Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Main Text

The plasma membrane is structurally asymmetric; the outer monolayer of plasma membrane is enriched in phospholipids, such as phosphatidylcholine and sphingomyelin, whereas the inner monolayer is enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) (see Fig. 1). The lateral asymmetry of the plasma membrane is retained for long periods of time because of the following: 1) a high free energy barrier against transverse diffusion of constituents (flip-flops), 2) membranes are created and elongated from pre-existing asymmetric membranes, and 3) the active maintenance by protein groups such as flippases and floppases. The appearance of negatively charged PS lipids on the outer monolayer of the cell membrane is an indication of a loss of membrane integrity. Extracellular expression of PS lipids targets the cell for engulfment by macrophages and is widely used as a diagnostic marker for apoptosis. Surprisingly, malignant (cancer) cells expose ∼7 times more PS lipids on the outer (extracellular) monolayer than healthy cells (1, 2). Exploiting this structural difference therefore allows for a promising and selective targeting of tumor cells by recognition of extracellular PS lipids or the concomitant redistribution of PE lipids. Furthermore, a recent molecular simulation suggested that the loss of lipid asymmetry decreases the ability of widely used chemotherapies such as cisplatin to travel through the membrane (2). Thus, the loss of membrane asymmetry may partly explain how cancer cells gain resistance to chemotherapies.

Figure 1.

Snapshot of a coarse-grained molecular simulation of the plasma membrane (9). The outer monolayer of the plasma membrane is enriched in phospholipids, such as phosphatidylcholine (PC) and sphingomyelin (SM), whereas the inner monolayer is enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS). Cholesterol is a molecule that can freely “shuttle” between monolayers. To see this figure in color, go online.

In contrast to phospholipids, sterols can “freely” redistribute themselves between the monolayers of the plasma membrane (i.e., their transverse diffusion between monolayers is relatively fast and occurs with a frequency of microseconds) (3, 4). Sterols regulate the plasma membrane’s permeability and fluidity and are an important precursor for sex hormones (steroids). The most abundant sterol in the mammalian plasma membrane is cholesterol. Commonly reported numbers in literature suggest that cholesterol can in fact comprise up to 50% of the overall composition of the plasma membrane. However, in which monolayer this—up to 50% cholesterol concentration—preferably hangs out in is a question that has puzzled the field for several decades and that has proven to be surprisingly hard to access experimentally (3). Hence, there is little reason to assume that cholesterol evenly distributes itself over the plasma membrane—despite being entropically more favorable—because the monolayers are asymmetric and of a rather different composition. For example, cholesterol is known to be strongly attracted to sphingomyelin because of its saturated tail and is most abundant (>90%) in the outer monolayer of the plasma membrane. Therefore, one would intuitively expect to find relatively more cholesterol in the outer monolayer than in the inner monolayer of the plasma membrane.

In their latest theoretical study, “Cholesterol-dependent bending energy is important in cholesterol distribution of the plasma membrane,” Allender et al. have calculated the preferred cholesterol concentration in each monolayer of the asymmetric plasma membrane (5). Indeed, the authors estimated the cholesterol concentration in the outer monolayer to be 63+/−6% of the total cholesterol concentration in the plasma membrane (which was assumed to be 40%). Thus, the outer monolayer is relatively enriched in cholesterol, whereas the inner monolayer is relatively depleted. Why?

Because the two monolayers of the plasma membrane are of a rather different composition, the free energy of each monolayer will change differently when absorbing a cholesterol molecule (the chemical potential). Therefore, to compensate for this inherent difference, cholesterol must distribute itself in such a way that both monolayers have the same chemical potential. The preferred cholesterol distribution—the distribution in which the chemical potential of both monolayers is equal—stems from a balance between three essential contributions to the chemical potential: 1) the intermolecular interactions between cholesterol and the different phospholipids in each monolayer (this drives cholesterol to the outer monolayer because of sphingomyelin), 2) the entropy (entropy favors an equal monolayer distribution), and 3) the bending stress in the membrane (cholesterol additionally introduces a negative spontaneous curvature within the monolayer).

Allender et al. derived the first contribution 1) from a simple mean field description by substituting experimentally derived values for the interaction energy between cholesterol and sphingomyelin molecules. The second contribution 2) is approximated by the mixing entropy of an ideal gas. For the third term (i.e., the contribution of cholesterol’s negative spontaneous curvature), 3) the authors conducted atomistic molecular dynamic simulations of preassembled, symmetric membranes that mimic the phospholipid composition of either the inner or outer monolayer of the plasma membrane. Based on the calculated local stress within the bilayer, the authors could estimate the effective, inherent shape that the monolayer prefers to adopt when being subjected to cholesterol and therefore gain an estimation of how the bending stress stored within each (flat) monolayer contributes to the chemical potential. Surprisingly, the contribution of bending stress opposes the attraction between cholesterol and sphingomyelin. In the absence of bending stress, the outer monolayer would in fact contain 72% rather than 63% of the total cholesterol concentration. Bending stresses thus tend to rather equalize the cholesterol distribution between the monolayers of the plasma membrane.

The effect of bending stress on cholesterol distribution does seem a bit counterintuitive, however. Hence, why would bending stress draw cholesterol—a molecule with an inverse cone shape—to the (inner) leaflet that is already enriched with inverse cone-shaped PE lipids. Cholesterol, however, does not always behave according to its presumed molecular shape. For example, the presence of a sufficiently large fraction of cholesterol in stacked POPE membranes is able to stabilize the lamellar phase with respect to the inverted hexagonal phase (6). In addition, cholesterol and PE lipids display quite opposite effects in coarse-grained simulations of membrane fusion (7). At a higher cholesterol concentration, the inner leaflet perhaps offers a relatively better “umbrella” for a hydrophobic molecule such as cholesterol to hide itself from the water phase (hydrophobic effect). Finally, the in vivo plasma membrane is additionally subject to an electric potential (ranging from −40 to −80 mV) because of a charge difference between the interior and the exterior of a biological cell. Molecular simulations have suggested that also the membrane potential may play a role in how cholesterol redistributes itself over the monolayers (8).

Editor: Ana-Suncana Smith.