profiles across the DMPC and DMPC/Chol bilayers in ref. [18], as well as from the molecular order parameter profile in ref. [56]. The order parameter is a cumulative quantity, even though it is measured locally. In the DMPC bilayer, the rigid ring of Chol acts as a “wall,” confining the wobbling of the chain fragments within its vicinity [48]; however, the “wall effect” is felt by all fragments of the chains due to the cumulative nature of the order parameter. In contrast are T 1 −1 reports on the local motional state of a chain fragment that is practically independent of the motional states of other fragments. In the vicinity of the steroid ring, the packing of DMPC chains is tighter [54], so both the rate and the amplitude of the wobbling is diminished. However, in the less-dense environment of the DMPC/Chol bilayer center, the terminal fragments of the DMPC acyl chains rotate faster because they have a greater motional freedom. Thus, the increased mobility induced by Chol in the membrane center, indicated by the dynamic parameters describing the rate of motion, is consistent with an increased order parameter, describing the amplitude of motion.
Hydrophobicity of The Membrane Interior
The hydrophobicity of the membrane interior is largely determined by the extent of water penetration into the membrane. The membrane environment becomes increasingly nonpolar proceeding from the membrane surface to the termini of the lipid acyl chains. The pioneering work of Griffith et al. [57, 58] demonstrated, using EPR spin-labeling approach, the existence of a hydrophobicity gradient across the membrane. Since then, this method has been used intensively to study the hydrophobicity of the membrane interior [59, 60]. The EPR spin-labeling method is based on the solvent-polarity dependence of the unpaired electron’s spin density at the nitrogen atom. Polar solvents enhance the density, thereby increasing the hyperfine interaction between the unpaired electron spin and the nitrogen nuclear spin. With an increase in solvent polarity, the z component of the hyperfine interaction, A Z, increases. In membranes, this parameter, measured as a maximum splitting (2A Z), may also be influenced by spin-label motion that averages the anisotropy of the A and g tensors. Because of that, and to eliminate the motional contribution, the hydrophobicity measurements are typically performed on samples in the frozen state, using 2A Z (Fig. 5a) as a convenient experimental observable.
The hydrophobicity profiles for the DMPC and DMPC with 50 mol% Chol bilayers are shown in Fig. 5b. In the absence of Chol, bilayer hydrophobicity gradually increases toward the bilayer center and has a typical bell-shaped profile. Chol has dramatic effects on the hydrophobicity profile as it induces a decrease in hydrophobicity in the region close to the membrane surface, and a significant increase in hydrophobicity in the central region of the bilayer [59]. The hydrophobicity sharply increases between the C9 and C10 carbon atoms and then becomes flat. This very sharp increase occurs within a narrow range of one carbon-carbon bond (i.e., 1.34–1.53 Å). At positions below (i.e., deeper than) the C10 atom, 2A Z values are almost constant, indicating a region of homogeneously high hydrophobicity. These give the hydrophobicity profile an overall rectangular shape.
Effects of Chol on membrane hydrophobicity close to the membrane surface (at the C5 position) and in the membrane center (at the C14 position) are summarized in Fig. 5c. Close to the surface, Chol decreases membrane hydrophobicity up to the Chol/DMPC molar ratio of 1. However, in the membrane center, Chol increases membrane hydrophobicity up to the Chol/DMPC molar ratio of 0.5 (~ 30 mol%). The further increase of Chol content does not affect membrane hydrophobicity, or slightly (within the error of measurement) decreases it. Measured values, both close to the membrane surface and in the membrane center, stabilize at the Chol/DMPC molar ratio of 1.
To give a physical meaning to the hydrophobicity measured as 2A Z and indicated in Fig. 5a, its values for selected depths in the membrane were related to the polarity (or dielectric constant, ε) of bulk organic solvents in which 2A Z of the dissolved spin label were similar, as shown in Fig. 2 in ref. [59]. Thus, for the DMPC bilayer, the addition of 50 mol% Chol increased polarity (decreased hydrophobicity) near the membrane surface (at the C5 positions) from that of 2-propanol (ε = 15) to that of methanol (ε = 50); the latter is high but indicates that this region is still considerably less polar than the bulk aqueous phase (ε = 80). Conversely, incorporation of 50 mol% Chol into the DMPC bilayer decreases polarity (increases hydrophobicity) in the middle of the bilayer from that of 2-pentanol or 1-octanol (ε = 10–20) to the level of hexane (ε = 2). Here, we would like to stress that, in the PL bilayer, the hydrophobicity of the membrane interior depends on PL species, especially on PL unsaturation [59]. Saturated bilayers do not form high hydrophobic barriers; the hydrophobicity in their center is close to that of 2-pentanol or 1-octanol (ε = 10–20) while that in the center of unsaturated bilayers is close to that of 1-decanol (ε = 10, 1-palmitoyl-2-oleoylphosphatidylcholine, POPC). All these differences are canceled at saturating Chol contents because, for all bilayers, the hydrophobicity in the bilayer center is close to that of hexane (ε = 2).
OTP (Discrimination of Membrane Phases and Domains)
Bimolecular collisions of a nitroxide moiety of the spin label with molecular oxygen induce spin exchange, enhancing spin-lattice relaxation of the nitroxide. This effect can be measured directly using SR EPR (Fig. 6a). An OTP was introduced as a convenient quantitative measure of the collision rate between the spin label and molecular oxygen by Kusumi et al. [61] and is determined by calculating the difference between the spin-lattice relaxation rates of spin labels in samples equilibrated with air and nitrogen. The parameter is proportional to the product of the local translational diffusion coefficient and the local concentration of oxygen at the bilayer depth where the nitroxide moiety is located. By convention, the OTP is normalized to an oxygen concentration corresponding to the sample equilibrated with atmospheric air. To obtain the OTP, in principle, two SR measurements have to be performed: one for the sample equilibrated with nitrogen and the other for the sample equilibrated with air (see Fig. 6a).
We will demonstrate the ability of this approach using OTP profiles obtained with PL-analog spin labels in the DMPC and DMPC with 50 mol% Chol bilayers as an example (Fig. 6b). The effect of Chol on the OTP is very different at different membrane depths (Fig. 6b). Chol changes the bell-shaped profile, observed for PL bilayers, to a rectangular-shaped profile. OTP abruptly increases between the positions of the C9 and C10 carbon atoms, that is, at the depth to which the rigid tetracyclic Chol structure is immersed. This abrupt increase is as large as ~threefold, and the overall change of the OTP across the DMPC/Chol bilayer becomes as large as ~fivefold. In the presence of Chol, the OTP in the region between the membrane surface and the position of the C9 carbon atom becomes as low as in the gel-phase PC bilayers; at locations deeper than the C9 carbon atom, it becomes as high as in the fluid-phase bilayers [40, 62]. The OTP profiles, similar to the hydrophobicity profiles, are significantly different for different PL membranes. However, saturation of PL membranes with Chol makes these profiles very similar in shape (rectangular) and values of the OTP.
The OTP makes it possible to discriminate different membrane domains because even small differences in the lipid packing of these domains can affect the partitioning and diffusion of molecular oxygen. Information about different packings of lipids is recorded in the SR signal. For membranes equilibrated with air and consisting of two lipid environments with different oxygen diffusion-concentration products, the SR signal is a simple double-exponential curve with two time constants. This is the basis of the SR EPR discrimination by oxygen transport (DOT) method [63, 64], which is used to detect and characterize coexisting phases and domains in the membrane [26, 39, 65–67]. Because membranes, and thus membrane domains, do not have uniform properties across themselves (and may be viewed as three-dimensional structures), knowledge of the molecular events in the depth dimension is important. The DOT method not only shows the lateral organization of lipid membranes or differentiate between membrane domains, but also provides information about molecular dynamics and structure in the third dimension, namely, in the direction perpendicular to the bilayer surface. We demonstrated that the DOT method, with the use of PL-analog spin labels, can distinguish and characterize l o, l d, and s o phases in different regions of a phase diagram when they form a single phase or when two phases coexist [39, 65]. When Chol-analog spin labels are used, the DOT method can discriminate the l o phase and the CBD [26, 68]. This case is described in this issue by Justyna Widomska et al.
Values of the OTP as a function of the overall Chol content for the DMPC/Chol bilayer at 27 °C (above the main phase transition temperature for the DMPC bilayer), measured close to the membrane surface with 5-PC and close to the membrane center with 14-PC, are shown in Fig. 6c. According to the phase diagram (Fig. 1), at an overall Chol content between 0 and ~ 10 mol%, the bilayer phase remains l d (the Chol mole fraction in the l d phase is the same as the experimental mixing ratio). Surprisingly, in the presence of Chol, the l d phase exhibits higher OTP values across the entire membrane than without Chol. Variations of the overall Chol content between ~ 10 and ~ 30 mol% will affect the fractions of l d and l o phases. (It follows from Fig. 1 that, at 27 °C, each phase contains constant 6 and 27 mol% Chol, respectively.) In the l o phase, the OTP’s slight dependence on the overall Chol content in the range of ~ 10 to ~ 30 mol% for both 5-PC and 14-PC suggests that, in this Chol range, not only do changes of the relative amounts of l d and l o phases take place but also the size of the individual l o-phase domain increases, reducing the effects of the surrounding l d phases. With an overall Chol content between ~ 30 and 50%, the phase remains l o. (The Chol mole fraction in the DMPC bilayer is the same as the experimental mixing ratio.) The major result for this Chol range is that the OTP profile in the l o phase containing 50 mol% Chol differs dramatically from that in the phase containing 30 mol% Chol [39]. At 50 mol% Chol, the OTP in the bilayer center in the l o phase is similar to that in the l d phase without Chol, but the OTP near the membrane surface (up to the C9 carbon atom) is substantially smaller in the l o phase (i.e., the Chol-based l o phase is only ordered near the membrane surface, but it retains a high-level disorder in the bilayer center). This property may facilitate lateral mobility of small, lipid-soluble molecules even in the bilayer-ordered domains. As shown in the phase diagram (Fig. 1), at a Chol content greater than 50 mol%, CBDs are formed in the DMPC bilayer. In Fig. 6c, this is indicated by the values of the OTP obtained with ASL for a Chol content greater than 50 mol%. The DOT method with ASL not only discriminates CBDs formed within the l o phase but also characterizes these two environments by the values of the OTP. At a Chol content greater than 66 mol% Chol, Chol crystals are formed. The OTP, measured using PL-analog spin labels located in the PL/Chol portion of the membrane, is not affected by the presence of CBDs formed within the Chol-saturated PL bilayer, nor by the Chol crystals formed presumably outside the membrane (Fig. 6c).
Discussion
Low Cholesterol Content
As follows from Figs 4b and c, 6b and c, bilayers in the liquid-disordered phase (without Chol or with a Chol content below 10 mol%) are highly fluid at all depths. Interestingly, their fluidity, sensed by the mobility of molecular oxygen (Fig. 6c), is even enhanced by the presence of Chol. These bilayers also exhibit a moderate hydrophobic barrier (Fig. 5b and c). During evolution, PL membranes with a low Chol content were chosen to separate subcellular compartments as a nuclear envelope, membranes of rough and smooth endoplasmic reticulum, and membranes of Golgi apparatus.
The nuclear envelope, which is made of two lipid bilayers, separates the nucleus from the cytoplasm. It contains multiple pores that regulate the passage of macromolecules, such as proteins and RNA, between these two compartments but permit polar small molecules, such as water, ions, and ATP, to pass freely. Lipid bilayer membranes of rough endoplasmic reticulum provide the basic surface to which (on its outside surface) ribosomes are attached to synthetize specific proteins destined for secretion. These membranes are also involved in the intracellular transport of the products of the rough endoplasmic reticulum to the Golgi apparatus, where, after modification, proteins are packed into secretory vesicles and then transported to their destinations: lysosomes, the plasma membrane, or secretion. Smooth endoplasmic reticulum is devoted almost exclusively to the manufacture of lipids and steroid hormones. The l d lipid bilayer membranes fulfill properties that are needed for these organelles to function properly. They form a physical, moderate hydrophobic barrier to keep genetic nucleus material separate from cytoplasm and to wrap macromolecules to form cargoes that can be safely transported through the cytoplasm. In the rough endoplasmic reticulum, they concentrate ribosomes (apparatus for protein synthesis) adjacent to the nucleus. It seems that a simple PL bilayer can provide the base for ribosome attachments.
Mitochondria membranes also belong to the low Chol content group. They are, however, involved in a very complex process of energy transduction. An electron transport chain located in the inner mitochondrial membrane, through a series of three proton pumps, creates the electrochemical proton gradient across the inner mitochondrial membrane [69]. The protons can return to the matrix through the ATP synthase complex, and their potential energy is used to synthesize ATP from ADP and inorganic phosphate. To make this process efficient, the proton leak across the lipid matrix of the inner mitochondrial membrane has to be minimized. Usually, membranes utilize Chol to create a high hydrophobic barrier, but Chol also strongly affects (decreases) membrane fluidity, which in this case is an undesirable effect. This problem was solved during evolution by loading the mitochondrial membranes with a highly unsaturated PL, cardiolipin [70–72]. As previously mentioned, polyunsaturated acyl chains of PLs create a high hydrophobic barrier across the bilayer, and, at the same time, prevent its high fluidity. In particular, elevated fluidity in mitochondrial membranes ensures the high mobility of CoQ10 within the bilayer, during its function as an electron carrier from enzyme complex I and enzyme complex II to enzyme complex III [8].
Normal Cholesterol Content
We classified membranes containing between 10 and 30 mol% Chol as those with a normal Chol content, as this range is typical for most plasma membranes. PL bilayer membranes with this Chol content possess unique lateral organization, namely coexisting l d and l o phases. Such organization is observed for membranes made both of a single PL species and of a mixture of PLs. The Chol-dependent l o phase is attractive to researchers as it is connected to membrane microdomains, particularly so-called raft domains, or rafts. The interest in rafts spans a broad range of biomedical research areas related to cell-cell signaling and trafficking of lipids and proteins in cells [73–80]. Simons and Vaz [43] stated that, “rafts could be considered domains of a l o phase.” However, even though l o and l d phases are observed and measured in simple systems of model membranes, their concept cannot be directly applied to the living cell membranes. Nevertheless, it is likely that knowledge of how they form and their physico-chemical properties will contribute to our understanding of the formation mechanism, structure, lifetime, and dynamics of raft domains in biological membranes [67, 75, 78–83]. Results providing some insight into the structure and molecular interactions in l o and l d phases in PL bilayers are presented in Figs 3c, 4c, 5c, and 6c. (Look for Chol content between 10 and 30 mol% where properties of coexisting l o and l d phases are demonstrated.) These figures and our explanations show lateral segregation of the phases and also their depth-dependent properties; these indicate that the structure of the bilayers is nonuniform in three dimensions, which we refer to as the three-dimensional structure of the lipid bilayer [39, 75].
High Cholesterol Content
As follows from the phase diagram (Fig. 1), PL bilayers containing 30 to 50 mol% Chol form a single l o phase. This Chol content is characteristic for plasma membranes of red blood cells and for myelin membranes of Schwann cells isolating nerve axons. This high Chol content ensures the highest hydrophobicity of the membrane as well as high membrane rigidity understood as reduced trans-gauche isomerization (extended conformation) and translational and rotational motion, e.g., ref. [84]. It should be noted that the properties of l o-phase bilayers change significantly between 30 and 50 mol% Chol, as was shown using Chol-analog spin labels [85] and PL-analog spin labels (discussed previously). The latter additionally shows that the three-dimensional dynamic structure of the bilayers differs significantly: At ~ 30 mol% Chol (the lowest Chol content of the l o phase), the bell-shaped profile of the OTP is similar in shape (although shifted to lower values at all depths) to that for the l d phase without Chol. At 50 mol% Chol (the highest Chol content of the single l o phase), the profile becomes rectangular (Fig. 6b).
Incorporating saturating amounts of Chol into PL bilayers also ensures the rectangular shape of the hydrophobicity profile with the hydrophobicity in the bilayer center comparable to that of hexane (ε = 2) (Fig. 5b). This greatly increases the activation energy required for polar and small ionic molecules to pass through the membrane. Thus, the rate-limiting step for the permeability of small polar molecules is likely to be the process of crossing the hydrophobic barrier at the membrane center. Saturating amounts of Chol in the PL bilayer also creates resistance to the permeation of small hydrophobic molecules (like oxygen) across the membrane. As shown in Fig. 6b, a rather high permeability barrier to oxygen transport is located in the polar headgroup region, and in the hydrophobic core of the bilayer to the depth of the C9 carbon atom. Resistance to oxygen permeation in this region (we call it the “rigidity barrier”) is much higher than resistance in the water phase. Thus, the rate-limiting step for permeation of small, nonpolar molecules across the membrane (including molecular oxygen) is likely to be the process of crossing the rigidity barrier located near the membrane surface.
Red blood cells (~ 8 µm in diameter) must withstand strong shear forces in the arteries, and must be able to fold up when they pass through narrow capillaries (<3 µm in diameter). To avoid any leakage of intracellular molecules, including hemoglobin, during these processes, the red blood cell plasma membrane must contain high (saturating) Chol concentrations. Additionally, because the red blood cell plasma membrane supported by the cytoskeleton is the only supramolecular structure of the cell (all intracellular organelles are absent), mainly integral membrane proteins are involved in regulatory processes maintaining appropriate conditions of the cytoplasm. For this reason, the membrane barrier must be very high to block nonspecific permeation of small molecules and to protect the cytoplasm. Incorporating a saturating amount of Chol into the membrane serves this purpose well, because Chol simultaneously raises hydrophobic barriers for polar molecules and increases rigidity barriers for nonpolar molecules. The question Is the mammalian cell plasma membrane a barrier to oxygen transport? is carefully discussed in [40, 86].
Similarly, the saturating Chol content in myelin membranes of Schwann cells ensures the highest electrical isolation of nerve axons and enables an especially rapid mode of electrical impulse propagation called saltatory conduction. To enhance insulating properties, these membranes contain high amounts of sphingolipids. Sphingolipids form tight links with Chol, which cause the membrane to become more compact (cf. [87]). Thus, axons are not only electrically shielded but also tightly isolated from being accessed by polar and nonpolar small molecules. Lipid composition and physico-chemical properties of myelin membranes are crucial because the membranes contain an amount of proteins (15–30% by weight) too small to be able to correct nonspecific leakage through the lipid.
Very High Cholesterol Content
The highest amount of Chol that can dissolve in a PL bilayer is 50 mol% (i.e., when the Chol/PL molar ratio is 1 [see schematic drawing in Fig. 1]). At a higher Chol/PL molar ratio, Chol is distributed between the Chol-saturated fraction of the PL/Chol bilayer and the CBDs immersed in it. Among biological membranes, such a high Chol content is observed only in the fiber cell plasma membranes of the eye lens, and this is their most unique biochemical characteristic. In the membrane of the human eye fiber cell, the Chol/PL molar ratio ranges from 1–2 in the lens cortex and can be as high as 3 to 4 in the lens nucleus (see sect. Beyond the cholesterol solubility threshold).
Based on the results of our EPR spin-labeling experiments with lens-lipid membranes as well as those found in the literature, we have been able to identify the functions of Chol specific to the fiber cell plasma membrane [10]. The CBD plays a crucial role in these membranes (again, we direct readers to this issue’s article by Justyna Widomska et al. for more-detailed information) as it ensures that the surrounding PL bilayer is saturated with Chol. (See schematic drawing in Fig. 1.) CBDs start to form at 50 mol% Chol; an increase of the Chol content above 50 mol% does not change the properties of the surrounding PL bilayer (Figs 3c, 4c, 5c and 6c). This feature is independent of the bilayer PL composition as is clearly illustrated in Fig. 7, which shows profiles of the four measured properties across bilayers consisting of lipids obtained from human lens membranes from different donors. For all investigated age groups, appropriate profiles are identical.
One of the consequences of the unique growth pattern of the lens is that there is no protein turnover in the matured fiber cells [88, 89]. The lens proteins should perform the same functions in old lenses that they performed just after synthesis. The lack of protein turnover is seemingly in conflict with the drastic changes in the fiber cell membrane PL composition that occurs during aging [9, 27, 90–92]. Such changes would be expected to alter the functioning of membrane integral proteins. However, this is not the case; presented data indicate that the saturating Chol content in these membranes (and the presence of CBDs) helps to maintain fiber cell membrane homeostasis during age-related changes of the PL composition without altering functions of integral proteins [11–14] and without compromising too much lens transparency. This constant saturating Chol content in the membranes of the fiber cells fulfills another important function as it makes the surfaces of the membrane smoother than otherwise. The smoother surface of each cell membrane reduces light scattering by the human lens and helps to maintain lens transparency [84].
Beyond The Cholesterol Solubility Threshold
When the Chol content in the membrane exceeds the CST, Chol crystals form; though it is not clear where this formation takes place, it most likely happens outside the membrane [30, 32]. As follows from sect. Very high cholesterol content, a high Chol content and CBDs serve beneficial functions for eye lenses. Can we also call formation of Chol crystals beneficial? Probably not. Our results indicate, however, that formation of Chol crystals is not harmful for aged human lenses [93]. Formation of Chol crystals in other tissues and organs is a sign of pathology. It was shown that deposition of the minute Chol crystals found in artery cells can initiate and promote atherosclerosis by activating inflammasomes [94] and, in fact, is an early cause of atherosclerosis. Some possible pathways lead to the formation of Chol crystals in the artery. One pathway that already has been investigated is the uptake of oxidized low density lipoproteins by macrophages, which promotes the intracellular accumulation of free Chol and the formation of Chol crystals, inflammation, and disease [30]. The alternative pathway involves plasma membranes oversaturated with Chol. This pathway should be very efficient in aged fiber cell membranes where Chol crystals already were detected [30, 31, 95]. However, inflammation does not appear to play a role in cataract formation. The unique structure of the lens protects it from the harmful induction of inflammasomes, as mature fiber cells do not contain intracellular organelles, and from initiation of the inflammatory cascade, as the lens is avascular. Thus, Chol crystals can be formed without harmful effects on lens properties (clarity) and functions. All these factors suggest that high Chol content, formation of CBDs, and formation of Chol crystals should not be considered major predispositions for the development of age-related cataracts [93].
Concluding Remarks
The role of Chol in the biological membrane is a long-standing puzzle. Kusumi et al. [96] first introduced the expression “fluidity buffer,” stating that “cholesterol acts as a ‘fluidity buffer’ so that small changes in the amount and arrangement of membrane proteins may not affect the overall properties of the membranes.” Later, he and the co-authors proposed that the membrane mixture of saturated and unsaturated acyl chains of PLs with Chol also provides a fluidity buffer [97, 98]. Present results suggest that Chol also has other crucial functions specific to certain biological membranes. At different Chol contents, the effects of Chol on the membrane properties (fluidity, permeability) and organization (phase and domain formation) are very different. We would like to conclude this review with a few remarks about findings that have emerged from our EPR spin-labeling studies of lipid and biological membranes supported in some places by our molecular modeling studies.
Ordering Effects of Cholesterol
The ordering effect of Chol in saturated PC bilayers is very strong. The acyl chain order parameter, when plotted as a function of the mole fraction of Chol at the same temperature above the main phase transition temperature of a PC bilayer with saturated chains of 14–22 carbon atoms, is nearly the same for all PC bilayers. This occurs over a wide range of temperatures and Chol mole fractions [97]. Chol also strongly reduces the rotational and wobbling motion of saturated acyl chains [48]. Unsaturated acyl chains greatly reduce these Chol effects, e.g., [99], thus allowing us to make the following final conclusion: The “fluidizing” effect of unsaturated chains observed in biological membranes seems to manifest itself by moderating the “rigidifying” effect of Chol.
Hydrophobic Barrier Formed by Acyl Chain Unsaturation and Cholesterol
Since cellular plasma membranes face the cell’s external environment, the membrane barriers must be very high to block nonspecific permeation of small molecules. Incorporation of Chol into the membrane serves this purpose well, because Chol simultaneously raises hydrophobic barriers for polar molecules and increases rigidity barriers for nonpolar molecules. The locations of permeation barriers are different for polar and nonpolar molecules. For the former, the major resistance to permeation is the hydrophobic barrier in the central part of the membrane. For the latter, the major resistance to permeation is the rigidity barrier near the membrane surface.
Pure saturated PC bilayers (without Chol) exhibit low hydrophobicity across the bilayer; at the bilayer center, where hydrophobicity is the highest, it is comparable to that of propanol and octanol (ε = 10–20) [59]. Introduction of a double bond to the acyl chain does increase bilayer hydrophobicity at all locations. This effect is especially great for the cis conformation of the double bond, for which hydrophobicity in the bilayer center increases to the level of dipropylamine (ε = 3, dioleoylphosphatidylcholine, DOPC) [59]. This suggests that the lipid-bilayer portion of the biological membrane can be a significant hydrophobic barrier only when it includes unsaturated acyl chains and/or Chol. Nature sometimes replaces Chol with polyunsaturated PLs to create membranes with a high hydrophobic barrier, especially when prevention of high membrane fluidity was necessary.
Hydrophobic Channeling
The striking similarity between the OTP (like in Fig. 6b) and hydrophobicity (like in Fig. 5b) profiles, especially for PL bilayers saturated with Chol, suggests a possibility for lateral transport of molecular oxygen and other small, nonpolar molecules in the central region of the membrane (parallel to the membrane surface), which we named “hydrophobic channeling.” This hypothesis was first formulated by Skulachev [100] for the transport of small hydrophobic solutes within the cell using extended membranous systems. The resistance to the movement of oxygen and other small, hydrophobic molecules in the membrane center is much lower than in the water phase, whereas their solubility is much higher. To escape from these channels, they have to cross high barriers with low permeability existing on both sides of the membrane (see ref. [101] for more explanation). This supports our hypothesis that a high Chol membrane content is responsible for creating hydrophobic channels for the movement of small, hydrophobic molecules, and, at the same time, it is responsible for creating the rigidity barrier to transport these molecules across the membrane.
Fluid-Phase Microimmiscibility of Unsaturated Phospholipids and Cholesterol
EPR spin-labeling measurements indicated that, in the DOPC/Chol bilayer, Chol is segregated from the bulk DOPC phase, forming Chol-enriched domains or clustered Chol domains, due to the structural conflict between the rigid ring skeleton of Chol and the rigid bend of the C9–C10 cis double bond in each DOPC acyl chain. However, such a domain is small and likely to consist of only several Chol molecules, and it is short lived, of the order of 1–100 ns [38]. The following two points are our speculations: (1) In a biological membrane, which, in general, contains both saturated and unsaturated PLs, Chol-enriched domains may be stabilized by the presence of the saturated acyl chains of sphingomyelin, and also by the clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein-rich, Chol-rich domain was found to be of the order of 100 µs [67], again showing the small/unstable nature of rafts in unstimulated cells. (2) At variance with the general view (e.g., ref. [5]), the formation of rafts may not be due to the affinity of Chol and saturated acyl chains, but rather to the segregation of Chol from the liquid-crystalline unsaturated bulk-phase membrane. Saturated PL chains may be associated with both the Chol-enriched domains and the disordered bulk domain, optimizing both structures such as to obtain the free energy minimum (possibly local and temporal minimum) of the whole membrane (see [75] for more discussion).
Properties of The Liquid-Ordered Phase
Additionally, data obtained with EPR spectroscopy of spin-labels attached to PL acyl chains at specific depths and located in specific membrane domains allowed correcting the commonly accepted statement that the properties of the l o-phase domains are between those for the l d and s o domains [102]. This statement is true for membranes containing bulk Chol mole fractions below 30%, where the l o domain coexists with the l d or s o domain (see Fig. 1). However, at a higher Chol content, even though the membrane is in the l o phase, the OTP in the region near the membrane surface (from the surface to the depth of the C9 carbon atom) is like that in the s o phase, and in the central region of the membrane (at depths greater than that of the C9 carbon atom), it is like that in the l d phase.
Understanding Cholesterol’s Functions in Model Membranes Helps To Understand Its Functions in Biological Membranes
In our opinion, the amount of data collected for simple lipid bilayers and those made from total lipid extracts of biological membranes is sufficient to determine some significant functions of Chol specific to the biological membranes discussed above. We are aware of the limitations of the conclusions and hypotheses presented in this review, as the membranes studied by us do not contain proteins. Intact membranes are sometimes loaded with membrane proteins, which raises questions and concerns regarding extent to which the presented conclusions can apply. Nevertheless, the study of the functions of Chol in simple models is undoubtedly helpful to better understanding its functions in intact membranes. Also, it is indispensable in revealing the mechanisms by which intrinsic proteins affect the properties of the lipid-bilayer portion of a biological membrane. Additionally, in this review, we directed the readers’ attention to profiles of physical properties across membranes saturated and oversaturated with Chol, which have filled an existing gap in membrane research.
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Acknowledgements
This work was supported by grants EY015526, EB001980, and EY001931 from the National Institutes of Health, USA. Faculty of Biochemistry, Biophysics, and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education, Poland.
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Subczynski, W.K., Pasenkiewicz-Gierula, M., Widomska, J. et al. High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review. Cell Biochem Biophys 75, 369–385 (2017). https://doi.org/10.1007/s12013-017-0792-7
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DOI: https://doi.org/10.1007/s12013-017-0792-7