Membranes: Structure & Function - Biochemistry of Extracellular & Intracellular Communication - Harper’s Illustrated Biochemistry, 29th Edition (2012)

Harper’s Illustrated Biochemistry, 29th Edition (2012)

SECTION V. Biochemistry of Extracellular & Intracellular Communication

Chapter 40. Membranes: Structure & Function

Robert K. Murray, MD, PhD & Daryl K. Granner, MD

OBJECTIVES

After studying this chapter, you should be able to:

Image Know that biological membranes are mainly composed of a lipid bilayer and associated proteins and glycoproteins. The major lipids are phospholipids, cholesterol, and glycosphingolipids.

Image Appreciate that membranes are asymmetric, dynamic structures containing a mixture of integral and peripheral proteins.

Image Know the fluid mosaic model of membrane structure and that it is widely accepted, with lipid rafts, caveolae, and tight junctions being specialized features.

Image Understand the concepts of passive diffusion, facilitated diffusion, active transport, endocytosis, and exocytosis.

Image Recognize that transporters, ion channels, the Na+-K+-ATPase, receptors, and gap junctions are important participants in membrane function.

Image Know that a variety of disorders result from abnormalities of membrane structure and function, including familial hypercholesterolemia, cystic fibrosis, hereditary spherocytosis, and many others.

BIOMEDICAL IMPORTANCE

Membranes are highly fluid, dynamic structures consisting of a lipid bilayer and associated proteins. Plasma membranes form closed compartments around the cytoplasm to define cell boundaries. The plasma membrane has selective permeabilities and acts as a barrier, thereby maintaining differences in composition between the inside and outside of the cell. The selective permeabilities for substrates and ions are provided mainly by specific proteins named transporters and ion channels. The plasma membrane also exchanges material with the extracellular environment by exocytosis and endocytosis, and there are special areas of membrane structure—gap junctions—through which adjacent cells exchange material. In addition, the plasma membrane plays key roles in cell-cell interactions and in transmembrane signaling.

Membranes also form specialized compartments within the cell. Such intracellular membranes help shape many of the morphologically distinguishable structures (organelles), eg, mitochondria, ER, Golgi, secretory granules, lysosomes, and the nucleus. Membranes localize enzymes, function as integral elements in excitation-response coupling, and provide sites of energy transduction, such as in photosynthesis and oxidative phosphorylation.

Changes in membrane components can affect water balance and ion flux, and therefore many processes within the cell. Specific deficiencies or alterations of certain membrane components (eg, caused by mutations genes encoding membrane proteins) lead to a variety of diseases (see Table 40-7). In short, normal cellular function depends on normal membranes.

MAINTENANCE OF A NORMAL INTRA- & EXTRACELLULAR ENVIRONMENT IS FUNDAMENTAL TO LIFE

Life originated in an aqueous environment; enzyme reactions, cellular and subcellular processes, and so forth have therefore evolved to work in this milieu, encapsulated within a cell.

The Body’s Internal Water Is Compartmentalized

Water makes up about 60% of the lean body mass of the human body and is distributed in two large compartments.

Intracellular Fluid (ICF)

This compartment constitutes two-thirds of total body water and provides a specialized environment for the cell (1) to make, store, and utilize energy; (2) to repair itself; (3) to replicate; and (4) to perform cell-specific functions.

Extracellular Fluid (ECF)

This compartment contains about one-third of total body water and is distributed between the plasma and interstitial compartments. The extracellular fluid is a delivery system. It brings to the cells nutrients (eg, glucose, fatty acids, and amino acids), oxygen, various ions and trace minerals, and a variety of regulatory molecules (hormones) that coordinate the functions of widely separated cells. Extracellular fluid removes CO2, waste products, and toxic or detoxified materials from the immediate cellular environment.

The Ionic Compositions of Intracellular & Extracellular Fluids Differ Greatly

As illustrated in Table 40-1, the internal environment is rich in K+ and Mg2+, and phosphate is its major inorganic anion. The cytosol of cells contains a high concentration of protein that acts as a major intracellular buffer. Extracellular fluid is characterized by high Na+ and Ca2+ content, and Cl is the major anion. Why are there such differences? It is thought that the primordial sea in which life originated was rich in K+ and Mg2+. It, therefore, follows that enzyme reactions and other biologic processes evolved to function best in that environment—hence, the high concentration of these ions within cells. Vast changes would have been required for evolution of a completely new set of biochemical and physiologic machinery; instead, as it happened, cells developed barriers—membranes with associated “pumps” such as the Na+-K+-ATPase (see below)—to maintain the internal microenvironment.

TABLE 40–1 Comparison of the Mean Concentrations of Various Molecules Outside and Inside a Mammalian Cell

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MEMBRANES ARE COMPLEX STRUCTURES COMPOSED OF LIPIDS, PROTEINS, & CARBOHYDRATE-CONTAINING MOLECULES

We shall mainly discuss the membranes present in eukaryotic cells, although many of the principles described also apply to the membranes of prokaryotes. The various cellular membranes have different compositions, as reflected in the ratio of protein to lipid (Figure 40–1). This is not surprising, given their divergent functions. Membranes are sheet-like enclosed structures consisting of an asymmetric lipid bilayer with distinct inner and outer surfaces. These sheet-like structures are non-covalent assemblies that form spontaneously in water due to the amphipathic nature of lipids. Many different proteins are located in membranes, where they carry out specific functions.

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FIGURE 40–1 Ratio of protein to lipid in different membranes. Proteins equal or exceed the quantity of lipid in nearly all membranes. The outstanding exception is myelin, an electrical insulator found on many nerve fibers.

The Major Lipids in Mammalian Membranes Are Phospholipids, Glycosphingolipids & Cholesterol

Phospholipids

Of the two major phospholipid classes present in membranes, phosphoglycerides are the more common and consist of a glycerol backbone to which are attached two fatty acids in ester linkages and a phosphorylated alcohol (Figure 40–2). The fatty acid constituents are usually even-numbered carbon molecules, most commonly containing 16 or 18 carbons. They are unbranched and can be saturated or unsaturated with one or more cis double bonds. The simplest phosphoglyceride is phosphatidic acid, which is 1,2-diacylglycerol 3-phosphate, a key intermediate in the formation of other phosphoglycerides (Chapter 24). In most phosphoglycerides present in membranes, the 3-phosphate is esterified to an alcohol such as choline, ethanolamine, glycerol, inositol or serine (Chapter 15). Phosphatidylcholine is generally the major phosphoglyceride by mass in the membranes of human cells.

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FIGURE 40–2 A phosphoglyceride showing the fatty acids (R1 and R2), glycerol, and a phosphorylated alcohol component. Saturated fatty acids are usually attached to carbon 1 of glycerol, and unsaturated fatty acids to carbon 2. In phosphatidic acid, R3 is hydrogen.

The second major class of phospholipids comprises sphingomyelin (Figure 15–13), which contains a sphingosine backbone rather than glycerol. A fatty acid is attached by an amide linkage to the amino group of sphingosine, forming ceramide. When the primary hydroxyl group of sphingosine is esterified to phosphorylcholine, sphingomyelin is formed. As the name implies, sphingomyelin is prominent in myelin sheaths.

Glycosphingolipids

The glycosphingolipids (GSLs) are sugar-containing lipids built on a backbone of ceramide; they include galactosyl- and glucosylceramide (cerebrosides) and the gangliosides. Their structures are described in Chapter 15. They are mainly located in the plasma membranes of cells, displaying their sugar components to the exterior of the cell.

Sterols

The most common sterol in the membranes of animal cells is cholesterol (Chapter 15), which resides mainly in their plasma membranes, but can also be found in lesser quantities in mitochondria, Golgi complexes, and nuclear membranes. Cholesterol intercalates among the phospholipids of the membrane, with its hydroxyl group at the aqueous interface and the remainder of the molecule within the leaflet. Its effect on the fluidity of membranes will be discussed subsequently. From a nutritional viewpoint, it is important to know that cholesterol is not present in plants.

Lipids can be separated from one another and quantitated by techniques such as column, thin-layer, and gas-liquid chromatography and their structures can be established by mass spectrometry and other techniques.

Membrane Lipids Are Amphipathic

All major lipids in membranes contain both hydrophobic and hydrophilic regions and are therefore termed amphipathic. If the hydrophobic region were separated from the rest of the molecule, it would be insoluble in water but soluble in oil. Conversely, if the hydrophilic region were separated from the rest of the molecule, it would be insoluble in oil but soluble in water. The amphipathic nature of a phospholipid is represented in Figure 40–3 and also Figure 15–24. Thus, the polar head groups of the phospholipids and the hydroxyl group of cholesterol interface with the aqueous environment; a similar situation applies to the sugar moieties of the GSLs (see below).

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FIGURE 40–3 Diagrammatic representation of a phospholipid or other membrane lipid. The polar head group is hydrophilic, and the hydrocarbon tails are hydrophobic or lipophilic. The fatty acids in the tails are saturated (S) or unsaturated (U); the former are usually attached to carbon 1 of glycerol and the latter to carbon 2 (see Figure 40–2). Note the kink in the tail of the unsaturated fatty acid (U), which is important in conferring increased membrane fluidity.

Saturated fatty acids have straight tails, whereas unsaturated fatty acids, which generally exist in the cis form in membranes, make kinked tails (Figure 40–3). As more kinks are inserted in the tails, the lipids become less tightly packed and the membrane more fluid. The problem caused by the presence of trans fatty acids in membrane lipids is described in Chapter 15.

Detergents are amphipathic molecules that are important in biochemistry as well as in the household. The molecular structure of a detergent is not unlike that of a phospholipid. Certain detergents are widely used to solubilizemembrane proteins and in their purification. The hydrophobic end of the detergent binds to hydrophobic regions of the proteins, displacing most of their bound lipids. The polar end of the detergent is free, bringing the proteins into solution as detergent-protein complexes, usually also containing some residual lipids.

Membrane Lipids Form Bilayers

The amphipathic character of phospholipids suggests that the two regions of the molecule have incompatible solubilities; however, in a solvent such as water, phospholipids organize themselves into a form that thermodynamically serves the solubility requirements of both regions. A micelle (Figure 40–4 and Figure 15–24) is such a structure; the hydrophobic regions are shielded from water, while the hydrophilic polar groups are immersed in the aqueous environment. However, micelles are usually relatively small in size (eg, ~200 nm) and thus are limited in their potential to form membranes. Detergents commonly form micelles.

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FIGURE 40–4 Diagrammatic cross-section of a micelle. The polar head groups are bathed in water, whereas the hydrophobic hydrocarbon tails are surrounded by other hydrocarbons and thereby protected from water. Micelles are relatively small (compared with lipid bilayers) spherical structures.

As was recognized in 1925 by Gorter and Grendel, a bimolecular layer, or lipid bilayer, can also satisfy the thermodynamic requirements of amphipathic molecules in an aqueous environment. Bilayers are the key structures in biological membranes. A bilayer exists as a sheet in which the hydrophobic regions of the phospholipids are sequestered from the aqueous environment, while the hydrophilic regions are exposed to water (Figure 40–5 and Figure 15–24). The ends or edges of the bilayer sheet can be eliminated by folding the sheet back upon itself to form an enclosed vesicle with no edges. The closed bilayer provides one of the most essential properties of membranes. It is impermeable to most water-soluble molecules since they would be insoluble in the hydrophobic core of the bilayer.

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FIGURE 40–5 Diagram of a section of a bilayer membrane formed from phospholipid molecules. The unsaturated fatty acid tails are kinked and lead to more spacing between the polar head groups, hence to more room for movement. This in turn results in increased membrane fluidity. (Slightly modified and reproduced, with permission, from Stryer L: Biochemistry, 2nd ed. Freeman, 1981. Copyright ©1981 by W H. Freeman and Company.)

Lipid bilayers are formed by self-assembly, driven by the hydrophobic effect (Chapter 2). When lipid molecules come together in a bilayer, the entropy of the surrounding solvent molecules increases due to the release of immobilized water.

Two questions arise from consideration of the above. First, how many biologic materials are lipid-soluble and can therefore readily enter the cell? Gases such as oxygen, CO2, and nitrogen—small molecules with little interaction with solvents—readily diffuse through the hydrophobic regions of the membrane. The permeability coefficients of several ions and of a number of other molecules in a lipid bilayer are shown in Figure 40–6. The three electrolytes shown (Na+, K+, and Cl) cross the bilayer much more slowly than water. In general, the permeability coefficients of small molecules in a lipid bilayer correlate with their solubilities in nonpolar solvents. For instance, steroidsmore readily traverse the lipid bilayer compared with electrolytes. The high permeability coefficient of water itself is surprising, but is partly explained by its small size and relative lack of charge. Many drugs are hydrophobic and can readily cross membranes and enter cells.

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FIGURE 40–6 Permeability coefficients of water, some ions, and other small molecules in lipid bilayer membranes. The permeability coefficient is a measure of the ability of a molecule to diffuse across a permeability barrier. Molecules that move rapidly through a given membrane are said to have a high permeability coefficient. (Slightly modified and reproduced, with permission, from Stryer L: Biochemistry, 2nd ed. Freeman, 1981. Copyright © 1981)

The second question concerns molecules that are not lipid-soluble: How are the transmembrane concentration gradients for nonlipid-soluble molecules maintained? The answer is that membranes contain proteins, many of which span the lipid bilayer. Such proteins form channels for the movement of ions and small molecules or serve as transporters for molecules that otherwise could not pass the bilayer. These structures are described below.

Membrane Proteins Are Associated with the Lipid Bilayer

Membrane phospholipids act as a solvent for membrane proteins, creating an environment in which the latter can function. As described in Chapter 5, the α-helical structure of proteins minimizes the hydrophilic character of the peptide bonds themselves. Thus, proteins can be amphipathic and form an integral part of the membrane by having hydrophilic regions protruding at the inside and outside faces of the membrane but connected by a hydrophobic region traversing the hydrophobic core of the bilayer. In fact, those portions of membrane proteins that traverse membranes do contain substantial numbers of hydrophobic amino acids and almost invariably have a high α-helical content. For many membranes, a stretch of ~20 amino acids in an α-helix will span the bilayer.

It is possible to calculate whether a particular sequence of amino acids present in a protein is consistent with a transmembrane location. This can be done by consulting a Table that lists the hydrophobicities of each of the 20 common amino acids and the free energy values for their transfer from the interior of a membrane to water. Hydrophobic amino acids have positive values; polar amino acids have negative values. The total free energy values for transferring successive sequences of 20 amino acids in the protein are plotted, yielding a so-called hydropathy plot. Values of over 20 kcal mol-1 are consistent with—but do not prove—the interpretation that the hydrophobic sequence is a transmembrane segment.

Another aspect of the interaction of lipids and proteins is that some proteins are anchored to one leaflet of the bilayer by covalent linkages to certain lipids. Palmitate and myristate are fatty acids involved in such linkages to specific cytosolic proteins. A number of cell surface proteins (see Chapter 47) are linked to the plasma membrane via glycophosphatidylinositol (GPI) structures.

Different Membranes Have Different Protein Compositions

The number of different proteins in a membrane varies from less than a dozen in the sarcoplasmic reticulum of muscle cells to over 100 in plasma membranes. Membrane proteins can be separated from one another using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique that separates proteins based on their molecular mass. Using standard proteins of known molecular mass as a comparison, one can estimate the approximate molecular mass of an unknown protein via SDS-PAGE. SDS is a powerful detergent that disrupts protein-lipid interactions and thereby solubilizes membrane proteins. SDS also disrupts protein-protein interactions and unfolds or denatures proteins. In the absence of SDS, few membrane proteins would remain soluble.

Proteins are the major functional molecules of membranes and consist of enzymes, pumps and transporters, channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every type of membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymatic properties of several different membranes are shown in Table 40-2.

TABLE 40–2 Enzymatic Markers of Different Membranes1

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Membranes Are Dynamic Structures

Membranes and their components are dynamic structures. The lipids and proteins in membranes undergo turnover, just as they do in other compartments of the cell. Different lipids have different turnover rates, and the turnover rates of individual species of membrane proteins may vary widely. The membrane itself can turn over even more rapidly than any of its constituents. This is discussed in more detail in the section on endocytosis.

Another indicator of the dynamic nature of membranes is that a variety of studies have shown that lipids and certain proteins exhibit lateral diffusion in the plane of their membranes. Some proteins do not exhibit lateral diffusion because they are anchored to the underlying actin cytoskeleton. In contrast, the transverse movement of lipids across the membrane (flip-flop) is extremely slow (see below) and does not occur at all in the case of membrane proteins.

Membranes Are Asymmetric Structures

Proteins have unique orientations in membranes, making the outside surfaces different from the inside surfaces. An inside-outside asymmetry is also provided by the external location of the carbohydrates attached to membrane proteins. In addition, specific proteins are located exclusively on the outsides or insides of membranes.

There are also regional heterogeneities in membranes. Some, such as occur at the villous borders of mucosal cells, are almost macroscopically visible. Others, such as those at gap junctions, tight junctions, and synapses, occupy much smaller regions of the membrane and generate correspondingly smaller local asymmetries.

There is also inside-outside asymmetry of the phospholipids. The choline-containing phospholipids (phosphatidylcholine and sphingomyelin) are located mainly in the outer leaflet; the aminophospholipids(phosphatidylserine and phosphatidylethanolamine) are preferentially located in the inner leaflet. Obviously, if this asymmetry is to exist at all, there must be limited transverse mobility (flip-flop) of the membrane phospholipids. In fact, phospholipids in synthetic bilayers exhibit an extraordinarily slow rate of flip-flop; the half-life of the asymmetry can be measured in several weeks.

The mechanisms involved in the establishment of lipid asymmetry are not well understood. The enzymes involved in the synthesis of phospholipids are located on the cytoplasmic side of microsomal membrane vesicles. Translocases (flip-pases) exist that transfer certain phospholipids (eg, phosphatidylcholine) from the inner to the outer leaflet. Specific proteins that preferentially bind individual phospholipids also appear to be present in the two leaflets, contributing to the asymmetric distribution of these lipid molecules. In addition, phospholipid exchange proteins recognize specific phospholipids and transfer them from one membrane (eg, the endoplasmic reticulum [ER]) to others (eg, mitochondrial and peroxisomal). A related issue is how lipids enter membranes. This has not been studied as intensively as the topic of how proteins enter membranes (see Chapter 46) and knowledge is still relatively meager. Many membrane lipids are synthesized in the ER. At least three pathways have been recognized. (1) Transport from the ER in vesicles, which then transfer the contained lipids to the recipient membrane. (2) Entry via direct contact of one membrane (eg, the ER) with another, facilitated by specific proteins. (3) Transport via the phospholipid exchange proteins (also known as lipid transfer proteins) mentioned above. This only exchanges lipids, but does not cause net transfer.

There is further asymmetry with regard to glycosphingolipids and glycoproteins; the sugar moieties of these molecules all protrude outward from the plasma membrane and are absent from its inner face. Thus, cells are “sugar coated”.

Membranes Contain Integral & Peripheral Proteins

It is useful to classify membrane proteins into two types: integral and peripheral (Figure 40–7). Most membrane proteins fall into the integral class, meaning that they interact extensively with the phospholipids and require the use of detergents for their solubilization. Also, they generally span the bilayer as a bundle of α-helical transmembrane segments. Integral proteins are usually globular and are themselves amphipathic. They consist of two hydrophilic ends separated by an intervening hydrophobic region that traverses the hydrophobic core of the bilayer. As the structures of integral membrane proteins were being elucidated, it became apparent that certain ones (eg, transporter molecules, ion channels, various receptors, and G proteins) span the bilayer many times (see Figure 46–7), whereas other simple membrane proteins (eg, glycophorin A) span the membrane only once, Integral proteins are asymmetrically distributed across the membrane bilayer. This asymmetric orientation is conferred at the time of their insertion in the lipid bilayer during biosynthesis in the ER. The molecular mechanisms involved in insertion of proteins into membranes and the topic of membrane assembly are discussed in Chapter 46.

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FIGURE 40–7 The fluid mosaic model of membrane structure. The membrane consists of a bimolecular lipid layer with proteins inserted in it or bound to either surface. Integral membrane proteins are firmly embedded in the lipid layers. Some of these proteins completely span the bilayer and are called transmembrane proteins, while others are embedded in either the outer or inner leaflet of the lipid bilayer. Loosely bound to the outer or inner surface of the membrane are the peripheral proteins. Many of the proteins and all the glycolipids have externally exposed oligosaccharide chains. (Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas,10th ed., McGraw-Hill, 2003.)

Peripheral proteins do not interact directly with the hydrophobic cores of the phospholipids in the bilayer and thus do not require use of detergents for their release. They are bound to the hydrophilic regions of specific integral proteins and head groups of phospholipids and can be released from them by treatment with salt solutions of high ionic strength. For example, ankyrin, a peripheral protein, is bound to the inner aspect of the integral protein “band 3” of erythrocyte membrane. Spectrin, a cytoskeletal structure within the erythrocyte, is in turn bound to ankyrin and thereby plays an important role in maintenance of the biconcave shape of the erythrocyte.

ARTIFICIAL MEMBRANES MODEL MEMBRANE FUNCTION

Artificial membrane systems can be prepared by appropriate techniques. These systems generally consist of mixtures of one or more phospholipids of natural or synthetic origin that can be treated (eg, by using mild sonication) to form spherical vesicles in which the lipids form a bilayer. Such vesicles, surrounded by a lipid bilayer with an aqueous interior, are termed liposomes (see Figure 15–24).

Some of the advantages and uses of artificial membrane systems in the study of membrane function are as follows:

1. The lipid content of the membranes can be varied, allowing systematic examination of the effects of varying lipid composition on certain functions.

2. Purified membrane proteins or enzymes can be incorporated into these vesicles in order to assess what factors (eg, specific lipids or ancillary proteins) the proteins require to reconstitute their function.

3. The environment of these systems can be rigidly controlled and systematically varied (eg, ion concentrations and ligands).

4. When liposomes are formed, they can be made to entrap certain compounds inside themselves, eg, drugs and isolated genes. There is interest in using liposomes to distribute drugs to certain tissues, and if components (eg, antibodies to certain cell surface molecules) could be incorporated into liposomes so that they would be targeted to specific tissues or tumors, the therapeutic impact would be considerable. DNA entrapped inside liposomes appears to be less sensitive to attack by nucleases; this approach may prove useful in attempts at gene therapy.

THE FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE IS WIDELY ACCEPTED

The fluid mosaic model of membrane structure proposed in 1972 by Singer and Nicolson (Figure 40–7) is now widely accepted. The model is often likened to icebergs (membrane proteins) floating in a sea of predominantly fluid phospholipid molecules. Early evidence for the model was the finding that certain integral proteins (detected by fluorescent labeling techniques) rapidly and randomly redistributed in the plasma membrane of a hybrid cell formed by the artificially induced fusion of two different (mouse and human) parent cells. Biophysical studies of integral proteins showed that they spanned the membrane and had a globular nature. It has subsequently been demonstrated that phospholipids undergo even more rapid redistribution in the plane of the membrane. This diffusion within the plane of the membrane, termed lateral diffusion, can be quite rapid for a phospholipid; in fact, within the plane of the membrane, one molecule of phospholipid can move several micrometers per second.

The phase changes—and thus the fluidity of membranes—are largely dependent upon the lipid composition of the membrane. In a lipid bilayer, the hydrophobic chains of the fatty acids can be highly aligned or ordered to provide a rather stiff structure. As the temperature increases, the hydrophobic side chains undergo a transition from the ordered state (more gel-like or crystalline phase) to a disordered one, taking on a more liquid-like or fluid arrangement. The temperature at which the structure undergoes the transition from ordered to disordered (ie, melts) is called the “transition temperature” (Tm). The longer and more saturated fatty acid chains interact more strongly with each other via their longer hydrocarbon chains and thus cause higher values of Tm—ie, higher temperatures are required to increase the fluidity of the bilayer. On the other hand, unsaturated bonds that exist in the cisconfiguration tend to increase the fluidity of a bilayer by decreasing the compactness of the side chain packing without diminishing hydrophobicity (Figure 40–3). The phospholipids of cellular membranes generally contain at least one unsaturated fatty acid with at least one cis double bond.

Cholesterol modifies the fluidity of membranes. At temperatures below the Tm, it interferes with the interaction of the hydrocarbon tails of fatty acids and thus increases fluidity. At temperatures above the Tm, it limits disorder because it is more rigid than the hydrocarbon tails of the fatty acids and cannot move in the membrane to the same extent, thus limiting fluidity. At high cholesterol-phospholipid ratios, transition temperatures are altogether indistinguishable.

The fluidity of a membrane significantly affects its functions. As membrane fluidity increases, so does its permeability to water and other small hydrophilic molecules. The lateral mobility of integral proteins increases as the fluidity of the membrane increases. If the active site of an integral protein involved in a given function is exclusively in its hydrophilic regions, changing lipid fluidity will probably have little effect on the activity of the protein; however, if the protein is involved in a transport function in which transport components span the membrane, lipid-phase effects may significantly alter the transport rate. The insulin receptor is an excellent example of altered function with changes in fluidity. As the concentration of unsaturated fatty acids in the membrane is increased (by growing cultured cells in a medium rich in such molecules), fluidity increases. This alters the receptor so that it binds more insulin. At normal body temperature (37°C), the lipid bilayer is in a fluid state. Bacteria can modify the composition of their membrane lipids to adapt to changes in temperature.

Lipid Rafts, Caveolae, & Tight Junctions Are Specialized Features of Plasma Membranes

Plasma membranes contain certain specialized structures whose biochemical natures have been investigated in some detail.

Lipid rafts are specialized areas of the exoplasmic leaflet of the lipid bilayer enriched in cholesterol, sphingolipids, and certain proteins (see Figure 40–8). They are involved in signal transduction and other processes. It is thought that clustering certain components of signaling systems closely together may increase the efficiency of their function.

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FIGURE 40–8 Schematic diagram of a lipid raft. Lipid rafts are somewhat thicker than the remainder of the bilayer. They are enriched in sphingolipids (eg, sphingomyelin), glycosphingolipids (eg, the ganglioside GM1), saturated phospholipids, and cholesterol. They also contain certain GPI-linked proteins (outer leaflet) and acylated and prenylated proteins (inner leaflet). GPI-linked proteins are discussed in Chapter 47. Acylation and prenylation are post-translational modifications of certain membrane proteins.

Caveolae may derive from lipid rafts. Many, if not all, contain the protein caveolin-1, which may be involved in their formation from rafts. Caveolae are observable by electron microscopy as flask-shaped indentations of the cell membrane facing the cytosol (Figure 40–9). Proteins detected in caveolae include various components of the signal transduction system (eg, the insulin receptor and some G proteins), the folate receptor, and endothelial nitric oxide synthase (eNOS). Caveolae and lipid rafts are active areas of research, and ideas concerning them and their possible roles in various disorders are rapidly evolving.

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FIGURE 40–9 Schematic diagram of a caveola. A caveola is an invagination in the plasma membrane. The protein caveolin appears to play an important role in the formation of caveolae and occurs as a dimer. Each caveolin monomer is anchored to the inner leaflet of the plasma membrane by three palmitoyl molecules (not shown).

Tight junctions are other structures found in surface membranes. They are often located below the apical surfaces of epithelial cells and prevent the diffusion of macromolecules between cells. They are composed of various proteins, including occludin, various claudins, and junctional adhesion molecules.

Yet other specialized structures found in surface membranes include desmosomes, adherens junctions, and microvilli; their chemical natures and functions are not discussed here. The nature of gap junctions is described below.

MEMBRANE SELECTIVITY ALLOWS ADJUSTMENTS OF CELL COMPOSITION & FUNCTION

If the plasma membrane is relatively impermeable, how do most molecules enter a cell? How is selectivity of this movement established? Answers to such questions are important in understanding how cells adjust to a constantly changing extracellular environment. Metazoan organisms also must have means of communicating between adjacent and distant cells, so that complex biologic processes can be coordinated. These signals must arrive at and be transmitted by the membrane, or they must be generated as a consequence of some interaction with the membrane. Some of the major mechanisms used to accomplish these different objectives are listed in Table 40-3.

TABLE 40–3 Transfer of Material and Information Across Membranes

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Passive Diffusion Involving Transporters & Ion Channels Moves Many Small Molecules Across Membranes

Molecules can passively traverse the bilayer down electrochemical gradients by simple diffusion or by facilitated diffusion. This spontaneous movement toward equilibrium contrasts with active transport, which requires energybecause it constitutes movement against an electrochemical gradient. Figure 40–10 provides a schematic representation of these mechanisms. We shall first describe various aspects of passive transport, and then discuss aspects of active transport.

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FIGURE 40–10 Many small, uncharged molecules pass freely through the lipid bilayer by simple diffusion. Larger uncharged molecules, and some small uncharged molecules, are transferred by specific carrier proteins (transporters) or through channels or pores. Passive transport is always down an electrochemical gradient, toward equilibrium. Active transport is against an electrochemical gradient and requires an input of energy, whereas passive transport does not. (Redrawn and reproduced, with permission, from Alberts B et al: Molecular Biology of the Cell. Garland, 1983.)

First, let us define the various terms. Simple diffusion is the passive flow of a solute from a higher to a lower concentration due to random thermal movement. Facilitated diffusion is passive transport of a solute from a higher to a lower concentration mediated by a specific protein transporter. Active transport is transport of a solute across a membrane against a concentration gradient, and thus requires energy (frequently derived from the hydrolysis of ATP); a specific transporter (pump) is involved.

As mentioned earlier in this chapter, some solutes such as gases can enter the cell by diffusing down an electrochemical gradient across the membrane and do not require metabolic energy. The simple diffusion of a solute across the membrane is limited by the thermal agitation of that specific molecule, by the concentration gradient across the membrane, and by the solubility of that solute (the permeability coefficient, Figure 40–6) in the hydrophobic core of the membrane bilayer. Solubility is inversely proportionate to the number of hydrogen bonds that must be broken in order for a solute in the external aqueous phase to become incorporated in the hydrophobic bilayer. Electrolytes, poorly soluble in lipid, do not form hydrogen bonds with water, but they do acquire a shell of water from hydration by electrostatic interaction. The size of the shell is directly proportionate to the charge density of the electrolyte. Electrolytes with a large charge density have a larger shell of hydration and thus a slower diffusion rate. Na+. for example, has a higher charge density than K+. Hydrated Na+ is therefore larger than hydrated K+;hence, the latter tends to move more easily through the membrane.

The following factors affect net diffusion of a substance. (1) Its concentration gradient across the membrane: solutes move from high to low concentration. (2) The electrical potential across the membrane: solutes move toward the solution that has the opposite charge. The inside of the cell usually has a negative charge. (3) The permeability coefficient of the substance for the membrane. (4) The hydrostatic pressure gradient across the membrane: increased pressure will increase the rate and force of the collision between the molecules and the membrane. (5) Temperature: increased temperature will increase particle motion and thus increase the frequency of collisions between external particles and the membrane.

Facilitated diffusion involves either certain transporters or ion channels (see Figure 40–11). Other transporters (mostly ATP-driven) are involved in active transport. A multitude of transporters and channels exist in biological membranes that route the entry of ions into and out of cells. They are described in the following sections. Table 40-4 summarizes some important points of difference between transporters and ion channels.

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FIGURE 40–11 A schematic diagram of the two types of membrane transport of small molecules.

TABLE 40–4 Comparison of Transporters and Ion Channels

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Transporters Are Specific Proteins Involved in Facilitated Diffusion & Also Active Transport

Transport systems can be described in a functional sense according to the number of molecules moved and the direction of movement (Figure 40–12) or according to whether movement is toward or away from equilibrium. The following classification depends primarily on the former. A uniport system moves one type of molecule bidirectionally. In cotransport systems, the transfer of one solute depends upon the stoichiometric simultaneous or sequential transfer of another solute. A symport moves two solutes in the same direction. Examples are the proton-sugar transporter in bacteria and the Na+-sugar transporters (for glucose and certain other sugars) and Na+-amino acid transporters in mammalian cells. Antiport systems move two molecules in opposite directions (eg, Na+ in and Ca2+ out).

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FIGURE 40–12 Schematic representation of types of transport systems. Transporters can be classified with regard to the direction of movement and whether one or more unique molecules are moved. A uniport can also allow movement in the opposite direction, depending on the concentrations inside and outside a cell of the molecule transported. (Redrawn and reproduced, with permission, from Alberts B et al: Molecular Biology of the Cell. Garland, 1983.)

Hydrophilic molecules that cannot pass freely through the lipid bilayer membrane do so passively by facilitated diffusion or by active transport. Passive transport is driven by the transmembrane gradient of substrate. Active transport always occurs against an electrical or chemical gradient, and so it requires energy, usually ATP. Both types of transport involve specific carrier proteins (transporters) and both show specificity for ions, sugars, and amino acids. Passive and active transports resemble a substrate-enzyme interaction. Points of resemblance of both to enzyme action are as follows: (1) There is a specific binding site for the solute. (2) The carrier is saturable, so it has a maximum rate of transport (Vmax; Figure 40–13). (3) There is a binding constant (Km for the solute, and so the whole system has a Km (Figure 40–13). (4) Structurally similar competitive inhibitors block transport. Transporters are thus like enzymes, but generally do not modify their substrates.

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FIGURE 40–13 A comparison of the kinetics of carriermediated (facilitated) diffusion with passive diffusion. The rate of movement in the latter is directly proportionate to solute concentration, whereas the process is saturable when carriers are involved. The concentration at half-maximal velocity is equal to the binding constant (Km) of the carrier for the solute. (Vmax, maximal rate.)

Cotransporters use the gradient of one substrate created by active transport to drive the movement of the other substrate. The Na+ gradient produced by the Na+-K+-ATPase is used to drive the transport of a number of important metabolites. The ATPase is a very important example of primary transport, while the Na+-dependent systems are examples of secondary transport that rely on the gradient produced by another system. Thus, inhibition of the Na+-K+-ATPase in cells also blocks the Na+-dependent uptake of substances like glucose.

Facilitated Diffusion Is Mediated by a Variety of Specific Transporters

Some specific solutes diffuse down electrochemical gradients across membranes more rapidly than might be expected from their size, charge, or partition coefficient. This is because specific transporters are involved. This facilitated diffusion exhibits properties distinct from those of simple diffusion. The rate of facilitated diffusion, a uniport system, can be saturated; ie, the number of sites involved in diffusion of the specific solutes appears finite. Many facilitated diffusion systems are stereospecific but, like simple diffusion, are driven by the transmembrane electrochemical gradient.

A “ping-pong” mechanism (Figure 40–14) helps explain facilitated diffusion. In this model, the carrier protein exists in two principal conformations. In the “ping” state, it is exposed to high concentrations of solute, and molecules of the solute bind to specific sites on the carrier protein. Binding induces a conformational change that exposes the carrier to a lower concentration of solute (“pong” state). This process is completely reversible, and net flux across the membrane depends upon the concentration gradient. The rate at which solutes enter a cell by facilitated diffusion is determined by the following factors: (1) the concentration gradient across the membrane; (2) the amount of carrier available (this is a key control step); (3) the affinity of the solute-carrier interaction; (4) the rapidity of the conformational change for both the loaded and the unloaded carrier.

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FIGURE 40–14 The “ping-pong” model of facilitated diffusion. A protein carrier (blue structure) in the lipid bilayer associates with a solute in high concentration on one side of the membrane. A conformational change ensues (“ping” to “pong”), and the solute is discharged on the side favoring the new equilibrium. The empty carrier then reverts to the original conformation (“pong” to “ping”) to complete the cycle.

Hormones can regulate facilitated diffusion by changing the number of transporters available. Insulin via a complex signaling pathway increases glucose transport in fat and muscle by recruiting glucose transporters (GLUT) from an intracellular reservoir. Insulin also enhances amino acid transport in liver and other tissues. One of the coordinated actions of glucocorticoid hormones is to enhance transport of amino acids into liver, where the amino acids then serve as a substrate for gluconeogenesis. Growth hormone increases amino acid transport in all cells, and estrogens do this in the uterus. There are at least five different carrier systems for amino acids in animal cells. Each is specific for a group of closely related amino acids, and most operate as Na+-symport systems (Figure 40–12).

Ion Channels Are Transmembrane Proteins That Allow the Selective Entry of Various Ions

Natural membranes contain transmembrane channels, porelike structures composed of proteins that constitute selective ion channels. Cation-conductive channels have an average diameter of about 5-8 nm. The permeability of a channel depends upon the size, the extent of hydration, and the extent of charge density on the ion. Specific channels for Na+, K+, Ca2+, and Cl have been identified. One such Na+ channel is illustrated in Figure 40–15. It is seen to consist of four subunits. Each subunit consists of six α-helical transmembrane domains. The amino and carboxyl terminals are located in the cytoplasm, with both extracellular and intracellular loops being present. The actual pore in the channel through which the ions pass is not shown. A pore constitutes the center (diameter about 5-8 nm) of a structure formed by apposition of the subunits. Ion channels are very selective, in most cases permitting the passage of only one type of ion (Na+, Ca2+, etc). The selectivity filter of K+ channels is made up of a ring of carbonyl groups donated by the subunits. The carbonyls displace bound water from the ion, and thus restrict its size to appropriate precise dimensions for passage through the channel. Many variations on the above structural theme are found, but all ion channels are basically made up of transmembrane subunits that come together to form a central pore through which ions pass selectively.

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FIGURE 40–15 Diagrammatic representation of the structures of an ion channel (a Na+ channel of rat brain). The Roman numerals indicate the four subunits of the channel and the Arabic numerals the α-helical transmembrane domains of each subunit. The actual pore through which the ions (Na+) pass is not shown, but is formed by apposition of the various subunits. The specific areas of the subunits involved in the opening and closing of the channel are also not indicated. (Catterall WA: Structure and function of voltage-sensitive ion channel. Science 1988;242(4875):50–61.)

The membranes of nerve cells contain well-studied ion channels that are responsible for the generation of action potentials. The activity of some of these channels is controlled by neurotransmitters; hence, channel activity can be regulated.

Ion channels are open transiently and thus are “gated.” Gates can be controlled by opening or closing. In ligand-gated channels, a specific molecule binds to a receptor and opens the channel. Voltage-gated channels open (or close) in response to changes in membrane potential. Mechanically gated channels respond to mechanical stimuli (pressure and touch).

Some properties of ion channels are listed in Tables 40-4 and 40-5 ; other aspects of ion channels are discussed briefly in Chapter 48.

TABLE 40–5 Some Properties of Ion Channels

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Detailed Studies of a K+ Channel & of a Voltage-Gated Channel Have Yielded Major Insights into Their Actions

There are at least four features of ion channels that must be elucidated: (1) their overall structures; (2) how they conduct ions so rapidly; (3) their selectivity; and (4) their gating properties. As described below, considerable progress in tackling these difficult problems has been made.

Especial progress has been made by Roderick MacKinnon, who received the Nobel Prize for elucidating the structure and function of a K+ channel (KvAP) present in Streptomyces lividans. A variety of techniques were used, including site-directed mutagenesis and x-ray crystallography. The channel is an integral membrane protein composed of four identical subunits, each with two transmembrane segments, creating an inverted teepee-like structure (Figure 40–16). The part of the channels that confers ion selectivity (the selectivity filter) measures 12 Å long (a relatively short length of the membrane, so K+ does not have far to travel in the membrane) and is situated at the wide end of the inverted teepee. The large, water-filled cavity and helical dipoles shown in Figure 40–16 help overcome the relatively large electrostatic energy barrier for a cation to cross the membrane. The selectivity filter is lined with carbonyl oxygen atoms (contributed by a TVGYG sequence), providing a number of sites with which K+ can interact. K+ ions, which dehydrate as they enter the narrow selectivity filter, fit with proper coordination into the filter, but Na+ is too small to interact with the carbonyl oxygen atoms in correct alignment and is rejected. Two K+ ions, when close to each other in the filter, repel one another. This repulsion overcomes interactions between K+ and the surrounding protein molecule and allows very rapid conduction of K+ with high selectivity.

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FIGURE 40–16 Schematic diagram of the structure of a K+ channel (KvAP) from Streptomyces lividans. A single K+ is shown in a large aqueous cavity inside the membrane interior. Two helical regions of the channel protein are oriented with their carboxylate ends pointing to where the K+ is located. The channel is lined by carboxyl oxygen. (Modified, with permission, from Doyle DA, et al, (1998), “The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity”. Science 280:69. Reprinted with permission from AAAS.)

Other studies on a voltage-gated ion channel (HvAP) in Aeropyrum pernix have revealed many features of its voltage-sensing and voltage-gating mechanisms. This channel is made up of four subunits, each with six transmembrane segments. One of the six segments (S4 and part of S3) is the voltage sensor. It behaves like a charged paddle (Figure 40–17), in that it can move through the interior of the membrane transferring four positive charges (due to four Arg residues in each subunit) from one membrane surface to the other in response to changes in voltage. There are four voltage sensors in each channel, linked to the gate. The gate part of the channel is constructed from S6 helices (one from each of the subunits). Movements of this part of the channel in response to changing voltage effectively close the channel or reopen it, in the latter case allowing a current of ions to cross.

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FIGURE 40–17 Schematic diagram of the voltage-gated K+ channel of Aeorpyrum pernix. The voltage sensors behave like charged paddles that move through the interior of the membrane. Four voltage sensors (only two are shown here) are linked mechanically to the gate of the channel. Each sensor has four positive charges contributed by arginine residues. (Modified, with permission, from Sigworth FJ: Nature 2003;423:21. Copyright © 2003. Macmillan Publishers Ltd.)

Ionophores Are Molecules That Act as Membrane Shuttles for Various Ions

Certain microbes synthesize small cyclic organic molecules, ionophores, such as valinomycin that function as shuttles for the movement of ions (K+ in the case of valinomycin) across membranes. These ionophores contain hydrophilic centers that bind specific ions and are surrounded by peripheral hydrophobic regions; this arrangement allows the molecules to dissolve effectively in the membrane and diffuse transversely therein. Others, like the well-studied polypeptide gramicidin (an antibiotic), fold up to form hollow channels.

Microbial toxins such as diphtheria toxin and activated serum complement components can produce large pores in cellular membranes and thereby provide macromolecules with direct access to the internal milieu. The toxin α-hemolysin (produced by certain species of Streptococcus) consists of seven subunits which come together to form a β-barrel that allows metabolites like ATP to leak out of cells, resulting in cell lysis.

Aquaporins Are Proteins That Form Water Channels in Certain Membranes

In certain cells (eg, red cells and cells of the collecting ductules of the kidney), the movement of water by simple diffusion is augmented by movement through water channels. These channels are composed of tetrameric transmembrane proteins named aquaporins. At least 10 distinct aquaporins (AP-1 to AP-10) have been identified. Crystallographic and other studies have revealed how these channels permit passage of water but exclude passage of ions and protons. In essence, the pores are too narrow to permit passage of ions. Protons are excluded by the fact that the oxygen atom of water binds to two asparagine residues lining the channel, making the water unavailable to participate in a H+ relay, and thus preventing entry of protons. Mutations in the gene encoding AP-2 have been shown to be the cause of one type of nephrogenic diabetes insipidus, a condition in which there is an inability to concentrate urine. Peter Agre won a Nobel Prize for his work on the structure and function of aquaporins.

ACTIVE TRANSPORT SYSTEMS REQUIRE A SOURCE OF ENERGY

The process of active transport differs from diffusion in that molecules are transported against concentration gradients; hence, energy is required. This energy can come from the hydrolysis of ATP, from electron movement, or from light. The maintenance of electrochemical gradients in biologic systems is so important that it consumes approximately 30% of the total energy expenditure in a cell.

As shown in Table 40-6, four major classes of ATP-driven active transporters (P, F, V, and ABC transporters) have been recognized. The nomenclature is explained in the legend to the Table. The first example of the P class, the Na+-K+-ATPase, is discussed below. The Ca2+ ATPase of muscle is discussed in Chapter 48. The second class is referred to as F-type. The most important example of this class is the mt ATP synthase, described in Chapter 13. V-type active transporters pump protons into lysosomes and other structures. ABC transporters include the CFTR protein, a chloride channel involved in the causation of cystic fibrosis (described later in this chapter and in Chapter 54). Another important member of this class is the multidrug resistance-1 protein (MDR-1 protein). This transporter will pump a variety of drugs, including many anti-cancer agents, out of cells. It is a very important cause of cancer cells exhibiting resistance to chemotherapy, although many other mechanisms are also implicated.

TABLE 40–6 Major Types of ATP-Driven Active Transporters

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The Na+-K+-ATPase of the Plasma Membrane Is a Key Enzyme in Regulating Intracellular Concentrations of Na+ and K+

In general, cells maintain a low intracellular Na+ concentration and a high intracellular K+ concentration (Table 40-1), along with a net negative electrical potential inside. The pump that maintains these ionic gradients is an ATPasethat is activated by Na+ and K+ (Na+- K+-ATPase; see Figure 40–18). It pumps three Na+ out and two K+ into cells. The ATPase is an integral membrane protein that contains a transmembrane domain allowing the passage of ions, and cytosolic domains that couple ATP hydrolysis to transport. It has catalytic centers for both ATP and Na+ on the cytoplasmic (inner) side of the plasma membrane (PM), with K+ binding sites located on the extracellular side of the membrane. Phosphorylation by ATP induces a conformational change in the protein leading to transfer of three Na+ ions from the inner to the outer side of the PM. Two molecules of K+ bind to sites on the protein on the external surface of the PM, resulting in dephosphorylation of the protein and transfer of the K+ ions across the membrane to the interior. Thus, three Na+ ions are transported out for every two K+ ions entering. This creates a charge imbalance between the inside and the outside of the cell, making the inside more negative (an electrogenic effect). Ouabain or digitalis (two important cardiac drugs) inhibit this ATPase by binding to the extracellular domain. This enzyme can consume ~30% of cellular energy. The Na+-K+-ATPase can be coupled to various other transporters, such as those involved in transport of glucose (see below).

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FIGURE 40–18 Stoichiometry of the Na+-K+-ATPase pump. This pump moves three Na+ ions from inside the cell to the outside and brings two K+ ions from the outside to the inside for every molecule of ATP hydrolyzed to ADP by the membrane-associated ATPase. Ouabain and other cardiac glycosides inhibit this pump by acting on the extracellular surface of the membrane. (Courtesy of R Post.)

TRANSMISSION OF NERVE IMPULSES INVOLVES ION CHANNELS AND PUMPS

The membrane enclosing neuronal cells maintains an asymmetry of inside-outside voltage (electrical potential) and is also electrically excitable due to the presence of voltage-gated channels. When appropriately stimulated by a chemical signal mediated by a specific synaptic membrane receptor (see discussion of the transmission of biochemical signals, below), channels in the membrane are opened to allow the rapid influx of Na+ or Ca2+ (with or without the efflux of K+), so that the voltage difference rapidly collapses and that segment of the membrane is depolarized. However, as a result of the action of the ion pumps in the membrane, the gradient is quickly restored.

When large areas of the membrane are depolarized in this manner, the electrochemical disturbance propagates in wavelike form down the membrane, generating a nerve impulse. Myelin sheets, formed by Schwann cells, wrap around nerve fibers and provide an electrical insulator that surrounds most of the nerve and greatly speeds up the propagation of the wave (signal) by allowing ions to flow in and out of the membrane only where the membrane is free of the insulation (at the nodes of Ranvier). The myelin membrane has a high content of lipid, accounting for its excellent insulating property. Relatively few proteins are found in the myelin membrane; those present appear to hold together multiple membrane bilayers to form the hydrophobic insulating structure that is impermeable to ions and water. Certain diseases, eg, multiple sclerosis and the Guillain-Barré syndrome, are characterized by demyelination and impaired nerve conduction.

TRANSPORT OF GLUCOSE INVOLVES SEVERAL MECHANISMS

A discussion of the transport of glucose summarizes many of the points made in this chapter. Glucose must enter cells as the first step in energy utilization. A number of different glucose transporters (GLUTs) are involved, varying in different tissues (see Table 20-2). In adipocytes and skeletal muscle, glucose enters by a specific transport system (GLUT4) that is enhanced by insulin. Changes in transport are primarily due to alterations of Vmax (presumably from more or fewer transporters), but changes in Km may also be involved.

Glucose transport in the small intestine involves some different aspects of the principles of transport discussed above. Glucose and Na+ bind to different sites on a Na+-glucose symporter located at the apical surface. Na+moves into the cell down its electrochemical gradient and “drags” glucose with it (Figure 40–19). Therefore, the greater the Na+ gradient, the more glucose enters; and if Na+ in extracellular fluid is low, glucose transport stops. To maintain a steep Na+ gradient, this Na+-glucose symporter is dependent on gradients generated by the Na+-K+-ATPase, which maintains a low intracellular Na+ concentration. Similar mechanisms are used to transport other sugarsas well as amino acids across the apical lumen in polarized cells such as are found in the intestine and kidney. The transcellular movement of glucose in this case involves one additional component: a uniport (Figure 40–19) that allows the glucose accumulated within the cell to move across the basolateral membrane and involves a glucose uniporter (GLUT2).

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FIGURE 40–19 The transcellular movement of glucose in an intestinal cell. Glucose follows Na+ across the luminal epithelial membrane. The Na+ gradient that drives this symport is established by Na+-K+ exchange, which occurs at the basal membrane facing the extracellular fluid compartment via the action of the Na+-K+-ATPase. Glucose at high concentration within the cell moves “downhill” into the extracellular fluid by facilitated diffusion (a uniport mechanism), via GLUT2 (a glucose transporter, see Table 20-2). The sodium-glucose symport actually carries 2 Na+ for each glucose.

The treatment of severe cases of diarrhea (such as is found in cholera) makes use of the above information. In cholera (see Chapter 57), massive amounts of fluid can be passed as watery stools in a very short time, resulting in severe dehydration and possibly death. Oral rehydration therapy, consisting primarily of NaCl and glucose, has been developed by the World Health Organization (WHO). The transport of glucose and Na+ across the intestinal epithelium forces (via osmosis) movement of water from the lumen of the gut into intestinal cells, resulting in rehydration. Glucose alone or NaCl alone would not be effective.

CELLS TRANSPORT CERTAIN MACROMOLECULES ACROSS THE PLASMA MEMBRANE BY ENDOCYTOSIS AND EXOCYTOSIS

The process by which cells take up large molecules is called endocytosis. Some of these molecules (eg, polysaccharides, proteins, and polynucleotides), when hydrolyzed inside the cell, yield nutrients. Endocytosis also provides a mechanism for regulating the content of certain membrane components, hormone receptors being a case in point. Endocytosis can be used to learn more about how cells function. DNA from one cell type can be used to transfect a different cell and alter the latter’s function or phenotype. A specific gene is often employed in these experiments, and this provides a unique way to study and analyze the regulation of that gene. DNA transfection depends upon endocytosis, which is responsible for the entry of DNA into the cell. Such experiments commonly use calcium phosphate since Ca2+ stimulates endocytosis and precipitates DNA, which makes the DNA a better object for endocytosis. Cells also release macromolecules by exocytosis. Endocytosis and exocytosis both involve vesicle formation with or from the plasma membrane.

Endocytosis Involves Ingestion of Parts of the Plasma Membrane

Almost all eukaryotic cells are continuously re-cycling parts of their plasma membranes. Endocytotic vesicles are generated when segments of the plasma membrane invaginate, enclosing a small volume of extracellular fluid and its contents. The vesicle then pinches off as the fusion of plasma membranes seals the neck of the vesicle at the original site of invagination (Figure 40–20). This vesicle fuses with other membrane structures and thus achieves the transport of its contents to other cellular compartments or even back to the cell exterior. Most endocytotic vesicles fuse with primary lysosomes to form secondary lysosomes, which contain hydrolytic enzymes and are therefore specialized organelles for intracellular disposal. The macromolecular contents are digested to yield amino acids, simple sugars, or nucleotides, and they are transported out of the vesicles to be reused by the cell. Endocytosis requires (1) energy, usually from the hydrolysis of ATP; (2) Ca2+; and (3) contractile elements in the cell (probably the microfilament system) (Chapter 48).

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FIGURE 40–20 Two types of pinocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase pinocytosis (A) is random and nondirected. Absorptive (receptor-mediated endocytosis) (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (brown symbols) specific for a variety of molecules. This results in the formation of an internalized coated vesicle (CV).

There are two general types of endocytosis. Phagocytosis occurs only in specialized cells such as macrophages and granulocytes. Phagocytosis involves the ingestion of large particles such as viruses, bacteria, cells, or debris. Macrophages are extremely active in this regard and may ingest 25% of their volume per hour. In so doing, a macrophage may internalize 3% of its plasma membrane each minute or the entire membrane every 30 min.

Pinocytosis (“cell drinking”) is a property of all cells and leads to the cellular uptake of fluid and fluid contents. There are two types. Fluid-phase pinocytosis is a nonselective process in which the uptake of a solute by formation of small vesicles is simply proportionate to its concentration in the surrounding extracellular fluid. The formation of these vesicles is an extremely active process. Fibroblasts, for example, internalize their plasma membrane at about one-third the rate of macrophages. This process occurs more rapidly than membranes are made. The surface area and volume of a cell do not change much, so membranes must be replaced by exocytosis or by being recycled as fast as they are removed by endocytosis.

The other type of pinocytosis, absorptive pinocytosi or receptor-mediated endocytosis, is primarily responsible for the uptake of specific macromolecules for which there are binding sites on the plasma membrane. These high-affinity receptors permit the selective concentration of ligands from the medium, minimize the uptake of fluid or soluble unbound macromolecules, and markedly increase the rate at which specific molecules enter the cell. The vesicles formed during absorptive pinocytosis are derived from invaginations (pits) that are coated on the cytoplasmic side with a filamentous material and are appropriately named coated pits. In many systems, the protein clathrinis the filamentous material. It has a three-limbed structure (called a triskelion), with each limb being made up of one light and one heavy chain of clathrin. The polymerization of clathrin into a vesicle is directed by assembly particles, composed of four adapter proteins. These interact with certain amino acid sequences in the receptors that become cargo, ensuring selectivity of uptake. The lipid phophatidylinositol 4.5-bisphosphate (PIP2) (see Chapter 15) also plays an important role in vesicle assembly. In addition, the protein dynamin, which both binds and hydrolyzes GTP, is necessary for the pinching off of clathrincoated vesicles from the cell surface. Coated pits may constitute as much as 2% of the surface of some cells. Other aspects of vesicles are discussed in Chapter 46.

As an example, the low-density lipoprotein (LDL) molecule and its receptor (Chapter 25) are internalized by means of coated pits containing the LDL receptor. These endocytotic vesicles containing LDL and its receptor fuse to lysosomes in the cell. The receptor is released and recycled back to the cell surface membrane, but the apoprotein of LDL is degraded and the cholesteryl esters metabolized. Synthesis of the LDL receptor is regulated by secondary or tertiary consequences of pinocytosis, eg, by metabolic products—such as cholesterol—released during the degradation of LDL. Disorders of the LDL receptor and its internalization are medically important and are discussed in Chapters 25 & 26.

Absorptive pinocytosis of extracellular glycoproteins requires that the glycoproteins carry specific carbohydrate recognition signals. These recognition signals are bound by membrane receptor molecules, which play a role analogous to that of the LDL receptor. A galactosyl receptor on the surface of hepatocytes is instrumental in the absorptive pinocytosis of asialoglycoproteins from the circulation (Chapter 47). Acid hydrolases taken up by absorptive pinocytosis in fibroblasts are recognized by their mannose 6-phosphate moieties. Interestingly, the mannose 6-phosphate moiety also seems to play an important role in the intracellular targeting of the acid hydrolases to the lysosomes of the cells in which they are synthesized (Chapter 47).

There is a dark side to receptor-mediated endocytosis in that viruses which cause such diseases as hepatitis (affecting liver cells), poliomyelitis (affecting motor neurons), and AIDS (affecting T cells) initiate their damage by entering cells by this mechanism. Iron toxicity also begins with excessive uptake due to endocytosis.

Exocytosis Releases Certain Macromolecules from Cells

Most cells release macromolecules to the exterior by exocytosis. This process is also involved in membrane remodeling, when the components synthesized in the ER and Golgi are carried in vesicles that fuse with the plasma membrane. The signal for exocytosis is often a hormone which, when it binds to a cell-surface receptor, induces a local and transient change in Ca2+ concentration. Ca2+ triggers exocytosis. Figure 40–21 provides a comparison of the mechanisms of exocytosis and endocytosis.

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FIGURE 40–21 A comparison of the mechanisms of endocytosis and exocytosis. Exocytosis involves the contact of two inside-surface (cytoplasmic side) monolayers, whereas endocytosis results from the contact of two outer-surface monolayers.

Molecules released by exocytosis have at least three fates. (1) They are membrane proteins and remain associated with the cell surface. (2) They can become part of the extracellular matrix, eg, collagen and glycosaminoglycans. (3) They can enter extracellular fluid and signal other cells. Insulin, parathyroid hormone, and the catecholamines are all packaged in granules and processed within cells, to be released upon appropriate stimulation.

VARIOUS SIGNALS ARE TRANSMITTED ACROSS MEMBRANES

Specific biochemical signals such as neurotransmitters, hormones, and immunoglobulins bind to specific receptors (integral proteins) exposed to the outside of cellular membranes and transmit information through these membranes to the cytoplasm. This process, called transmembrane signaling (see Chapter 42), involves the generation of a number of signaling molecules, including cyclic nucleotides, calcium, phosphoinositides, and diacylglycerol. Many of the steps involve phosphorylation of receptors and downstream proteins.

GAP JUNCTIONS ALLOW DIRECT FLOW OF MOLECULES FROM ONE CELL TO ANOTHER

Gap junctions are structures that permit direct transfer of small molecules (up to ~1200 Da) from one cell to its neighbor. They are composed of a family of proteins called connexins that form a hexagonal structure consisting of 12 such proteins. Six connexins form a connexin hemichannel and join to a similar structure in a neighboring cell to make a complete connexon channel (Figure 40–22). One gap junction contains several connexons. Different connexins are found in different tissues. Mutations in genes encoding connexins have been found to be associated with a number of conditions, including cardiovascular abnormalities, one type of deafness, and the X-linked form of Charcot-Marie-Tooth disease (a demyelinating neurologic disorder).

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FIGURE 40–22 Schematic diagram of a gap junction. One connexon is made from two hemiconnexons. Each hemiconnexon is made from six connexin molecules. Small solutes are able to diffuse through the central channel, providing a direct mechanism of cell-cell communication.

MUTATIONS AFFECTING MEMBRANE PROTEINS CAUSE DISEASES

In view of the fact that membranes are located in so many organelles and are involved in so many processes, it is not surprising that mutations affecting their protein constituents should result in many diseases or disorders. While some mutations directly affect the function of membrane proteins, the majority of mutations cause protein misfolding and impair traffic (see Chapter 46) of the membrane proteins from their site of synthesis in the ER to the plasma membrane or other intracellular sites. Examples of diseases or disorders due to abnormalities in membrane proteins are listed in Table 40-7. These mainly reflect mutations in proteins of the plasma membrane, with one affecting lysosomal function (I-cell disease).

TABLE 40-7 Some Diseases or Pathologic States Resulting From or Attributed to Abnormalities of Membranes1

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Proteins in plasma membranes can be classified as receptors, transporters, ion channels, enzymes, and structural components. Members of all of these classes are often glycosylated, so that mutations affecting this process (see Chapter 47) may alter their function. Mutations in receptors can cause defects in transmembrane signaling, a common occurrence in cancer (see Chapter 55). Many genetic diseases or disorders have been ascribed to mutations affecting various proteins involved in the transport of amino acids, sugars, lipids, urate, anions, cations, water, and vitamins across the plasma membrane.

Mutations in genes encoding proteins in other membranes can also have harmful consequences. For example, mutations in genes encoding mitochondrial membrane proteins involved in oxidative phosphorylation can cause neurologic and other problems (eg, Leber hereditary optic neuropathy; LHON, a condition in which some success with gene therapy was reported in 2008).

Membrane proteins can also be affected by conditions other than mutations. Formation of autoantibodies to the acetylcholine receptor in skeletal muscle causes myasthenia gravis. Ischemia can quickly affect the integrity of various ion channels in membranes. Overexpression of P-glycoprotein (MDR-1), a drug pump, results in multidrug resistance (MDR) in cancer cells. Abnormalities of membrane constituents other than proteins can also be harmful. With regard to lipids, excess of cholesterol (eg, in familial hypercholesterolemia), of lysophospholipid (eg, after bites by certain snakes, whose venom contains phospholipases), or of glycosphingolipids (eg, in a sphingolipidosis), can all affect membrane function.

Cystic Fibrosis Is Due to Mutations in the Gene Encoding CFTR, a Chloride Transporter

Cystic fibrosis (CF) is a recessive genetic disorder prevalent among whites in North America and certain parts of northern Europe. It is characterized by chronic bacterial infections of the airways and sinuses, fat maldigestion due to pancreatic exocrine insufficiency, infertility in males due to abnormal development of the vas deferens, and elevated levels of chloride in sweat (>60 mmol/L). In 1989, it was shown that mutations in a gene encoding a protein named cystic fibrosis transmembrane regulator protein (CFTR) were responsible for CF. CFTR is a cyclic AMP-regulated Cl transporter. The major clinical features of CF and further information about the gene responsible for CF and about CFTR are presented in Case 5, Chapter 57.

SUMMARY

Image Membranes are complex structures composed of lipids, proteins, and carbohydrate-containing molecules.

Image The basic structure of all membranes is the lipid bilayer. This bilayer is formed by two sheets of phospholipids in which the hydrophilic polar head groups are directed away from each other and are exposed to the aqueous environment on the outer and inner surfaces of the membrane. The hydrophobic nonpolar tails of these molecules are oriented toward each other, in the direction of the center of the membrane.

Image Membranes are dynamic structures. Lipids and certain proteins show rapid lateral diffusion. Flip-flop is very slow for lipids and nonexistent for proteins.

Image The fluid mosaic model forms a useful basis for thinking about membrane structure.

Image Membrane proteins are classified as integral if they are firmly embedded in the bilayer and as peripheral if they are attached to the outer or inner surface.

Image The 20 or so membranes in a mammalian cell have different compositions and functions and they define compartments, or specialized environments, within the cell that have specific functions (eg, lysosomes).

Image Certain hydrophobic molecules freely diffuse across membranes, but the movement of others is restricted because of their size or charge.

Image Various passive and active (usually ATP-dependent) mechanisms are employed to maintain gradients of such molecules across different membranes.

Image Certain solutes, eg, glucose, enter cells by facilitated diffusion along a downhill gradient from high to low concentration using specific carrier proteins (transporters).

Image The major ATP-driven pumps are classified as P (phosphorylated), F (energy factors), V (vacuolar), and ABC transporters. Member of these classes include the Na+-K+-ATPase and the Ca2+ ATPase of the sarcoplasmic reticulum; the mt ATP synthase; the ATPase acidifying lysosomes; and the CFTR protein and the MDR-1 protein.

Image Ligand-or voltage-gated ion channels are often employed to move charged molecules (Na+, K+, Ca2+, etc.) across membranes down their electrochemical gradients

Image Large molecules can enter or leave cells through mechanisms such as endocytosis or exocytosis. These processes often require binding of the molecule to a receptor, which affords specificity to the process.

Image Receptors may be integral components of membranes (particularly the plasma membrane). The interaction of a ligand with its receptor may not involve the movement of either into the cell, but the interaction results in the generation of a signal that influences intracellular processes (transmembrane signaling).

Image Mutations that affect the structure of membrane proteins (receptors, transporters, ion channels, enzymes, and structural proteins) may cause diseases; examples include cystic fibrosis and familial hypercholesterolemia.

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