The plasma membrane retains the contents of the cell and acts as a permeability barrier. That is, it allows only certain substances to enter or leave the cell, and the rate of entry is strictly controlled. Early researchers recognised that hydrophobic materials entered cells easily and proposed that an oily or 'lipoidal' layer was present at the cell surface. Gorter and Grendel in 1925 estimated the thickness of this layer by extracting the oily membrane from erythrocytes with acetone and spreading it as a monomolecular film in a Langmuir trough1. By measuring the film area and calculating the surface area of the original red cells (chosen since their geometry is reasonably constant), they concluded that exactly two layers of molecules were present at the interface, and proposed a lipid bilayer as the major cell membrane element. We now know that their experiment was subject to a considerable number of errors2, but fortunately these cancelled out in the final analysis and hence they obtained the correct answer by the wrong route. Electron micrographs indicate a double layered lipid membrane with bands approximately 3 nm in width and an overall thickness of between 8 to 12 nm. Although this is consistent with the lipid bilayer view, electron micrographic evidence was held in doubt for many years due to the difficulty of preparing the samples and the possibility of artefacts at so small a scale.
Subsequent discovery of the incorporation of proteins and polysaccharides led to the fluid mosaic model of Singer and Nicholson3. This model tended to suggest that the membrane was a sea of tightly packed phospholipids interspersed with proteins, leading to a rather ill-defined mixed membrane. However, studies during the last decade have demonstrated that the membrane is a highly organised structure; proteins in specific
conformations act as structural elements, transport nutrients, and sample the cell environment. The bilayer is not a lipid 'sea' but a carefully designed liquid crystal whose composition is controlled by the cell to achieve a specific degree of fluidity and an optimum environment for the processes which occur within it.
The detailed chemistry of the cell membrane was not worked out for many years due to the very large number of components that occur in membranes from varying organs. The development of chromatography was pivotal in allowing the lipid mixtures to be separated into their numerous components for detailed analysis. We now know that the main 'scaffolding' of the bilayer consists of a range of surfactant molecules, of which phospholipids are the most important. Most membranes also contain other materials, most notably proteins and sterols, but the surfactant lipids themselves are sufficient to form the lipid bilayer.
Phospholipids are compounds of glycerol (propane-1, 2, 3-triol) in which two of the alcohol groups are joined to fatty acids, and the third to phosphoric acid (Figure 1.1). The phosphate group can additionally form a bond to a smaller organic molecule (generally a hydrophilic one). The resultant molecule thus has two oily tails, usually of 12-24 carbon atoms length, and a hydrophilic region around the charged phosphate ester, called the headgroup. Common headgroup molecules are choline, ethanolamine, serine and inositol, and the resulting phospholipids are termed phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol respectively. Due to the cumbersome names these are normally abbreviated to PC, PE, PS and PI. These molecules have a typical surfactant structure. In water surfactants usually aggregate to form micelles, small clusters in which the oily tails are turned towards a common centre, since it is energetically unfavourable for the oily tails to be surrounded by water molecules. However, in phospholipids and other membrane-forming surfactants, the molecules aggregate to a bilayer sheet in which the tails are in the centre of the bilayer and the polar headgroups are in contact with the external aqueous environment (Figure 1.2). Phospholipids are not the only surfactants that behave in this way; the distinction between whether a surfactant forms a closed micelle or a bilayer sheet depends purely on the
Figure 1.2 The phospholipid bilayer
Polar Headgroups iydrophobic Chains
Figure 1.2 The phospholipid bilayer geometry of the molecule. Phospholipids form bilayers spontaneously when dispersed in water, as this is their thermodynamically most stable configuration, that is, it has the lowest free energy. However the bilayers do not form infinite planar sheets, but generally close in on themselves to form spherical structures in which one layer is on the outside of the sphere, and the other on the inside, enclosing an aqueous space. Simply shaking phospholipids in water results in the formation of these microscopic structures, this are termed liposomes.
Although membranes are often depicted as regular structures, as in Figure 1.2, the reality is that the bilayer is much more disordered and dynamic. The most important dynamic processes are lateral diffusion, in which the lipid molecules can move in the plane of the bilayer, and transverse diffusion or flip-flop, where a lipid molecule switches from one side of the membrane to the other. Since this involves moving the headgroup through the oily core of the bilayer, this is an extremely slow process, and in natural systems is generally catalysed when required by specialised membrane proteins.
The most important factor in determining the dynamic behaviour of the membrane is the transition temperature of the bilayer. At low temperatures the lipid tails are held in a relatively ordered array in the bilayer core. As the temperature is raised, little movement takes place until the transition temperature is reached. At this point the lipid spacing increases slightly and the tails become much more disordered in arrangement. The transition is often thought of as a gel-liquid melting of the bilayer, and in the fluid state the lipid molecules are relatively mobile to lateral diffusion; they diffuse at a speed of several microns a second and can move around a typical cell membrane on a timescale of seconds. The transition temperature depends mainly on the structure of the fatty acid chains attached to the glycerol backbone, with unsaturated chains causing low transition temperatures (generally below 0°C) and saturated chains having higher transition temperatures. For example, when the fatty chains in PC are both formed from palmitic acid (C16 saturated fatty acid) the lipid bilayer melts at 42°C. It is thus evident that the cell can control the fluidity of its membrane by varying the fatty acid composition of the phospholipids.
Although cell membrane fluidity can be regulated by altering the phospholipid fatty acid content, this is not the organism's only means of control. Most cell membranes contain varying amounts of sterols. In plants the primary membrane sterol is sitosterol; in animals, cholesterol, and in fungi ergosterol, although many other similar compounds also occur. Sterols alter the fluidity of the cell membrane by 'broadening' the melting transition so that the membrane melts over a much wider temperature range than that observed for the lipid alone. This is illustrated in Figure 1.3, which is a thermogram for the melting of a typical lipid membrane, in this case dipalmitoyl phosphatidylcholine. A thermogram is a plot of the energy absorbed as the temperature of the system is raised; the peak is caused by the absorption of energy required to melt the lipid bilayer. In the absence of sterols the bilayer melts over a small temperature range, causing a sharp peak in the thermogram. In the presence of cholesterol the melting transition is much broader, and the thermogram peak spans several degrees. The membrane begins to melt at a lower temperature than in the absence of sterol, and retains some structure up to a temperature above the transition temperature of the pure lipid. The effect of this is to 'smear out' the melting of the membrane, so that the fluidity is not so dependent on temperature. Obviously, this is of considerable importance in allowing the cell to function over a range of temperatures.
Cell membranes are extremely complex structures, and it is difficult to untangle all the aspects of their behaviour by studying whole cells. Consequently, most of our understanding of membrane function has arisen from a study of membrane models, systems which display certain aspects of membrane behaviour without the complexity of the whole cell. We have already mentioned liposomes, which are the spherical structures formed when
phospholipids are dispersed in water. Liposomes made in this way are actually multilayered, in a concentric 'onion-like' structure, and unlike cells do not have a large central aqueous space. They are termed large multilamellar vesicles or MLV's. Exposure to shear fields (normally by ultrasound) breaks them into small unilamellar vesicles or SUV's which are, however, rather smaller than single cells. Better membrane models are provided by giant unilamellar vesicles (GUV's) which can be made by careful injection of ethanolic lipid solutions into water.
Although liposomes can be made from well-characterised lipid mixtures, it is often useful to study natural membranes which have a more 'natural' structure, without the complexity added by the cell contents. The most widely used model in this respect is the erythrocyte ghost, which is the membrane surrounding a red blood cell from which the contents have been removed. These are prepared by placing the cells in hypertonic saline, which causes pores to form in the membrane. The cell contents then equilibrate with the suspending medium, and since this is normally much larger in volume than the total cell interior, the cells effectively become washed clean. Markers such as dyes or radiolabels can then be added, and will equilibrate with the solution inside the cells. If the tonicity is then adjusted to normal, the cell pores will re-seal and the cells, now labelled in their inner space, can be washed free of unentrapped marker by repeated centrifugation. There have been several attempts to place drugs inside the cells which are then returned to the donor, the idea being that they will then not be recognised as foreign, the outer membrane having the donor's correct antigen profile4. This has proven only partly successful, the cell surfaces being easily damaged during the labelling process.
The most serious problem with these membrane models is that it is only possible to access the outside of the membrane, the interior being sealed, unless an invasive technique such as the insertion of a microelectrode is used. This problem can be avoided by the use of black lipid films, a technique in which a lipid bilayer is formed across a small hole in the partition of a two-compartment vessel (Figure 1.4). The technique allows access to both sides of the membrane so that electrical measurements can be made, and the composition of the fluid on either side of the membrane can be readily altered. Using this method it is, for example, possible to measure the ion current across the membrane caused by pore-forming antibiotics, and study the operation of ion pump proteins.
Figure 1.4 The black lipid film between 2 aqueous compartments
Figure 1.4 The black lipid film between 2 aqueous compartments
The cell membrane is home for a number of types of proteins, which are generally divided into integral proteins and peripheral proteins. Integral proteins contain a sequence of hydrophobic groups that are embedded in the lipid bilayer, while peripheral proteins are adsorbed on to the surface of the cell, generally attached to an integral protein. The majority of functional proteins are integral, the most important peripheral proteins being the spectrin and ankyrin proteins. These bind to the inside of the plasma membrane to form the cytoskeleton, a network of proteins which runs throughout the cell, and is involved in a range of structural and transport functions.
One of the most important groups of integral membrane proteins from a pharmacological viewpoint is the transport proteins. These are responsible for moving substances into and out of the cell; for example, ATPase proteins pump ions across the cell membranes to maintain the required Na+/K+ electrolyte imbalance, and secrete H+ from the gastric parietal cells. Proteins also recognise and transport nutrients such as small carbohydrates and amino acids into the cell, each protein transporting a small group of structurally similar compounds.
A second important group of membrane proteins are the cell surface receptors. Although many biochemical receptors are present in the cytosol, a number of important materials are recognised by membrane proteins. These include a range of pituitary hormones, histamine receptors on mast cells, prostaglandins, and gastric peptides in the intestine.
Glycoproteins are a group of integral proteins carrying polysaccharide chains which are responsible for cell recognition and immunological behaviour. The segment of the protein chain, which is external to the cell, consists of hydrophilic protein residues, many of which carry small carbohydrate groups such as sialic acid. In many cells, these hydrophilic oligosaccharides form a continuous coat around the cell, together with polysaccharides attached to lipids (glycolipids). This layer is termed the glycocalyx. A common component of this layer is a peripheral protein called fibronectin, which contains binding sites for many membrane proteins, extracellular structural proteins such as collagen, and polysaccharides, and is thus an important component in intercellular binding and tissue formation.
Although liposomes are normally made with a similar lipid composition on both the inside and outside, living cells are much more asymmetric since they perform a range of processes which are obviously directional. The phospholipid composition of the inside and outside layers of the membrane is different in most cells; for example in the erythrocyte membrane phosphatidylcholine occurs predominantly on the outside of the cell and phosphatidylethanolamine predominantly on the inside. Glycolipids are normally oriented so that the polysaccharide segment is outside the cell, since it is responsible for immunogenicity and tissue adhesion. Integral proteins always have a specific orientation in the membrane that depends on their function; all molecules of the protein point in the same direction.
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