15

Transport across Cell Membranes

Three-dimensional structure of a gap junction membrane channel connecting two adjacent cells.

he plasma membrane is a selectively permeable barrier between the cell and the extracellular environment. Its permeability properties ensure that essential molecules such as glucose, amino acids, and lipids readily enter the cell, metabolic intermediates remain in the cell, and waste compounds leave the cell. In short, the selective permeability of the plasma membrane allows the cell to maintain a constant internal environment. In several earlier chapters, we examined the components and structural organization of cell membranes (see Figures 3-32 and 5-30). The phospholipid bilayerthe basic structural unit of biomembranesis essentially impermeable to most water-soluble molecules, such as glucose and amino acids, and to ions. Transport of such molecules and ions across all cellular membranes is mediated by transport proteins associated with the underlying bilayer. Because different cell types require different mixtures of low-molecular-weight compounds, the plasma membrane of each cell type contains a specific set of transport proteins that allow only certain ions or molecules to cross. Similarly, organelles within the cell often have a different internal environment from that of the surrounding cytosol, and organelle membranes contain specific transport proteins that maintain this difference. In animals, sheets of epithelial cells line all the body cavities (e.g., the stomach, intestines, urinary bladder) and the skin (see Figure 6-4). Epithelial cells frequently transport ions or small molecules from one side to the other. Those lining the small intestine, for instance, transport products of digestion (e.g., glucose and amino acids)

OUTLINE

15.1 Diffusion of Small Molecules across Phospholipid Bilayers 579 15.2 Overview of Membrane Transport Proteins 580 15.3 Uniporter-Catalyzed Transport 582 15.4 Intracellular Ion Environment and Membrane Electric Potential 15.5 Active Transport by ATP-Powered Pumps 588

15.6 Cotransport by Symporters and Antiporters 597 15.7 Transport across Epithelia 602 608 15.8 Osmosis, Water Channels, and the Regulation of Cell Volume

MEDIA CONNECTIONS
585 Overview: Biological Energy Interconversions Classic Experiment 15.1: Stumbling upon Active Transport
MOHRIG: EXPER. ORGANIC CHEM. Figure: CD ICON 100% of size Fine Line Illustrations (516) 781-7200 5/29/97 6/9/97

Diffusion of Small Molecules across Phospholipid Bilayers

579

into the blood, and those lining the stomach secrete hydrochloric acid into the stomach lumen. In order for epithelial cells to carry out these transport functions, their plasma membrane must be organized into at least two discrete regions, each with different sets of transport proteins. In addition, specialized regions of the plasma membrane interconnect epithelial cells, imparting strength and rigidity to the sheet and preventing material on one side from moving between the cells to the other. In the first two sections of this chapter, we discuss the protein-independent movement of small hydrophobic molecules across phospholipid bilayers and present an overview of the various types of transport proteins present in cell membranes. We then describe each of the main types of transport proteins. We also explain how specific combinations of transport proteins in different subcellular membranes enable cells to carry out essential physiological processes, including the maintenance of cytosolic pH, the transport of glucose across the absorptive intestinal epithelium, the accumulation of sucrose and salts in plantcell vacuoles, and the directed flow of water in both plants and animals. Often the same type of transport protein is involved in quite different physiological processes.

Gases

CO2 N2 O2

Small uncharged polar molecules Water Urea Large uncharged polar molecules Ions

Ethanol

H 2O NH2

C O

NH2

Glucose K +, Mg2 +, Ca2 +, Cl , HCO3, HPO42 Amino acids ATP Glucose 6-phosphate

Charged polar molecules

15.1

Diffusion of Small Molecules across Phospholipid Bilayers

An artificial membrane composed of pure phospholipid or of phospholipid and cholesterol is permeable to gases, such as O2 and CO2, and small, uncharged polar molecules, such as urea and ethanol (Figure 15-1). Such molecules also can cross cellular membranes by passive diffusion unaided by transport proteins. No metabolic energy is expended because movement is from a high to a low concentration of the molecule, down its chemical concentration gradient. As noted in Chapter 2, such transport reactions have a positive S value (increase in entropy) and a negative G (decrease in free energy). The relative diffusion rate of a substance across the bilayer is proportional to its concentration gradient across the layer and to its hydrophobicity. There is little specificity to the process, in that any small hydrophobic molecule will be transported. The first step in transport by passive diffusion is movement of a molecule from the aqueous solution into the hydrophobic interior of the phospholipid bilayer. The hydrophobicity of a substance is measured by its partition coefficient, K, the equilibrium constant for its partition between oil and water. Since the composition of the interior of the phospholipid bilayer resembles that of oil, the partition coefficient of a substance moving across a bilayer equals the ratio of its concentration just inside the hydrophobic core of the bilayer C m to its concentration in the aqueous solution C aq:

v FIGURE 15-1 A pure artificial phospholipid bilayer is permeable to small hydrophobic molecules and small uncharged polar molecules. It is slightly permeable to water and urea and impermeable to ions and to large uncharged polar molecules. When a small phospholipid bilayer separates two aqueous compartments, membrane permeability can be easily determined by adding a small amount of radioactive material to one compartment and measuring its rate of appearance in the other compartment.

(15-1)

The partition coefficient is a measure of the relative affinity of a substance for lipid versus water: the higher a substances partition coefficient, the more lipid-soluble it is. For example, urea

has a K of 0.0002, whereas diethylurea (with two ethyl groups)

has a K of 0.01. Diethylurea, which is 50 times (0.01 0.0002) more hydrophobic than urea, will diffuse through

580

CHAPTER 15

Transport across Cell Membranes

phospholipid bilayer membranes about 50 times faster than urea. Diethylurea also enters cells about 50 times faster than urea. Once a molecule moves into the hydrophobic interior of a bilayer, it diffuses across it; finally, the molecule moves from the bilayer into the aqueous medium on the other side of the membrane. Because the hydrophobic core of a typical cell membrane is 1001000 times more viscous than water, the diffusion rate of all substances across a phospholipid membrane is very much slower than the diffusion rate of the same molecule in water. Thus, movement across the hydrophobic portion of a membrane is the rate-limiting step in the passive diffusion of molecules across cell membranes. Now lets consider the passive diffusion of small molecules through a membrane more quantitatively. Suppose a membrane of surface area A and thickness x separates two solutions of concentrations C1aq and C2aq, where C1aq C2aq (Figure 15-2). In this case, the diffusion rate dn/dt (in mol/s) is given by a modification of Ficks law, which states that the diffusion rate across the membrane is directly proportional to the permeability coefficient P, to the difference in solution concentrations C1aq C2aq, and to the area A, or
(15-2)

C1aq

C1m

C2m

C2aq

v FIGURE 15-2 A simple model for passive diffusion of small hydrophobic molecules directly across the hydrocarbon core of a pure phospholipid bilayer of thickness x in centimeters and area A in square centimeters. C1aq and C2aq are the concentrations of two solutions on sides 1 and 2 of the membrane; C1m and C2m are the corresponding concentrations just within the hydrocarbon core of the bilayer. Movement of a solute molecule is indicated by the blue arrow.

For any molecule, the value of P, and thus its rate of passive diffusion, is proportional to its partition coefficient K:
(15-3)

15.2

Overview of Membrane Transport Proteins

where D is the diffusion coefficient of the substance within the membrane and x is the membrane thickness. By substituting Equation 15-3 into 15-2, we obtain

Thus we can see that the rate of diffusion is proportional to both the partition coefficient and the diffusion constant and is inversely proportional to the membrane thickness. However, the thickness of the hydrophobic interior of all phospholipid bilayer membranes is approximately the same, about 2.5 to 3 nm, and the diffusion coefficient D is the same for most substances. Thus differences in the rate at which molecules passively diffuse across membranes depends largely on differences in their partition coefficients. The greater the hydrophobicity of a water-soluble molecule, the faster it diffuses across a phospholipid bilayer. Gases and some small, uncharged molecules, such as ethanol and urea, enter and leave cells by passive diffusion across the plasma membrane. This transport is described by Ficks law. In the following sections, we will see how movement of other molecules and ions across cell membranes differs from simple diffusion.

Very few molecules enter or leave cells, or cross organellar membranes, unaided by proteins. Even transport of molecules, such as water and urea, that can diffuse across pure phospholipid bilayers is frequently accelerated by transport proteins. The three major classes of membrane transport proteins are depicted in Figure 15-3a. All are integral transmembrane proteins and exhibit a high degree of specificity for the substance transported. The rate of transport by the three types differs considerably owing to differences in their mechanism of action. ATP-powered pumps (or simply pumps) are ATPases that use the energy of ATP hydrolysis to move ions or small molecules across a membrane against a chemical concentration gradient or electric potential. This process, referred to as active transport, is an example of a coupled chemical reaction (Chapter 2). In this case, transport of ions or small molecules uphill against a concentration gradient or electric potential across a membrane, which requires energy, is coupled to the hydrolysis of ATP to ADP and P i, which releases energy. The overall reactionATP hydrolysis and the uphill movement of ions or small moleculesis energetically favorable. Such pumps maintain the low calcium (Ca2) and sodium (Na ) ion concentrations inside virtually all animal cells relative to that in the medium, and generate the low pH inside animal-cell lysosomes, plant-cell vacuoles, and the lumen of the stomach.

Overview of Membrane Transport Proteins (a) Exterior

581

Cytosol ATP ADP + Pi

Closed Open

ATP-powered pump (100 103 ions/s)

Ion channel (107 108 ions/s)

Transporter (102 104 molecules/s)

(b)

Uniporter

Symporter

Antiporter

v FIGURE 15-3 Schematic diagrams illustrating action of membrane transport proteins. Gradients are indicated by triangles with the tip pointing toward lower concentration, electrical potential, or both. (a) The three major types of transport proteins. Pumps utilize the energy released by ATP hydrolysis to power movement of specific ions (red circles) or small molecules against their electrochemical gradient. Channels catalyze movement of specific ions (or water) down their electrochemical gradient. Transporters, which fall into three groups, facilitate movement of specific small molecules or ions (black circles). (b) The three groups of transporters. Uniporters, also shown in part (a), transport a single type of molecule down its concentration gradient. Cotransport proteins (symporters and antiporters) catalyze the movement of one molecule against its concentration gradient (black circles), driven by movement of one or more ions down an electrochemical gradient (red circles). The two types of cotransporters differ in the relative direction of movement of the transported molecule and cotransported ion.

bind only one (or a few) substrate molecules at a time; after binding substrate molecules, the transporter undergoes a conformational change such that the bound substrate molecules, and only these molecules, are transported across the membrane. Because movement of each substrate molecule (or small number of molecules) requires a conformational change in the transporter, transporters move only about 102 104 molecules per second, a lower rate than that associated with channel proteins. Three types of transporters have been identified (Figure 15-3b). Uniporters transport one molecule at a time down a concentration gradient. This type of transporter, for example, moves glucose or amino acids across the plasma membrane into mammalian cells. In contrast, antiporters and symporters couple the movement of one type of ion or molecule against its concentration gradient to the movement of a different ion or molecule down its concentration gradient. Like ATP pumps, antiporters and symporters mediate coupled reactions in which an energetically unfavorable reaction is coupled to an energetically favorable reaction. Because symporters and antiporters catalyze uphill movement of certain molecules, they are often referred to as active transporters, but unlike pumps, they do not hydrolyze ATP (or any other molecule) during transport. A better term for these proteins is cotransporters, referring to their ability to transport two different solutes simultaneously. To study the functional properties of the different kinds of membrane-transport proteins, researchers need experimental systems in which a particular transport protein predominates. In one common approach, a specific transport protein is extracted and purified; the purified protein then is reincorporated into pure phospholipid bilayer membranes, such as liposomes (Figure 15-4). Alternatively, the gene encoding a transport protein can be expressed at high levels in a cell normally not expressing it; the difference in transport of a substance by the transfected and nontransfected cells will be due to the expressed transport protein. In these systems, the functional properties of the various membrane proteins can be examined without ambiguity.

Channel proteins transport water or specific types of ions down their concentration or electric potential gradients, an energetically favorable reaction. They form a protein-lined passageway across the membrane through which multiple water molecules or ions move simultaneously, single file at a very rapid rateup to 108 per second. As discussed in a later section, the plasma membrane of all animal cells contains potassium-specific channel proteins that are generally open and are critical to generating the normal, resting electric potential across the plasma membrane. Many other types of channel proteins are usually closed, and open only in response to specific signals. Because these types of ion channels play a fundamental role in the functioning of nerve cells, they will be discussed in detail in Chapter 21. Transporters, a third class of membrane transport proteins, move a wide variety of ions and molecules across cell membranes. In contrast to channel proteins, transporters

S U M M A R Y Overview of Membrane Transport Proteins

The plasma membrane regulates the traffic of molecules into and out of the cell. Gases and small hydrophobic molecules diffuse directly across the phospholipid bilayer at a rate proportional to their ability to dissolve in a liquid hydrocarbon. Ions, sugars, amino acids, and sometimes water cannot diffuse across the phospholipid bilayer at sufficient rates to meet the cells needs and must be transported by a group of integral membrane proteins including channels, transporters, and ATP-powered ion pumps (see Figure 15-3).

582

CHAPTER 15

Transport across Cell Membranes

Glucose transport protein

Other transport protein

Intact erythrocyte membrane

Disrupt membrane, solubilize protein with detergents, and purify

Detergent molecules

Phospholipids Mix with phospholipids

Glucose

Dialyze or dilute to remove detergent

Glucose

Liposome with glucose transport protein

Two common experimental systems for studying the functions of transport proteins are liposomes containing a purified transport protein (see Figure 15-4) and cells transfected with the gene encoding a particular transport protein.

15.3

Uniporter-Catalyzed Transport

We begin our discussion of membrane transport proteins with the simplest type, which catalyze uniport transport. The plasma membrane of most cells contains several uniporters that enable amino acids, nucleosides, sugars, and other small molecules to enter and leave cells down their concentration gradients. Similar to enzymes, uniporters accelerate a reaction that is already thermodynamically favored, and the

FIGURE 15-4 Liposomes containing a single type of transport protein can be used to investigate properties of the transport process. Here, all the integral proteins of the erythrocyte membrane are solubilized by a nonionic detergent, such as octylglucoside. The glucose transport protein, a uniporter, can be purified by chromatography on a column containing a specific monoclonal antibody and then incorporated into liposomes made of pure phospholipids.

movement of a substance across a membrane down its concentration gradient will have the same negative G value whether or not a protein transporter is involved. This type of movement sometimes is referred to as facilitated transport (or facilitated diffusion). As we stressed in Chapter 2, many chemical reactions that are thermodynamically favored will not occur unless an appropriate enzyme is present; such is also the case with movement of hydrophilic molecules across biological membranes. Unlike the substrates of enzymatic reactions, however, transported substances undergo no chemical change during movement across a membrane.

Three Main Features Distinguish Uniport Transport from Passive Diffusion

Three properties of uniporter-catalyzed movement of glucose and other small hydrophilic molecules across a membrane distinguish this type of transport from passive diffusion: 1. The rate of facilitated transport by uniporters is far higher than predicted by Ficks equation describing passive diffusion (Figure 15-5). Because the transported molecules never enter the hydrophobic core of the phospholipid bilayer, the partition coefficient K is irrelevant. 2. Transport is specific. Each uniporter transports only a single species of molecule or a single group of closely related molecules. 3. Transport occurs via a limited number of uniporter molecules, rather than throughout the phospholipid bilayer. Consequently, there is a maximum transport rate Vmax that is achieved when the concentration gradient across the membrane is very large and each uniporter is working at its maximal rate. Figure 15-5 shows the initial rate of glucose uptake by erythrocytes at different external glucose concentrations. Since the concentration of glucose is usually higher in the extracellular medium than in the cell, the plasma-membrane glucose transporters usually catalyze net movement of glucose in one direction: from the medium into the cell. Under this condition, Vmax is achieved at high external glucose concentrations. However, if the concentration gradient is reversed, the glucose transporter, like all uniporters, is equally able to catalyze net movement in the reverse direction: from the cell into the medium. Such a situation occurs in liver cells during periods of starvation, when these cells synthe-

Uniporter-Catalyzed Transport

583

500 Rate of glucose uptake (v )

Vmax

Facilitated transport

250

1/2V

max

v FIGURE 15-6 Normal human erythrocytes, viewed by

Passive diffusion 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 External concentration of glucose (mM)

differential interference light microscopy, are disk shaped and contain no internal membranes. The opposite surface also is concave. [Courtesy of M. Murayama, Biological Photo Service.]

Km

v FIGURE 15-5 Comparison of the observed uptake rate of

glucose by erythrocytes (red curve) with the calculated rate if glucose were to enter solely by passive diffusion through the phospholipid bilayer (blue curve). The rate of glucose uptake (measured as micromoles per milliliter of cells per hour) in the first few seconds is plotted against the glucose concentration in the extracellular medium. In this experiment the initial concentration of glucose in the erythrocyte is zero, so that the concentration gradient of glucose across the membrane is the same as the external concentration. The glucose transporter in the erythrocyte membrane clearly increases the rate of glucose transport, compared with that associated with passive diffusion, at all glucose concentrations. Like enzymes, the transporter-catalyzed uptake of glucose exhibits a maximum transport rate Vmax and is said to be saturable. The Km is the concentration at which the rate of glucose uptake is half-maximal.

ing site faces the inside. Figure 15-7 depicts the sequence of events occurring during the unidirectional transport of glucose from the cell exterior inward to the cytosol. GLUT1 also can catalyze the net movement of glucose from the cytosol outward by reversal of steps 14 shown in Figure 15-7. Experimental support for this model, which is thought to apply to other uniport proteins as well, has come from kinetic experiments discussed below.
Kinetics of GLUT1-Catalyzed Movement of Glucose As noted previously, a plot of the entry rate of glucose into erythrocytes versus external glucose concentration is not linear; rather, it is a curve that levels off at Vmax at high external glucose concentrations (see Figure 15-5). The kinetics of the unidirectional transport of glucose (and other small molecules) from the outside of a cell inward via a uniporter can be described by the same type of equation used to describe a simple enzyme-catalyzed chemical reaction. For simplicity, lets assume that a substance S (say, glucose) is present initially only on the outside of the membrane. In this case, we can write

size glucose (from fatty acids, amino acids, and other small molecules) and release it into the blood, and in intestinal epithelial cells during transport of glucose from the intestine to the blood.

GLUT1 Transports Glucose into Most Mammalian Cells

Virtually all mammalian cells use blood glucose as the major source of cellular energy, and most express GLUT1, a plasmamembrane uniporter that catalyzes movement of glucose down its concentration gradient. The properties of GLUT1, as well as of many other transport proteins, have been extensively studied in the mammalian erythrocyte, since this cell has no nucleus and no internal membranes; it is essentially a bag of hemoglobin containing relatively few other intracellular proteins (Figure 15-6). We discuss GLUT1 in some detail as an example of the uniport type of transport protein. The glucose transporter GLUT1 alternates between two conformational states: in one, a glucose-binding site faces the outside of the membrane; in the other, a glucose-bindwhere Km is the substance-transporter binding constant and Vmax is the maximum transport rate of S into the cell. By a similar derivation used to arrive at the Michaelis-Menten equation in Chapter 3, we can derive the following expression for v, the transport rate for S into the cell:
(15-4)

where C is the concentration of Sout (initially, the concentration of Sin 0); Vmax is the rate of transport if all molecules of the transporter contain a bound S, which occurs at high

584

CHAPTER 15 Glucose

Transport across Cell Membranes

Outward-facing glucose-binding site Exterior Plasma membrane Cytosol 1 2

Bound glucose

Inward-facing glucose-binding site

Glucose

v FIGURE 15-7 Model of the mechanism of uniport transport by GLUT1, which is believed to shuttle between two conformational states. In one conformation ( 1, 2 , and 5 ), the glucose-binding site faces outward; in the other ( 3 , 4 ), the binding site faces inward. Binding of glucose to the outward-facing binding site ( 1 n 2 ) triggers a conformational change in the transporter ( 2 n 3 ), moving the bound glucose through the protein such that it is now bound to the inward-facing binding

site. Glucose can then be released to the inside of the cell ( 3 n 4 ). Finally, the transporter undergoes the reverse conformational change ( 4 n 5 ), inactivating the inward-facing glucose binding site and regenerating the outward-facing one. If the concentration of glucose is higher inside the cell than outside, the cycle will work in reverse ( 4 n 1 ), catalyzing net movement of glucose from inside to out.

Sout concentrations; and Km is the substrate concentration at which half-maximal transport occurs across the membrane. The lower the value of Km, the more tightly the substrate binds to the transporter, and the greater the transport rate. Equation 15-4 describes the curve for glucose uptake shown in Figure 15-5. For GLUT1 in the erythrocyte membrane, the Km for glucose transport is 1.5 millimolar (mM); at this concentration roughly half the transporters with outward-facing binding sites would have a bound glucose. Blood glucose is normally 5 mM, or 0.9 g/L. At this concentration, the erythrocyte glucose transporter is functioning at 77 percent of the maximal rate Vmax, as can be seen from Figure 15-5. The kinetics of glucose transport are more complex and more revealing than this simple analysis suggests. For instance, if [14C]glucose is added to a suspension of erythrocytes whose intracellular glucose concentration is zero, the labeled glucose is transported inward at a particular initial rate proportional to the concentration of labeled glucose, as described by Equation 15-4. This initial rate of [14C]glucose transport is accelerated severalfold if unlabeled glucose is present inside the cells before addition of the labeled glucose. This unexpected experimental observation indicates that the slow (rate-determining) step in the inward transport of glucose is the change in GLUT1 from a conformation with an unoccupied inward-facing glucose-binding site to a conformation with an unoccupied outward-facing binding site (step 4 n step 5 in Figure 15-7). This conformational change is accelerated severalfold when an unlabeled glucose molecule binds to the inward-facing site and is transported outward. This result adds strong support to the conformationalchange model of GLUT1 depicted in Figure 15-7.
Specificity and Structure of GLUT1 As noted above, the

Km for glucose transport by GLUT1 is 1.5 mM. The Km for the nonbiological L-isomer of glucose is 3000 mM; thus at concentrations at which D-glucose is readily transported

into the erythrocyte, L-glucose does not enter at a measurable rate. The isomeric sugars D-mannose and D-galactose, which differ from D-glucose in the configuration at only one carbon atom (see Figure 2-8), also are transported by GLUT1 at measurable rates. However, the Km for D-mannose is 20 mM and for D-galactose is 30 mM, so that considerably higher concentrations of these substrates than of D-glucose are needed to half-saturate the transport reaction. Thus GLUT1 is quite specific, having a much higher affinity (indicated by a lower Km) for the normal substrate D-glucose than for other substrates. After glucose is transported into the erythrocyte, it is rapidly phosphorylated, forming glucose 6-phosphate, which cannot leave the cell (see Figure 16-3). Because this reaction is the first step in the metabolism of glucose, the intracellular concentration of free glucose does not increase as glucose is taken up by the cell. Consequently, the glucose concentration gradient across the membrane is maintained, as is the rate of glucose entry into the cell. GLUT1 is an integral, transmembrane protein with a molecular weight of 45,000. It accounts for 2 percent of the protein in the plasma membrane of erythrocytes. Insertion of purified GLUT1 into artificial liposomes dramatically increases their permeability to D-glucose (see Figure 15-4). This artificial system exhibits all the properties of glucose entry into erythrocytes: in particular, D-glucose, D-mannose, and D-galactose are taken up, but L-glucose is not. Amino acid sequence and biophysical studies on the glucose transporter indicate that it contains 12 helices that span the phospholipid bilayer. Although the amino acid residues in the transmembrane helices are predominantly hydrophobic, several helices bear amino acid residues (e.g., serine, threonine, asparagine, and glutamine) whose side chains can form hydrogen bonds with the hydroxyl groups on glucose. These residues are thought to form the inward-facing and outwardfacing glucose-binding sites in the interior of the protein.

Intracellular Ion Environment and Membrane Electric Potential

585

S U M M A R Y Uniporter-Catalyzed Transport

Uniport-type membrane transport proteins operate to import many types of molecules into the cell driven only by a concentration gradient, a process termed facilitated transport or facilitated diffusion. Three main features distinguish uniport transport from passive diffusion: the rate of transport is far higher than predicted by Ficks equation, transport is specific, and transport occurs via a limited number of transporter proteins rather than throughout the phospholipid bilayer. The kinetics of uniporter-catalyzed transport reactions, similar to those of simple enzyme-catalyzed reactions, are characterized by a Km and a Vmax (see Figure 15-5). The glucose transporter GLUT1, a uniport protein in the plasma membrane of most mammalian cells, allows only glucose and closely related sugars to cross the bilayer down their concentration gradients. GLUT1 shuttles between two conformational states, one in which the glucose-binding site faces outward and one in which the binding site faces inward (see Figure 15-7). Transport by other uniporters is thought to involve a similar conformational-change mechanism.

15.4

Intracellular Ion Environment and Membrane Electric Potential

2040 times higher in cells than in the blood, while the concentration of Na is 812 times lower in cells than in the blood (Table 15-1). The concentration of Ca2 free in the cytosol is generally less than 0.2 micromolar (2 107 M), a thousand or more times lower than that in the blood. Plant cells and many microorganisms maintain similarly high cytosolic concentrations of K and low concentrations of Ca2 and Na even if the cells are cultured in very dilute salt solutions. The ATP-driven ion pumps that generate and maintain these ionic gradients are discussed later. In addition to ion pumps, which transport ions against their concentration gradients, the plasma membrane contains channel proteins that allow the principal cellular ions (Na, K, Ca2, and Cl) to move through it at different rates down their concentration gradients. Ion concentration gradients and selective movements of ions through channels create a difference in voltage across the plasma membrane. The magnitude of this electric potential is 70 millivolts (mV) with the inside of the cell always negative with respect to the outside. This value does not seem like much until we realize that the plasma membrane is only about 3.5 nm thick. Thus the voltage gradient across the plasma membrane is 0.07 V per 3.5 107 cm, or 200,000 volts per centimeter! (To appreciate what this means, consider that high-voltage transmission lines for electricity utilize gradients of about 200,000 volts per kilometer!) As explained below, the plasma membrane, like all biological membranes, acts like a capacitor a device consisting of a thin sheet of nonconducting material (the hydrophobic interior) surrounded on both sides by electrically conducting material (the polar head

TABLE 15-1

Typical Ion Concentrations in Invertebrates and Vertebrates Cell (mM) 400 50 40150 0.0003 300400 139 12 4 12 138 0.8 0.0002 Blood (mM) 20 440 560 10 510 4 145 116 29 9 1.5 1.8

The movement of ions across the plasma membrane and organelle membranes is mediated by several types of transport proteins: all symporters and certain antiporters cotransport ions simultaneously along with specific small molecules, whereas ion channels, ion pumps, and some antiporters transport only ions. In all cases, the rate and extent of ion transport across membranes is influenced not only by the ion concentrations on the two sides of the membrane but also by the voltage (i.e., the electric potential) that exists across the membrane. Here we discuss the origin of the electric potential across the plasma membrane and its relationship to ion channels within the membrane.

Ion SQUID AXON* K Na Cl Ca2 X MAMMALIAN CELL K Na Cl HCO3 X Mg2 Ca2

Ionic Gradients and an Electric Potential Are Maintained across the Plasma Membrane
The specific ionic composition of the cytosol usually differs greatly from that of the surrounding fluid. In virtually all cellsincluding microbial, plant, and animal cellsthe cytosolic pH is kept near 7.2 and the cytosolic concentration of K is much higher than that of Na. In addition, in both invertebrates and vertebrates, the concentration of K is

*The large nerve axon of the squid, an invertebrate cell, has been widely used in studies of the mechanism of conduction of electric impulses. X represents proteins, which have a net negative charge at the neutral pH of blood and cells.

586

CHAPTER 15

Transport across Cell Membranes

(a) Membrane impermeable to Na+, K +, and Cl (a) Membrane impermeable to Na+, K +, and Cl Potentiometer 0 (a) Membrane impermeable to Na+, K +, and Cl Potentiometer 0 Zero potential 0 + 60Potentiometer 60 difference Zero potential + 60 60 difference Zero potential + 60 60 difference 15 mM Cl Na+ 15 mM + Cl Na 15 mM 150 mM + Cl Na K + Cl 150 mM + K Cl 150 mM K + Cl Cell cytosol Cell cytosol Cell cytosol 150 mM Na+ Cl 150 mM + Cl Na 150 mM 15 + mM Na Cl K + Cl 15 mM + K Cl 15 mM K + Cl Extracellular medium Extracellular medium Extracellular medium

groups and the ions in the surrounding aqueous medium) that can store positive charges on one side and negative charges on the other. The ionic gradients and electric potential across the plasma membrane drive many biological processes. Opening and closing of Na, K, and Ca2 channels are essential to the conduction of an electric impulse down the axon of a nerve cell (Chapter 21). In many animal cells, the Na concentration gradient and the membrane electric potential power the uptake of amino acids and other molecules against their concentration gradient; this transport is catalyzed by ionlinked symport and antiport proteins. In most cells, a rise in the cytosolic Ca2 concentration is an important regulatory signal, initiating contraction in muscle cells and triggering secretion of digestive enzymes in the exocrine pancreatic cells. Here we discuss the role of ion channels in generating the membrane electric potential. Later we examine the ATPpowered ion pumps that generate ion concentration gradients, and ion-linked cotransport proteins.

The Membrane Potential in Animal Cells Depends Largely on Resting K Channels

In the experimental system outlined in Figure 15-8a, the distribution of K, Na, and Cl ions is similar to that between an animal cell and its aqueous environment. A membrane separates a 15 mM KCl/150 mM NaCl solution on the right side (representing the outside of the cell) from a 150 mM KCl/15 mM NaCl solution on the left side (the inside). A potentiometer (voltmeter) is connected to the solution on each side to measure any difference in electric potential across the membrane. If the membrane is impermeable to all ions, no ions will flow across it; there will be no electric potential across it.

(b) Membrane permeable to Na+ only (b) Membrane 0 permeable to Na+ only (b) Membrane permeable to Na+ only 0 At equilibrium, potential is 59 mV, 0 + 60 At with the right side negative 60 equilibrium, potential is 59 mV, respect to thenegative left is 59 mV, + 60 At with the right side equilibrium, potential 60 respect to thenegative left + 60 with the right side 60 with respect to the left 15 mM Na+ Cl 15 mM + Cl Na 15 mM 150 mM + Cl Na K + Cl 150 mM + K Cl 150 mM K + Cl 150 mM Na+ Cl 150 mM + Cl Na 150 mM 15 + mM Na Cl K + Cl 15 mM + K Cl 15 mM + + Cl KNa Na+ + Na Na+ Na+ Na+ Na+ Na+ Na+

FIGURE 15-8 Experimental system for generating a transmembrane voltage potential across a membrane separating a 150 mM KCl/15 mM NaCl solution (a similar composition to that of the cell cytosol) from a 15 mM KCl/150 mM NaCl solution (concentrations similar to those in blood). (a) An impermeable membrane prevents ion movement across the membrane, and thus no difference in electric potential is registered on the potentiometer connecting the two solutions. (b) If the membrane is selectively permeable only to Na, then Na ions diffuse from right to left, through Na channels. As a consequence, a net positive charge builds up on the left side and a net negative charge builds up on the right side of the membrane. At equilibrium, the membrane potential caused by the charge separation becomes equal to the Nernst potential ENa registered on the potentiometer, and the movement of Na ions in the two directions becomes equal. (c) If the membrane is selectively permeable only to K, diffusion of K ions from left to right through K channels causes accumulation of a net negative charge on the left side and a net positive charge on the right side. At equilibrium, the membrane electric potential is equal to EK.

Na+ channel + Na+ channel + + + Na+ channel + + + + Net + charge Net charge Net charge (c) Membrane permeable to K + only (c) Membrane 0 permeable to K + only (c) Membrane permeable to K + only 0 At equilibrium, potential is +59 mV, 0 with the right side positive + 60 At 60 equilibrium, potential is +59 mV, respect to thepositive left is +59 mV, with the right side + 60 At equilibrium, potential 60 respect to thepositive left right side + 60 with the 60 with respect to the left 15 mM Na+ Cl 15 mM + Cl Na 15 mM 150 mM + Cl Na K + Cl 150 mM + K Cl 150 mM + K + ClK K+ K+ K+ K+ K+ K+ K+ K+ 150 mM Na+ Cl 150 mM + Cl Na 150 mM 15 + mM Na Cl K + Cl 15 mM + K Cl 15 mM K + Cl

+ + + + + + + + Net charge + Net charge Net charge

K + channel K + channel K + channel

Intracellular Ion Environment and Membrane Electric Potential

587

Now suppose that the membrane contains Na-channel proteins that accommodate Na ions but exclude K and Cl ions. Na ions then tend to move down their concentration gradient from the right side to the left, leaving an excess of negative Cl ions compared with Na ions on the right side and generating an excess of positive Na ions compared with Cl ions on the left side. The excess Na on the left and Cl on the right remain near the respective surfaces of the membrane, since, as in a capacitor, the excess positive charges on one side of the membrane are attracted to the excess negative charges on the other side. The resulting separation of charge across the membrane can be measured by a potentiometer as an electric potential, or voltage, with the right side of the membrane negative (having excess negative charge) with respect to the left (Figure 15-8b). As more and more Na ions move through channels across the membrane, the magnitude of this charge difference (i.e., voltage) increases. However, continued right-to-left movement of the Na ions eventually is inhibited by the mutual repulsion between the excess positive (Na) charges accumulated on the left side of the membrane and by the attraction of Na ions to the excess negative charges built up on the right side. The system soon reaches an equilibrium point at which the two opposing factors that determine the movement of Na ionsthe membrane electric potential and the ion concentration gradientbalance each other out. At equilibrium, no net movement of Na ions occurs across the membrane. Thus the excess negative (Cl) charges bound to the right surface of the membrane are separated from and attracted to the excess positive (Na) ones on the left. In this way, the phospholipid membrane, with its nonconducting hydrophobic interior bounded by the conducting polar head groups and adjacent aqueous medium, stores the charge across it exactly as does a capacitor in an electric circuit. If a membrane is permeable only to Na ions, then the measured electric potential across the membrane equals the sodium equilibrium potential in volts, ENa. The magnitude of ENa is given by the Nernst equation, which is derived from basic principles of physical chemistry:
(15-5)

If [Nal]/[Nar] 0.1, as in Figure 15-8b, then ENa 0.059 V (59 mV), with the right side negative with respect to the left. If the membrane is permeable only to K ions and not to Na or Cl ions, then a similar equation describes the potassium equilibrium potential EK:
(15-7)

where R (the gas constant) 1.987 cal/(degree mol), or 8.28 joules/(degree mol); T (the absolute temperature) 293 K at 20 C, Z (the valency) 1, F (the Faraday constant) 23,062 cal/(mol V), or 96,000 coulombs/(mol V), and [Nal] and [Nar] are the Na concentrations on the left and right sides, respectively, at equilibrium. The Nernst equation is similar to the equations used to calculate the voltage change associated with oxidation or reduction reactions (Chapter 2), which also involve movement of electric charges. At 20 C, Equation 15-5 reduces to
(15-6)

The magnitude of the membrane electric potential is the same (59 mV), except that the right side is now positive with respect to the left (Figure 15-8c), opposite to the polarity obtained with selective Na permeability. As noted earlier, the membrane potential across the plasma membrane of animal cells is about 70 mV; that is, the cytosolic face is negative with respect to the exoplasmic (outside) face. These membranes contain many open K channels but few open Na or Ca2 channels. As a result, the major ionic movement across the plasma membrane is that of K from the inside outward, leaving an excess of negative charge on the inside and creating an excess of positive charge on the outside. Thus the flow of K ions through these open channels, called K leak channels or resting K channels, is the major determinant of the inside-negative membrane potential. Quantitatively, the usual resting membrane potential of 70 mV is close to but less than that of the potassium equilibrium potential calculated from the Nernst equation. The K concentration gradient that drives the flow of ions through resting K channels is generated by an ion pump that transports K ions into the cytosol from the extracellular medium and Na ions out. In the absence of this pump, which is discussed later, the K concentration gradient could not be maintained and eventually the membrane potential would fall. Recent cloning and molecular characterization of resting K channels show that the channel protein is built of four identical subunits. Each subunit contains two membranespanning helices, which partially line the ion-conducting pore in the middle of the protein, and a shorter looped P segment, which acts as a filter to allow K but not other ions to enter the pore and cross the membrane. As we discuss in Chapter 21, the structure of resting K channels is generally similar to the structures of other ion channels that are critical to the function of nerve cells. Although resting K channels play the dominant role in generating the electric potential across the plasma membrane of animal cells, this is not the case in plant and fungal cells. The inside-negative membrane potential in these cells is generated by transport of H ions out of the cell by an ATPpowered proton pump.

Na Entry into Mammalian Cells Has a Negative G

As weve seen, two forces govern the movement of such ions as K, Cl, and Na across selectively permeable membranes:

588

CHAPTER 15

Transport across Cell Membranes

the voltage and the ion concentration gradient across the membrane. These forces may act in the same direction or in opposite directions. To calculate the free-energy change G corresponding to the transport of any ion across a membrane, we need to consider the contribution from each of these forces independent of the other. For example, in a reaction where Na moves from outside to inside the cell, the free-energy change generated from the Na concentration gradient is given by
(15-8)

namically favored transport of 1 mol of Na ions from outside to inside the cell if there were no Na concentration gradient. Given both forces acting on Na ions, the total G will be the sum of the two partial values:

At the concentrations of Nain and Naout shown in Figure 15-9, which are typical for many mammalian cells, Gc would be 1.45 kcal/mol, the change in free energy for the thermodynamically favored transport of 1 mol of Na ions from outside to inside the cell if there were no membrane electric potential. The free-energy change generated from the membrane electric potential is given by
(15-9)

In this typical example, the Na concentration gradient and the membrane electric potential contribute almost equally to the total G for transport of Na ions. Since G is 0, the inward movement of Na ions is thermodynamically favored. As discussed later, certain cotransport proteins use the inward movement of Na to power the uphill movement of several ions and small molecules into or out of animal cells.

S U M M A R Y Intracellular Ion Environment and Membrane Electric Potential

ATP-driven ion pumps generate and maintain ionic gradients across the plasma membrane. As a result, the ionic composition of the cytosol usually differs greatly from that of the surrounding fluid (see Table 15-1). In both invertebrates and vertebrates, the K concentration is higher and the Na concentration is lower in cells than in the blood. The cytosolic Ca2 concentration is maintained at less than 0.2 M. An inside-negative electric potential (voltage) of 5070 mV exists across the plasma membrane of all cells; this is equivalent to a voltage gradient of 200,000 volts per centimeter. In animal cells, the electric potential across the plasma membrane is generated primarily by movement of cytosolic K ions through resting K channels to the external medium. Unlike most other ion channels, which open only in response to various signals, these K channels are usually open. In plants and fungi, the membrane potential is maintained by the ATP-driven pumping of protons from the cytosol across the membrane. Two forces govern the movement of ions across selectively permeable membranes: the membrane electric potential and the ion concentration gradient, which may act in the same or opposite directions. For the thermodynamically favored inward movement of Na into animal cells, these forces act in the same direction (see Figure 15-9).

where F is the Faraday constant and E is the membrane electric potential. If E 70 mV, then Gm would be 1.6 kcal/mol, the change in free energy for the thermody-

Biological Energy Interconversions

Ion concentration gradient Inside 12 mM Na+ Outside 145 mM Na+ Na+ Gc = 1.45 kcal/mol

Membrane electrical potential Inside Outside + + + +

MEDIA CONNECTIONS

Gm = 1.61 kcal/mol

Free-energy change during transport of Na+ from outside to inside

MOHRIG: EXPER. ORGANIC CHEM. Figure: CD ICON 100% of size Fine Line Illustrations (516) 781-7200 5/29/97 6/9/97

Inside

Outside + + Na+

+ +

G = Gc + Gm = 3.06 kcal/mol

15.5

v FIGURE 15-9 Transmembrane forces acting on Na ions. As with all ions, the movement of Na ions across the plasma membrane is governed by the sum of two separate forcesthe membrane electric potential and the ion concentration gradient. In the case of Na ions, these forces usually act in the same direction.

Active Transport by ATP-Powered Pumps

We turn now to the ATP-powered pumps that transport ions and various small molecules against their concentration gradients. The general structures of the four principal classes

Active Transport by ATP-Powered Pumps Exterior

589

c c c a Cytosol b P-class pump F- and V-class pump ATP-binding region ABC superfamily A A T T

ATP-binding region

v FIGURE 15-10 The four classes of ATP-powered transport

proteins. P-class pumps are composed of two different polypeptides, and , and become phosphorylated as part of the transport cycle. The sequence around the phosphorylated residue, located in the larger subunits, is homologous among different pumps. F-class and V-class pumps do not form phosphoprotein intermediates. Their structures are similar and contain similar proteins, but none of their subunits are related to those of P-class pumps. All members of the large ABC

superfamily of proteins contain four core domains: two transmembrane (T) domains and two cytosolic ATP-binding (A) domains that couple ATP hydrolysis to solute movement. These core domains are present as separate subunits in some ABC proteins (depicted here), but are fused into a single polypeptide in other ABC proteins. [Adapted from C. H. Higgins, 1995, Cell 82:693;
P. Zhang et al., 1998, Nature 392:835; Y. Zhou, T. Duncan, and R. Cross, 1997, Proc. Natl. Acad. Sci. USA 94:10583; and T. Elston, H. Wang, and G. Oster, 1998, Nature 391:510.]

of these transport proteins are depicted in Figure 15-10, and their properties are summarized in Table 15-2. Note that the P, F , and V classes transport ions only, whereas the ABC superfamily class transports small molecules as well as ions. P-class ion pumps contain a transmembrane catalytic subunit, which contains an ATP-binding site, and usually a smaller subunit, which may have regulatory functions. Many of these pumps are tetramers composed of two and two subunits. During the transport process, at least one of the subunits is phosphorylated (hence the label P), and the transported ions are thought to move through the phosphorylated subunit. This class includes the Na/K ATPase in the plasma membrane, which maintains the Na and K gradients typical of animal cells, and several Ca2 ATPases, which pump Ca2 ions out of the cytosol into the external medium or into the lumen of the sarcoplasmic reticulum (SR) of muscle cells. Another member of the P class, found in acid-secreting cells of the mammalian stomach, transports protons (H ions) out of and K ions into the cell. The H pump that maintains the membrane electric potential in plant, fungal, and bacterial cells also belongs to this class. The structures of F-class and V-class ion pumps are similar to each other but unrelated to and more complicated than P-class pumps. F- and V-class pumps contain at least three kinds of transmembrane proteins and five kinds of extrinsic polypeptides that form the cytosolic domain. Several of the transmembrane and extrinsic subunits in F-class and

V-class pumps exhibit sequence homology, and each pair of homologous subunits is thought to have evolved from a common polypeptide. All known V and F pumps transport only protons in a process that does not involve a phosphoprotein intermediate. V-class pumps generally function to maintain the low pH of plant vacuoles and of lysosomes and other acidic vesicles in animal cells by using the energy released by ATP hydrolysis to pump protons from the cytosolic to the exoplasmic face of the membrane against the proton electrochemical gradient. F-class pumps are found in bacterial plasma membranes and in mitochondria and chloroplasts. In contrast to V pumps, they generally function to power the synthesis of ATP from ADP and Pi by movement of protons from the exoplasmic to the cytosolic face of the membrane down the proton electrochemical gradient. Because of their importance in ATP synthesis in chloroplasts and mitochondria, F-class proton pumps are treated separately in the next chapter. The final class of ATP-powered transport proteins is larger and more diverse than the other classes. Referred to as the ABC (ATP-binding cassette) superfamily, this class includes more than 100 different transport proteins found in organisms ranging from bacteria to humans. Each ABC protein is specific for a single substrate or group of related substrates including ions, sugars, peptides, polysaccharides, and even proteins. All ABC transport proteins share a common organization consisting of four core domains: two transmembrane (T) domains, forming the passageway through

590

CHAPTER 15

Transport across Cell Membranes

TABLE 15-2

Comparison of Major Classes of ATP-Powered Ion and Small-Molecule Pumps F Class V Class Substances Transported ABC Class

P Class

H, Na, K, Ca2

H only

H only Structural and Functional Features

Ions and various small molecules

Large catalytic subunits (often two) become phosphorylated during solute transport; smaller subunits may regulate transport.

Multiple transmembrane and cytosolic subunits generally function to synthesize ATP on cytosolic subunits powered by movement of H down an electrochemical gradient.

Multiple transmembrane and cytosolic subunits generally use energy released by ATP hydrolysis to pump H ions from cytosol to organelle lumens, acidifying them.

Two transmembrane domains form the pathway for solute; two cytosolic ATP-binding domains couple ATP hydrolysis to solute movement. Domains may be in one or separate subunits.

Location of Specific Pumps Plasma membrane of plants, fungi, bacteria (H pump) Plasma membrane of higher eukaryotes (Na/K pump) Bacterial plasma membranes Vacuolar membranes in plants, yeast, other fungi Bacterial plasma membranes (amino acid, sugar, and peptide transporters) Mammalian endoplasmic reticulum (transporters of peptides associated with antigen presentation by MHC proteins)

Inner mitochondrial membrane

Endosomal and lysosomal membrane in animal cells

Apical plasma membrane of mammalian stomach cells (H/K pump) Plasma membrane of all eukaryotic cells (Ca2 pump)

Thylakoid membrane of chloroplast

Plasma membrane of certain acid-secreting animal cells (e.g., osteoclasts and some kidney tubule cells) Mammalian plasma membranes (transporters of small molecules, phospholipids, small lipidlike drugs)

Sarcoplasmic reticulum membrane in muscle cells (Ca2 pump)

which transported molecules cross the membrane, and two cytosolic ATP-binding (A) domains. In some ABC proteins, the core domains are present in four separate polypeptides; in others, the core domains are fused into one or two multidomain polypeptides. All classes of ATP-powered pumps have one or more binding sites for ATP, and these are always on the cytosolic face of the membrane (see Figure 15-10). Although these proteins are often called ATPases, they normally do not hydrolyze ATP into ADP and Pi unless ions or other molecules are simultaneously

transported. Because of the tight coupling between ATP hydrolysis and transport, the energy stored in the phosphoanhydride bond is not dissipated. Thus ATP-powered transport proteins are able to collect the free energy released during ATP hydrolysis and use it to move ions or other molecules uphill against a potential or concentration gradient. The energy expended by cells to maintain the concentration gradients of Na, K, H, and Ca2 across the plasma and intracellular membranes is considerable. In nerve and kidney cells, for example, up to 25 percent of the ATP produced

Active Transport by ATP-Powered Pumps

591

by the cell is used for ion transport; in human erythrocytes, up to 50 percent of the available ATP is used for this purpose. In cells treated with poisons that inhibit the aerobic production of ATP (e.g., 2,4-dinitrophenol), the ion concentration inside the cell gradually approaches that of the exterior environment as the ions move through plasma membrane channels down their electric and concentration gradients. Eventually treated cells die: partly because protein synthesis requires a high concentration of K ions and partly because in the absence of a Na gradient across the cell membrane, a cell cannot import certain nutrients such as amino acids. Studies on the effects of such poisons provided early evidence for the existence of ion pumps. In this section, we discuss in some detail examples of the P, V, and ABC classes of ATPpowered pumps.

Plasma-Membrane Ca2 ATPase Exports Ca2 Ions from Cells

As discussed in Chapter 20, small increases in the concentration of free Ca2 ions in the cytosol trigger a variety of cellular responses. In order for Ca2 to function in intracellular signaling, its cytosolic concentration usually must be kept below 0.10.2 M. (Although some cytosolic Ca2 is bound to negatively charged groups, it is the concentration of free, unbound Ca2 that is critical to its signaling function.) The plasma membranes of animal, yeast, and probably plant cells contain Ca2 ATPases that transport Ca2 out of the cell against its electrochemical gradient. These P-class ion pumps help maintain the concentration of free Ca2 ions in the cytosol at a low level. In addition to a catalytic subunit containing an ATPbinding site, as found in other P-class pumps, plasmamembrane Ca2 ATPases also contain the Ca2-binding regulatory protein calmodulin. A rise in cytosolic Ca2 induces the binding of Ca2 ions to calmodulin, which triggers an allosteric activation of the Ca2 ATPase; as a result, the export of Ca 2 ions from the cell accelerates, and the original low cytosolic concentration of free Ca2 is restored rapidly.

Muscle Ca2 ATPase Pumps Ca2 Ions from the Cytosol into the Sarcoplasmic Reticulum
Besides the plasma-membrane Ca2 ATPase, muscle cells contain a second, different Ca2 ATPase that transports Ca2 from the cytosol into the lumen of the sarcoplasmic reticulum (SR), an internal organelle that concentrates and stores Ca2 ions. As discussed in Chapter 18, the SR and its calcium pump (referred to as the muscle calcium pump) are critical in muscle contraction and relaxation: release of Ca2 ions from the SR into the muscle cytosol causes contraction, and the rapid removal of Ca 2 ions from the cytosol by the muscle calcium pump induces relaxation. Because the muscle calcium pump constitutes more than 80 percent of the integral protein in SR membranes, it is easily purified and characterized. Each transmembrane catalytic

subunit has a molecular weight of 100,000 and transports two Ca2 ions per ATP hydrolyzed. In the cytosol of muscle cells, the free Ca2 concentration ranges from 107 M (resting cells) to more than 106 M (contracting cells), whereas the total Ca2 concentration in the SR lumen can be as high as 102 M. Sites on the cytosolic surface of the muscle calcium pump have a very high affinity for Ca2 (Km 107 M), allowing the pump to transport Ca2 efficiently from the cytosol into the SR against the steep concentration gradient. The concentration of free Ca 2 within the sarcoplasmic reticulum is actually much less than the total concentration of 102 M. Two soluble proteins in the lumen of SR vesicles bind Ca2 and serve as a reservoir for intracellular Ca2, thereby reducing the concentration of free Ca 2 ions in the SR vesicles, and consequently decreasing the energy needed to pump Ca2 ions into them from the cytosol. The activity of the muscle Ca2 ATPase is so regulated that if the free Ca2 concentration in the cytosol becomes too high, the rate of calcium pumping increases until the cytosolic Ca2 concentration is reduced to less than 1 M. Thus in muscle cells, the calcium pump in the SR membrane can supplement the activity of the plasma-membrane pump, assuring that the cytosolic concentration of free Ca2 remains below 1 M. The current model of the mechanism of action of the Ca2 ATPase in the SR membrane is outlined in Figure 15-11. Coupling of ATP hydrolysis with ion pumping involves several steps that must occur in a defined order. When the protein is in one conformation, termed E1, two Ca2 ions bind in sequence to high-affinity sites on the cytosolic surface (step 1). Then an ATP binds to its site on the cytosolic surface; in a reaction requiring that a Mg2 ion be tightly complexed to the ATP, the bound ATP is hydrolyzed to ADP and the liberated phosphate is transferred to a specific aspartate residue in the protein, forming a high-energy acyl phosphate bond, denoted by E1P (step 2). The protein then changes its conformation to E2P, generating two lowaffinity Ca2-binding sites on the exoplasmic surface, which faces the SR lumen; this conformational change simultaneously propels the two Ca2 ions through the protein to these sites (step 3) and inactivates the high-affinity Ca2-binding sites on the cytosolic face. The Ca2 ions then dissociate from the exoplasmic surface of the protein (step 4). Following this, the aspartyl-phosphate bond in E2P is hydrolyzed, causing E2 to revert to E1, a change that inactivates the exoplasmicfacing Ca2-binding sites and regenerates the cytosolicfacing Ca2-binding sites (step 5). Thus phosphorylation of the muscle calcium pump by ATP favors conversion of E1 to E2, and dephosphorylation favors the conversion of E2 to E1. While only E2P, not E1P, is actually hydrolyzed, the free energy of hydrolysis of the aspartyl-phosphate bond in E1P is greater than that for E2 P. The reduction in free energy of the aspartyl-phosphate bond in E2P, relative to E1P, can be said to power the E1 n E2 conformational change. The affinity of Ca2 for the cytosolic-facing binding sites in E1 is a thousandfold greater than the affinity of Ca2 for the exoplasmic-facing

592

CHAPTER 15

Transport across Cell Membranes

Low-affinity Ca+-binding sites

Ca2+

E1

E1

E2

E2

E1

High-affinity Ca+-binding sites Cytosol ATP site 2 Ca2+ ATP

P P ADP P

v FIGURE 15-11 Model of the mechanism of action of muscle

Ca2 ATPase, which is located in the sarcoplasmic reticulum (SR) membrane. Only one of the two subunits of this P-class pump is depicted. E1 and E2 are alternate conformational forms of the protein in which the Ca2-binding sites are on the cytosolic and exoplasmic faces, respectively. An ordered sequence of steps,

as diagrammed here, is essential for coupling ATP hydrolysis and the transport of Ca2 ions (red circles) across the membrane. P indicates a high-energy acyl phosphate bond; P indicates a low-energy phosphoester bond. See the text for more details.
[Adapted from W. P. Jencks, 1980, Adv. Emzymol. 51:75; W. P. Jencks, 1989, J. Biol. Chem. 264:18855; and P. Zhang et al., 1998, Nature 392:835.]

sites in E2; this difference enables the protein to transport Ca2 unidirectionally from the cytosol, where it binds tightly to the pump, to the exoplasm, where it is released. Much evidence supports the model depicted in Figure 15-11. For instance, the muscle calcium pump has been isolated with phosphate linked to an aspartate residue, and spectroscopic studies have detected slight alterations in protein conformation during the E1 n E2 conversion. On the basis of the proteins amino acid sequence and various biochemical studies, investigators proposed the structural model

for the catalytic subunit shown in Figure 15-12. The membrane-spanning helices are thought to form the passageway through which Ca2 ions move. The bulk of the subunit consists of cytosolic globular domains that are involved in ATP binding, phosphorylation of aspartate, and energy transduction. These domains are connected by stalks to the membrane-embedded domain. As noted previously, all P-class ion pumps, regardless of which ion they transport, are phosphorylated during the transport process. The amino acid sequences around the

FIGURE 15-12 Schematic structural model for the catalytic () subunit of muscle Ca2 ATPase. The 10 transmembrane helices are thought to form a channel through which Ca2 ions move. Site-specific mutagenesis studies have identified four residues (red dots), located in four of the transmembrane helices, that participate in Ca2 binding. Trypsin digestion releases three cytosolic globular domains, which constitute the bulk of the protein. One cytosolic domain functions in ATP binding; a second bears the aspartate that is phosphorylated / dephosphorylated; and the third is involved in energy transduction. [After D. H.
MacLennan et al., 1985, Nature 316:696; T. Toyofuku et al., 1992, J. Biol. Chem. 267:14490.]

Transmembrane helix SR lumen

residue

H3+N

Energy transduction Phosphorylation of aspartate Globular domains ATP binding

12nm

10

Cytosol COO Ca2+-binding

Active Transport by ATP-Powered Pumps

593

phosphorylated aspartate in the catalytic subunit are highly conserved in all proteins of this type. Thus the mechanistic model in Figure 15-11 probably is generally applicable to all these ATP-powered ion pumps. In addition, the subunits of all the P pumps examined to date have a similar molecular weight and, as deduced from their amino acid sequences derived from cDNA clones, have a similar arrangement of transmembrane helices (see Figure 15-12). These findings strongly suggest that all these proteins evolved from a common precursor, although they now transport different ions.

Na/K ATPase Maintains the Intracellular Na and K Concentrations in Animal Cells

A second P-class ion pump that has been studied in considerable detail is the Na/K ATPase present in the plasma membrane of all animal cells. This ion pump is a tetramer of subunit composition 22. (Classic Experiment 15.1 describes the discovery of this enzyme.) The polypeptide is required for newly synthesized subunits to fold properly in the endoplasmic reticulum but apparently is not involved

directly in ion pumping. The subunit is a 120,000-MW nonglycosylated polypeptide whose amino acid sequence and predicted membrane structure are very similar to those of the muscle Ca2 ATPase. In particular, the Na/K ATPase has a stalk on the cytosolic face that links domains containing the ATP-binding site and the phosphorylated aspartate to the membrane-embedded domain. The overall process of transport moves three Na ions out of and two K ions into the cell per ATP molecule split (Figure 15-13a). Several lines of evidence indicate that the Na/K ATPase is responsible for the coupled movement of K and Na into and out of the cell, respectively. For example, the drug ouabain, which binds to a specific region on the exoplasmic surface of the protein and specifically inhibits its ATPase activity, also prevents cells from maintaining their Na/K balance. Any doubt that the Na /K ATPase is responsible for ion movement was dispelled by the demonstration that the enzyme, when purified from the membrane and inserted into liposomes, propels K and Na transport in the presence of ATP. The mechanism of action of the Na/K ATPase, outlined in Figure 15-13b, is similar to that of the muscle calcium

MOHRIG: EXPER. ORGANIC CHEM. Figure: CD ICON 100% of size Fine Line Illustrations (516) 781-7200 5/29/97 6/9/97

FIGURE 15-13 Models for the structure and function of the Na/K ATPase in the plasma membrane. (a) This P-class pump comprises two copies each of a small glycosylated subunit and a large subunit, which performs ion transport. Hydrolysis of one molecule of ATP to ADP and Pi is coupled to export of three Na ions (blue circles) and import of two K ions (dark red triangles) against their concentration gradients (large triangles). It is not known whether only one subunit, or both, in a single ATPase molecule transports ions. (b) Ion pumping by the Na/K ATPase involves a high-energy acyl phosphate intermediate (E1P) and conformational changes, similar to transport by the muscle Ca2 ATPase. In this case, hydrolysis of the E2P intermediate powers transport of a second ion (K) inward. Na ions are indicated by blue circles; K ions, by red triangles. See text for details. [Adapted from P. Luger,
1991, Electrogenic Ion Pumps, Sinauer Associates, p. 178.]

(a)

Oligosaccharide 3 Na+ Na+ K+ 2 K+ ATP ADP + Pi

Exterior

Biological Energy Interconversions

MEDIA CONNECTIONS

Cytosol

(b)

Low-affinity Na+ -binding sites

High-affinity K + -binding site

E1 ATP site High-affinity Na+ -binding site Low-affinity K + -binding sites

E1 P ATP ADP

E2 P

Binding of 3 Na+ ions

Binding of ATP, phosphorylation of aspartate

E1 E2 conformational change, outward transport of Na+

E2 P Dissociation of Na+ , binding of K +

E2

E1

E1

Pi Hydrolysis of aspartyl phosphate E2 E1 conformational change, inward transport of K + Dissociation of K + ions

594

CHAPTER 15

Transport across Cell Membranes

pump, except that ions are pumped in both directions across the membrane. In its E1 conformation, the Na/K ATPase has three high-affinity Na-binding sites and two low-affinity K-binding sites on the cytosolic-facing surface of the protein. The Km for binding of Na to these cytosolic sites is 0.6 mM, a value considerably lower than the intracellular Na concentration of 12 mM; as a result, Na ions normally will fill these sites. Conversely, the affinity of the cytosolic K-binding sites is low enough that K ions, transported inward through the protein, dissociate from E1 into the cytosol despite the high intracellular K concentration. During the E1 n E2 transition, the three bound Na ions move outward through the protein. Transition to the E2 conformation also generates two high-affinity K sites and three low-affinity Na sites on the exoplasmic face. Because the Km for K binding to these sites (0.2 mM) is considerably lower than the extracellular K concentration (4 mM), these sites will fill quickly with K ions. In contrast, the three Na ions, transported outward through the protein, will dissociate into the extracellular medium from the low-affinity Na sites on the exoplasmic surface despite the high extracellular Na concentration. Similarly, during the E2 n E1 transition, the two bound K ions are transported inward.

V-Class H ATPases Pump Protons across Lysosomal and Vacuolar Membranes

All V-class ATPases transport H ions only. These proton pumps, present in the membranes of lysosomes, endosomes, and plant vacuoles, function to acidify the lumen of these organelles. The acidity of the lysosomal lumen, usually 4.55.0, can be measured precisely in living cells by use of particles labeled with a pH-sensitive fluorescent dye. Cells phagocytose these particles (see Figure 5-44a) and transfer them to the lysosomes. The ability of different wavelengths of visible light to excite fluorescence is highly dependent on pH, and the lysosomal pH can be calculated from the spectrum of the fluorescence emitted. Maintenance of the 100-fold or more proton gradient between the lysosomal lumen (pH 4.55.0) and the cytosol (pH 7.0) depends on ATP production by the cell. The ATP-powered proton pumps in lysosomal and vacuolar membranes have been isolated, purified, and incorporated into liposomes. As illustrated in Figure 15-10, these V-class proton pumps contain two discrete domains: a cytosolic-facing hydrophilic domain (V1) composed of five different polypeptides and a transmembrane domain (V0) containing 912 copies of proteolipid c, one copy of protein b, and one copy of protein a. The subunit composition of the cytosolic domain is 33; the and subunits contain the sites where ATP binding and hydrolysis occur. Each transmembrane c subunit is thought to span the membrane two times; the c and a subunits together form the protonconducting channel. Unlike P-class ion pumps, the V-class H ATPases are not phosphorylated and dephosphorylated during proton transport.

Similar V-class ATPases are found in the plasma membrane of certain acid-secreting cells. These include osteoclasts, bone-resorbing macrophagelike cells, which bind to a bone and seal off a small segment of extracellular space between the plasma membrane and the surface of the bone. HCl secreted into this space by osteoclasts dissolves the calcium phosphate crystals that give bone its rigidity and strength. Another example is the mitochondria-rich epithelial cells lining the toad bladder; the apical plasma membrane of these cells contain many V-class H ATPases, which function to acidify the urine (Figure 15-14). As we discuss later, the membrane of plant vacuoles contains two proton pumps: a typical V-class H ATPase and another one that utilizes the energy released by hydrolysis of inorganic pyrophosphate (PPi) to pump protons into the vacuole. This PPi-hydrolyzing proton pump, believed to be unique to plants, has an amino acid sequence different from any other ion-transporting proteins. ATP-powered proton pumps cannot acidify the lumen of an organelle (or the extracellular space) by themselves. The reason for this is that pumping of protons would rapidly cause a buildup of positive charge on the exoplasmic face of the membrane on the inside of the vesicle membrane and a corresponding buildup of negative charges on the cytosolic face. In other words, the pump would generate a voltage across the membrane, exoplasmic face positive, which would prevent movement of protons into the vesicle before a significant H concentration gradient had been established. In fact, this is the way that H pumps generate an insidenegative potential across plant and yeast plasma membranes. In order for an organelle lumen or an extracellular space

v FIGURE 15-14 The plasma membrane of certain acidsecreting cells contains an almost crystalline array of V-class H ATPases. This electron micrograph is of a platinum replica of the cytosolic surface of the apical plasma membrane of a toad bladder epithelial cell. Each stud is a single V-class H ATPase (600,000 MW) composed of several polypeptide subunits surrounding a central channel. [From D. Brown, S. Gluck, and
J. Hartwig, 1987, J. Cell Biol. 105:1637.]

Active Transport by ATP-Powered Pumps

595

(e.g., the outside of an osteoclast) to become acidic, movement of H up its concentration gradient must be accompanied by (1) movement of an equal number of anions in the same direction or (2) movement of equal numbers of a different cation in the opposite direction. The first process occurs in lysosomes and plant vacuoles whose membranes contain V-class H ATPases and ion channels through which accompanying anions (e.g., Cl) move. The second occurs in the lining of the stomach, which contains a P-class H/K ATPase that pumps one H outward and one K inward.

The ABC Superfamily Transports a Wide Variety of Substrates

As noted earlier, all members of the very large and diverse ABC superfamily of transport proteins contain two transmembrane (T) domains and two cytosolic ATP-binding (A) domains (see Figure 15-10). The T domains, each built of six membrane-spanning helices, form the pathway through which the transported substance (substrate) crosses the membrane and determine the substrate specificity of each ABC protein. The sequence of the A domains is 30 to 40 percent homologous in all members of this superfamily, indicating a common evolutionary origin. Some ABC proteins also contain a substrate-binding subunit or regulatory subunit.
Bacterial Plasma-Membrane Permeases

gradient. Bacterial permeases generally are inducible; that is, the quantity of a transport protein in the cell membrane is regulated by both the concentration of the nutrient in the medium and the metabolic needs of the cell. In E. coli histidine permease, a typical bacterial ABC protein, the two transmembrane domains and two cytosolic ATP-binding domains are formed by four separate subunits. In gram-negative bacteria such as E. coli, which have an outer membrane, a soluble histidine-binding protein in the periplasmic space assists in transport (Figure 15-15). This soluble protein binds histidine tightly and directs it to the T subunits, through which histidine crosses the membrane powered by ATP hydrolysis. Mutant E. coli cells that are defective in any of the histidine-permease subunits or the soluble binding protein are unable to transport histidine into the cell, but are able to transport other amino acids whose uptake is facilitated by other transport proteins. Such genetic analyses provide strong evidence that histidine permease and similar ABC proteins function to transport solutes into the cell.
Mammalian MDR Transport Proteins A series of rather unexpected observations led to discovery of the first eukaryotic ABC protein. Oncologists noted that tumor cells often became simultaneously resistant to several chemotherapeutic drugs with unrelated chemical structures; similarly, cell biologists observed that cultured cells selected for resistance to one toxic substance (e.g., colchicine, a microtubule inhibitor) frequently became resistant to several other drugs, including the anticancer drug adriamycin. Subsequent studies showed that this resistance is due to enhanced expression of a multidrug-resistance (MDR) transport protein known as MDR1. In this member of the ABC superfamily, all four domains are fused into a single 170,000-MW protein (Figure 15-16). This protein

The plasma membrane of many bacteria contain numerous permeases that belong to the ABC superfamily. These proteins use the energy released by hydrolysis of ATP to transport specific amino acids, sugars, vitamins, or even peptides into the cell. Since bacteria frequently grow in soil or pond water where the concentration of nutrients is low, these ABC transport proteins allow the cells to concentrate amino acids and other nutrients in the cell against a substantial concentration

Histidine Exterior Outer membrane

Porin

Periplasmic histidinebinding protein Histidine permease Plasma membrane Cytosol A A ATP A T T T

Periplasmic space

ADP + Pi

FIGURE 15-15 Gram-negative bacteria import many solutes by means of ABC proteins (permeases) that utilize a soluble substrate-binding protein present in the periplasmic space. Depicted here is the import of the amino acid histidine. After diffusing through porins in the outer membrane, histidine is bound by a specific periplasmic histidine-binding protein, which undergoes a conformational change. The histidineprotein complex binds to the exoplasmic surface of a T subunit in histidine permease located in the plasma membrane. Hydrolysis of ATP bound to the A subunit then powers movement of histidine through the protein into the cytosol. The transport process does not appear to involve a phosphoprotein intermediate.

596 v

CHAPTER 15

Transport across Cell Membranes

Transmembrane helix Exterior

FIGURE 15-16 Schematic structural model for mammalian MDR1 protein. In this member of the ABC superfamily, the two transmembrane domains and two cytosolic ATP-binding domains are part of a single polypeptide. Each transmembrane domain contains six helices. The two halves of this 1280-aa protein have similar amino acid sequences. A variety of lipid-soluble molecules that diffuse across the plasma membrane into the cell are transported outward by MDR1.

Cytosol H3+ N ATP binding ATP binding COO

uses the energy derived from ATP hydrolysis to export a large variety of drugs from the cytosol to the extracellular medium. The Mdr1 gene is frequently amplified in multidrugresistant cells, resulting in a large overproduction of the MDR1 protein. Most drugs transported by MDR1 are small hydrophobic molecules, which diffuse from the culture medium across the plasma membrane into the cell. The ATP-powered export of such drugs from the cytosol by MDR1 means a much higher extracellular drug concentration is required to kill cells. That MDR1 is an ATP-powered small-molecule pump has been demonstrated with liposomes containing the purified protein (see Figure 15-4). The ATPase activity of these liposomes is enhanced by different drugs in a dose-dependent manner corresponding to their ability to be transported by MDR1. Not only does MDR1 transport a varied group of molecules, but all these substrates compete with one another for transport by MDR1. Although the mechanism of action of MDR1-assisted transport has not been definitively demonstrated, the flippase model, depicted in Figure 15-17a, is a

likely candidate. Substrates of MDR1 are primarily planar, lipid-soluble molecules with one or more positive charges, and they move spontaneously from the cytosol into the cytosolic-facing leaflet of the plasma membrane. The hydrophobic portion of a substrate molecule is oriented toward the hydrophobic core of the membrane, and the charged portion toward the polar cytosolic face of the membrane and is still in the cytosol. The substrate diffuses laterally until encountering and binding to a site on the MDR1 protein that is within the bilayer. The protein then flips the charged substrate molecule into the exoplasmic leaflet, an energetically unfavorable reaction powered by the coupled ATPase activity of MDR1. Once in the exoplasmic face, the substrate diffuses into the aqueous phase on the outside of the cell. Support for the flippase model of transport by MDR1 comes from MDR2, a homologous protein present in the region of the liver cell plasma membrane that faces the bile duct. MDR2 has been shown to flip phospholipids from the cytosolic-facing leaflet of the plasma membrane to the exoplasmic leaflet, thereby generating an excess of phospholipids

(a) Flippase model Substrate Hydrophobic end Charged end Exterior 3 Cytosol A ATP ADP + Pi A 1 ATP 4 2

(b) Pump model

Concentration gradient T T

ADP + Pi
4 ). (b) According to the aqueous phase on the outside of the cell ( the pump model, MDR1 has a single multisubstrate binding site and transports molecules by a mechanism similar to that of other ATP-powered pumps. [Adapted from G. Ferro-Luzzi Ames and H. Legar,

v FIGURE 15-17 Possible mechanisms of action of the MDR1

protein. (a) The flippase model proposes that a lipid-soluble molecule first dissolves in the cytosolic-facing leaflet of the plasma membrane ( 1 ) and then diffuses in the membrane until binding to a site on the MDR1 protein that is within the bilayer ( 2 ). Powered by ATP hydrolysis, the substrate molecule flips into the exoplasmic leaflet ( 3 ), from which it can move directly into

1992, FASEB J. 6:2660; N. Nelson, 1992, Curr. Opin. Cell Biol. 4:654; C. F. Higgins and M. M. Gottesman, 1992, Trends Biochem. Sci. 17:18; and C. F. Higgins, 1995, Cell 82:693.]

Cotransport by Symporters and Antiporters

597

in the exoplasmic leaflet; these phospholipids peel off into the bile duct and form an essential part of the bile. An alternative pump model also has been proposed for MDR1 (Figure 15-17b). According to this model, drug molecules in the cytosol bind directly to a single small-molecule binding site on the cytosolic face of the MDR1 protein; subsequent ATP hydrolysis powers movement of the bound drug through the protein to the aqueous phase on the outside of the cell by a mechanism similar to that of other ATP-powered pumps. MDR1 protein is expressed in abundance in the liver, intestines, and kidneysites from which natural toxic products are removed from the body. Thus the natural function of MDR1 may be to transport a variety of natural and metabolic toxins into the bile, intestinal lumen, or forming urine. During the course of its evolution, MDR1 appears to have coincidentally acquired the ability to transport drugs whose structures are similar to those of these toxins. Tumors derived from these cell types, such as hepatomas (liver cancers), frequently are resistant to virtually all chemotherapeutic agents and thus difficult to treat, presumably because the tumors exhibit increased expression of the MDR1 or MDR2 proteins.
Cystic Fibrosis Transmembrane Regulator (CFTR) Protein Discovery of another ABC trans-

S U M M A R Y Active Transport by ATP-Powered Pumps

Four types of membrane transport proteins couple the energy-releasing hydrolysis of ATP with the energyrequiring transport of substances against their concentration gradient (see Figure 15-10 and Table 15-2). In P-class pumps, phosphorylation of the subunit and a change in conformational states are essential for coupled transport of H, Na, K, or Ca2 ions (see Figures 15-11 and 15-13). The P-class Na/K ATPase pumps three Na ions out of and two K ions into the cell per ATP hydrolyzed. A homolog, the Ca2 ATPase, pumps two Ca2 ions out of the cell or, in muscle, into the sarcoplasmic reticulum per ATP hydrolyzed. The combined action of these pumps in animal cells creates an intracellular ion milieu of high K, low Ca2, and low Na very different from the extracellular fluid milieu of high Na, high Ca2, and low K. In the multisubunit V-and F-class ATPases, which pump protons exclusively, a phosphorylated protein is not an intermediate in transport. A V-class H pump in animal lysosomal and endosomal membranes and plant vacuole membranes is responsible for maintaining a lower pH inside the organelles than in the surrounding cytosol. All members of the large and diverse ABC superfamily of transport proteins contain four core domains: two transmembrane domains, which form a pathway for solute movement and determine substrate specificity, and two cytosolic ATP-binding domains. The ABC superfamily includes bacterial amino acid and sugar permeases (see Figure 15-15); the mammalian MDR1 protein, which exports a wide array of drugs from cells; and CFTR protein, a Cl channel that is defective in cystic fibrosis. According to the flippase model of MDR1 activity, a substrate molecule diffuses into the cytosolic leaflet of the plasma membrane, then is flipped to the exoplasmic leaflet in an ATP-powered process, and finally diffuses from the membrane into the extracellular space (see Figure 15-17a).

port protein came from studies of cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease of Caucasians. This disease is caused by a mutation in the CFTR gene, which encodes a chloride-channel protein that is regulated by cyclic AMP (cAMP), an intracellular second messenger. These Cl channels are present in the apical plasma membranes of epithelial cells in the lung, sweat glands, pancreas, and other tissues. An increase in cAMP stimulates Cl transport by such cells from normal individuals, but not from CF individuals who have a defective CFTR protein. The sequence and predicted structure of the encoded CFTR protein, based on analysis of the cloned gene, are very similar to those of MDR1 protein except for the presence of an additional domain, the regulatory (R) domain, on the cytosolic face. The Cl-channel activity of CFTR protein clearly is enhanced by binding of ATP. Moreover, as detailed in Chapter 20, cAMP activates a protein kinase that phosphorylates, and thereby activates, CFTR. When purified CFTR protein is incorporated into liposomes, it forms Cl channels with properties similar to those in normal epithelial cells. And when the wild-type CFTR protein is expressed by recombinant techniques in cultured epithelial cells from CF patients, the cells recover normal Cl-channel activity. This latter result raises the possibility that gene therapy might reverse the course of cystic fibrosis. Since CFTR protein is similar to MDR1 in structure, it may also function as an ATP-powered pump of some as-yet unidentified molecule. In any case, much remains to be learned about this fascinating class of ABC transport proteins.

15.6

Cotransport by Symporters and Antiporters

Besides ATP-powered pumps, cells have a second, discrete class of proteins that import or export ions and small molecules, such as glucose and amino acids, against a concentration gradient. These proteins use the energy stored in the electrochemical gradient of Na or H ions to power the

598

CHAPTER 15

Transport across Cell Membranes

uphill movement of another substance, a process called cotransport. For instance, the energetically favored movement of a Na ion (the cotransported ion) into a cell across the plasma membrane, driven both by its concentration gradient and by the transmembrane voltage gradient (see Figure 15-9), can be coupled obligatorily to movement of the transported molecule (e.g., glucose) against its concentration gradient. When the transported molecule and cotransported ion move in the same direction, the process is called symport; when they move in opposite directions, the process is called antiport (see Figure 15-2b).

as defined previously. According to our previous calculations, the membrane electric potential and the Na concentration gradient each contribute about 1.5 kcal per mole of Na transported inward, or a total of about 3 kcal /mol (see Equations 15-8 and 15-9). Thus the change in free energy G for transport of two moles of Na inward is about 6 kcal. By substituting this value into the partial equation for glucose transport

Na-Linked Symporters Import Amino Acids and Glucose into Many Animal Cells
Many cells, such as those lining the small intestine and the kidney tubules, need to concentrate glucose against a very large concentration gradient. Such cells utilize a two Na/one-glucose symporter; a protein that couples transmembrane movement of one glucose molecule to the transport of two Na ions:

As discussed earlier, movement of Na from the external medium into the cell is driven by two forces: by the Na concentration gradient (the Na concentration is lower inside the cell than in the medium) and by the inside-negative membrane electric potential (see Figure 15-9). Quantitatively, the free-energy change for the symport transport of two Na ions and one glucose molecule can be written
(15-10)

we can calculate that a G of 6 kcal /mol can generate an equilibrium concentration of glucose inside the cell that is 30,000 times greater than the exterior concentration. By transporting two Na ions per glucose, this Na/glucose symport protein can accumulate glucose against a much steeper concentration gradient than if only one Na ion were transported per glucose. The Na/glucose symporter contains 14 transmembrane helices (Figure 15-18). A recombinant protein consisting of only the five C-terminal transmembrane helices has been shown to transport glucose across the plasma membrane, down its concentration gradient, independently of Na. This portion of the molecule thus functions as a glucosepermeation pathway. The N-terminal portion of the protein, including helices 19, is required to couple Na binding and glucose transport against a concentration gradient. Figure 15-19 depicts the current model of transport by Na/glucose symporters. This model, which has not yet been experimentally supported, entails conformational changes analogous to those that occur in uniport transporters such as GLUT1, which do not require a cotransported ion (see Figure 15-7).

Na-Linked Antiporter Exports Ca2 from Cardiac Muscle Cells

where F is the Faraday constant, E is the electric potential across the plasma membrane, and the other parameters are The plasma membrane of most cells contains one or more types of antiporters, which couple movement of a cotrans-

FIGURE 15-18 Structural model for the two-Na/one-glucose symporter. This 662-aa protein forms 14 transmembrane helices with the N-and C-termini facing the cytosol. The five C-terminal helices form the sugar-permeation pathway; the rest of the protein may be required to couple Na binding and glucose transport. The exoplasmic surface of the protein has binding sites for two Na ions and one glucose 3.5 nm apart, but the location of these sites has not yet been determined.
[Adapted from M. Panayotova-Heiermann et al., 1997, J. Biol. Chem. 272:20324.]

Exterior 1 Cytosol COO NH3+ Glucose-permeation pathway 2 3 4 5 6 7 8 9 10 11 12 13 14

Cotransport by Symporters and Antiporters

MOHRIG: EXPER. ORGANIC CHEM. Figure: CD ICON 100% of size Fine Line Illustrations (516) 781-7200 5/29/97 6/9/97

599

Na+ Exterior Na+

Glucose

Biological Energy Interconversions

MEDIA CONNECTIONS

Cytosol Glucose

v FIGURE 15-19 Proposed model for operation of the

two-Na/one-glucose symporter. The simultaneous binding of Na and glucose to sites on the exoplasmic surface induces a conformational change, generating a transmembrane pore or tunnel that allows both bound Na

and glucose to move through the protein to binding sites on the cytosolic domain and then to pass into the cytosol. After this passage, the protein reverts to its original conformation. [See
E. Wright, K. Hager, and E. Turk, 1992, Curr. Opin. Cell Biol. 4:696 for details on the structure and function of this and related transporters.]

ported ion (often Na) down its electrochemical gradient to movement of a different molecule in the opposite direction against a concentration gradient (see Figure 15-3b). In cardiac muscle cells, for example, a Na/Ca2 antiporter, rather than a plasma membrane Ca2 ATPase, plays the principal role in maintaining a low concentration of Ca2 in the cytosol. The reaction of this cation antiporter can be written

Note that the movement of three Na ions is required to power the export of one Ca2 ion against the greater than 10,000-fold concentration gradient between the cell interior (2 107 M) and cell exterior (2 103 M). As in other muscle cells, a rise in the intracellular Ca2 concentration in cardiac muscle triggers contraction. Thus the operation of the Na/Ca2 antiporter lowers the cytosolic concentration of Ca2 and reduces the strength of heart muscle contraction. The Na/K ATPase in the plasma membrane of cardiac cells, as in other body cells, creates the Na concentration gradient used to power export of Ca2 ions. The drugs ouabain and digoxin increase the force of heart muscle contraction and are widely used in the treatment of congestive heart failure. The primary effect of these drugs is to inhibit the Na/K ATPase, thereby raising the intracellular Na concentration (and lowering intracellular K). Because the Na/Ca2 antiporter functions less efficiently with a lower Na concentration gradient, fewer Ca2 ions are exported and the intracellular Ca2 concentration increases. This increase causes the muscle to contract more strongly.

transport only negative ions. An important example is AE1 protein, the predominant integral protein of the mammalian erythrocyte. This anion antiporter catalyzes the one-for-one exchange of Cl and HCO3 across the plasma membrane. Since one singly charged negative ion is exchanged for another, there is no net movement of electric charge and the reaction is not affected by the membrane potential. Thus, the direction of the reaction is dependent only on the concentration gradients of the transported ions. Transmembrane anion exchange is essential to an important function of the erythrocytethe transport of waste carbon dioxide (CO2), which is generated in peripheral tissues, to the lungs for excretion by respiratory exhalation (Figure 15-20). Waste CO 2 released from cells into the capillary blood diffuses across the erythrocyte membrane. In its gaseous form, CO2 dissolves poorly in aqueous solutions, such as the cytosol or blood plasma, but the potent enzyme carbonic anhydrase inside the erythrocyte converts CO2 to the water-soluble bicarbonate (HCO3) anion:

Since

we can write the overall reaction for carbonic anhydrase as

AE1 Protein, a Cl/HCO3 Antiporter, Is Crucial to CO2 Transport by Erythrocytes

In addition to cation antiporters, which transport only positive ions, many cells also contain anion transporters, which

The release of oxygen from hemoglobin into the peripheral capillaries induces a conformational change in the globin polypeptide that enables a histidine side chain to bind the proton produced by the carbonic anhydrase reaction. Meanwhile, the HCO3 formed by carbonic anhydrase is transported out of the erythrocyte in exchange for an entering Cl via AE1 protein (see Figure 15-20, top).

600

CHAPTER 15

Transport across Cell Membranes

CO2

In systemic capillaries High CO2 pressure Low O2 pressure

O2 Hemoglobin

H2O CO2 + OH

H+
Carbonic anhydrase

N C N Cl

C C H

HCO3

Erythrocyte plasma membrane

AE1 protein HCO3

In pulmonary capillaries Low CO2 pressure High O2 pressure CO2

O2

H2O CO2 + OH

H+
Carbonic anhydrase

N C N

C C H

HCO3

think of as equal to OH CO2) for Cl causes the cytosolic pH to remain near neutrality. The overall direction of this anion-exchange process is reversed in the lungs. CO2 diffuses out of the erythrocyte and is eventually expelled in breathing. The lowered concentration of CO2 within the cytosol drives the carbonic anhydrase reaction, as written above, from right to left: HCO3 reacts to yield CO2 and OH. At the same time, oxygen binding to hemoglobin causes a proton to be released from hemoglobin; the proton combines with the OH to form H2O. The lowered intracellular HCO3 concentration causes HCO3 to enter the erythrocyte in exchange for Cl (see Figure 15-20, bottom). AE1, which has been studied extensively, carries out the precise one-for-one sequential exchange of anions on opposite sides of the membrane required to preserve electroneutrality in the cell; only once every 10,000 or so transport cycles does an anion move unidirectionally from one side of the membrane to the other. AE1 has a large membraneembedded domain, folded into at least 12 transmembrane helices, which carry out anion transport, and a cytosolicfacing domain, which anchors certain cytoskeletal proteins to the membrane. Although the precise transport mechanism is not known, conformational changes most likely have a key role as in other membrane transport proteins.

Several Cotransporters Regulate Cytosolic pH

HCO3

Cl

v FIGURE 15-20 Schematic drawings showing anion transport across the erythrocyte membrane in systemic and pulmonary capillaries. AE1 protein (purple)an anion antiportercatalyzes the reversible exchange of Cl and HCO3 ions across the membrane and works in conjunction with carbonic anhydrase. In systemic capillaries, the overall reaction causes HCO3 to be released from the cell, which is essential for CO2 transport from the tissues to the lungs. In the lungs, the overall reaction is reversed. See the text for further discussion.

The entire anion-exchange process in peripheral-blood erythrocytes is completed within 50 milliseconds (ms), during which time 5 109 HCO3 ions are exported from the cell down its concentration gradient. If anion exchange did not occur, HCO3 would accumulate inside the erythrocyte to toxic levels during periods of exercise, when much CO2 is generated. About 80 percent of the CO2 in blood is transported as HCO3 generated inside erythrocytes; anion exchange allows about two-thirds of this HCO3 to be transported by blood plasma external to the cells, increasing the amount of CO2 that can be transported from tissues to the lungs. Also, without anion exchange, the increased HCO 3 concentration in the erythrocyte would cause the cytosol to become alkaline. The exchange of HCO3 (which we can

The anaerobic metabolism of glucose yields lactic acid, and aerobic metabolism yields CO2, which is hydrated by carbonic anhydrase to carbonic acid (H2CO3). These weak acids dissociate, yielding H ions (protons); if these protons were not exported from cells, the cytosolic pH would drop precipitously, endangering cellular functioning. Two types of cotransport proteins are employed to remove some of the excess protons generated during metabolism of animal cells. One is a NaHCO3/Cl cotransporter, which imports one Na ion down its concentration gradient, together with one HCO 3, in exchange for export of one Cl ion against its concentration gradient. The imported HCO3 ions combine with protons generated by metabolism to produce CO2, which diffuses out of the cell. Thus the overall action of this transporter raises the cytosolic pH (reduces the H concentration). Also important in removing excess protons is a Na/H antiporter, which couples entry of one Na ion into the cell down its concentration gradient to export one H ion. The plasma membranes of most animal cells also contain a Na-independent Cl/HCO3 antiporter similar to the erythrocyte AE1 protein discussed previously. This anionexchange protein functions to lower the cytosolic pH, in effect removing excess OH ions. Recall that a HCO3 ion can be viewed as a complex of OH and CO2, so export of HCO3 lowers the cytosolic pH. Exchange of cytosolic HCO3 for extracellular Cl is powered by the import of Cl down its concentration gradient (Clout Clin).

Cotransport by Symporters and Antiporters 100

601

Cl/HCO3 antiporter 50 Na+/H+ antiporter Na+ HCO3/Cl cotransporter

6.8

7.2 Intracellular pH

7.6

v FIGURE 15-21 Effect of intracellular pH on activity of membrane transport proteins that regulate the cytosolic pH of mammalian cells. See the text for discussion. [After
S. L. Alper, 1991, Ann. Rev. Physiol. 53:549.]

The activity of all three of these antiport proteins depends on pH, providing cells with a fine-tuned mechanism for controlling the cytosolic pH (Figure 15-21). The protonexporting transporters, which are activated when the pH of the cytosol falls, act to raise the cytosolic pH. Similarly, a rise in pH above 7 stimulates the Cl/HCO3 antiporter, leading to a more rapid export of HCO3 and decrease in the cytosolic pH. In this manner the cytosolic pH of growing cells is maintained very close to pH 7.4. Small changes in the cytosolic pH may have profound effects on the overall cellular metabolic rate. For instance, primary fibroblast cells grown to maximal density (confluence) in tissue culture generally become quiescent: DNA synthesis stops; the rates of RNA synthesis, glucose catabolism, and protein synthesis are reduced; and the cytosolic pH drops from the characteristic 7.4 of growing cells to 7.2. Treatment of quiescent cells with a mixture of serum growth factors restimulates cell growth and DNA synthesis. An early effect of these growth factors is a marked increase in the cytosolic pH to 7.4; this dramatic change is caused in part by stimulation of the Na/H antiport, which expels protons into the medium. The rise in cytosolic pH is believed to help activate certain metabolic pathways required for cell growth and division.

channels that transport these anions from the cytosol into the vacuole. Entry of these anions against their concentration gradients is driven by the inside-positive potential generated by the H pumps. Operation of both types of proton pumps in conjunction with these anion channels produces an inside-positive electric potential of about 20 mV across the vacuolar membrane and also a substantial pH gradient. The proton gradient and electric potential across the plant vacuole membrane are used in much the same way as the Na gradient and electric potential across the animal-cell plasma membrane: to power the selective uptake or extrusion of ions and small molecules. In the leaf, for example, excess sucrose generated during photosynthesis in the day is stored in the vacuole; during the night the stored sucrose moves into the cytoplasm and is metabolized to CO2 and H2O with concomitant generation of ATP from ADP and Pi. A protonsucrose antiporter in the vacuolar membrane operates to accumulate sucrose in plant vacuoles. The inward movement of sucrose is powered by the outward movement of H, which is favored by its concentration gradient (lumen cytosol) and by the outward-negative potential across the vacuolar membrane (see Figure 15-22). Uptake of Ca2 and Na into the vacuole from the cytosol against their concentration gradients is similarly mediated by proton antiporters.
H+-pumping proteins ADP + Pi PPi 2Pi (pH = 7.5) 2H+ Ion channel proteins Cl NO3 Na+ H+ + + + Sucrose 20 mV Plant vacuole membrane

Percent maximal rate

ATP

(pH = 3 6) Ca 2 +

H+

H+

H+

Proton antiport proteins

Numerous Transport Proteins Enable Plant Vacuoles to Accumulate Metabolites and Ions
The lumen of plant vacuoles is much more acidic (pH 3 to 6) than is the cytosol (pH 7.5). As noted earlier, the vacuolar membrane contains a V-class ATP-powered pump and a unique PPi-powered pump, both of which function to pump H ions into the vacuolar lumen against a concentration gradient. As illustrated in Figure 15-22, the vacuolar membrane also contains Cl and NO3

v FIGURE 15-22 Concentration of ions and sucrose by the plant vacuole. The vacuolar membrane contains two types of proton pumps: a V-class H ATPase (light green) and a unique pyrophosphate-hydrolyzing proton pump (dark green). These pumps generate a lowered luminal pH as well as an insidepositive electric potential across the vacuolar membrane owing to the inward pumping of H ions. The inside-positive potential powers the movement of Cl and NO3 from the cytosol through separate channel proteins (dark purple). Proton antiporters (light purple), powered by the H gradient, accumulate Na, Ca2, and sucrose inside the vacuole. [After P. Rea and D. Sanders, 1987,
Physiol. Plant 71:131; J. M. Maathuis and D. Sanders, 1992, Curr. Opin. Cell Biol. 4:661; P. A. Rea et al., 1992, Trends Biochem. Sci. 17:348.]

602

CHAPTER 15

Transport across Cell Membranes

S U M M A R Y Cotransport by Symporters and Antiporters

A small molecule or ion may be imported or exported against its concentration gradient by coupling its movement to that of another molecule or ion, usually H or Na, down its electrochemical gradient. Two forces power the movement of H or Na across a membrane: the electric potential and the ion concentration gradient. Entry of glucose and amino acids into certain cells against their concentration gradient is coupled by symport proteins to the energetically favorable entry of Na (see Figure 15-19). In cardiac muscle cells, the export of Ca2 is coupled to the import of Na by a cation antiporter, which transports 3 Na ions inward for each Ca2 ion exported. The erythrocyte membrane contains a Cl/HCO3 anion antiporter (AE1 protein) that facilitates transport of CO2 by the blood (see Figure 15-20). Two proton-exporting transportersa NaCl/HCO3 cotransporter and a Na/H antiportermaintain the cytosolic pH in animal cells very close to 7.4 despite metabolic production of carbonic and lactic acids. A Na-independent Cl/HCO3 antiporter, similar to AE1 protein, functions to export HCO3 when the cytosolic pH rises above normal, causing a decrease in pH. Uptake of sucrose, Na, Ca2, and other substances into plant vacuoles is carried out by proton antiporters in the vacuolar membrane. Ion channels in the membrane are critical in generating a proton concentration gradient large enough to power accumulation of ions and metabolites in vacuoles by these proton antiporters (see Figure 15-22).

across to the other side. In this section we first describe the polarized nature of epithelia and how different combinations of membrane proteins enable epithelial cells to carry out their transport or secretory functions. Then we discuss the structure and function of the junctions that interconnect epithelial cells.

The Intestinal Epithelium Is Highly Polarized

An epithelial cell is said to be polarized because one side differs in structure and function from the other. In particular, its plasma membrane is organized into at least two discrete regions, each with different sets of transport proteins. In the epithelial cells that line the intestine, for example, that portion of the plasma membrane facing the intestine, the apical surface, is specialized for absorption; the rest of the plasma membrane, the lateral and basal surfaces, often referred to as the basolateral surface, mediates transport of nutrients from the cell to the surrounding fluids which lead to the blood and forms junctions with adjacent cells and the underlying extracellular matrix called the basal lamina (Figure 15-23). Extending from the lumenal (apical) surface of intestinal epithelial cells are numerous fingerlike projections (100 nm in diameter) called microvilli (singular, microvillus). Often referred to collectively as the brush border because of their appearance, microvilli greatly increase the area of the apical surface and thus the number of transport proteins it can contain, enhancing the absorptive capacity of the intestinal epithelium. A bundle of actin filaments that runs down the center of each microvillus gives rigidity to the projection. Overlying the brush border is the glycocalyx, a loose network composed of the oligosaccharide side chains of integral membrane glycoproteins, glycolipids, and enzymes that catalyze the final stages in the digestion of ingested carbohydrates and proteins (Figure 15-24). The action of these enzymes produces monosaccharides and amino acids, which are transported across the intestinal epithelium and eventually into the bloodstream.

15.7

Transport across Epithelia

With few exceptions, all the internal and external body surfaces of animals, such as the skin, stomach, and intestines, are covered with a layer of epithelial cells called an epithelium (see Figure 6-4). Many epithelial cells transport ions or small molecules from one side to the other of the epithelium. Those lining the stomach, for instance, secrete hydrochloric acid into the stomach lumen, which after a meal becomes pH 1, while those lining the small intestine transport products of digestion (e.g., glucose and amino acids) into the blood. All epithelial cells in a sheet are interconnected by several types of specialized regions of the plasma membrane called cell junctions. These impart strength and rigidity to the tissue and prevent water-soluble material on one side of the sheet (as in the intestinal lumen) from moving

Transepithelial Movement of Glucose and Amino Acids Requires Multiple Transport Proteins
Movement of monosaccharides and amino acids from the intestinal lumen into the blood is a two-stage transcellular process. The first stage, import of substances from the lumen into intestinal epithelial cells, is carried out by membrane transport proteins in the microvilli on the apical surface of intestinal cells. The second stage, export of substances from the cells into the fluid surrounding the basolateral surface, is carried out by other transport proteins on the basolateral plasma membrane. In order for such transepithelial transport to occur, the epithelial cell must be polarized, with different sets of transport proteins localized in the basolateral and apical surfaces. To illustrate this process, we examine the membrane transport proteins required to move glucose

Transport across Epithelia

603

Apical surface Microvillus

Tight junctions Adherens junction Spot desmosome Lateral surface

Gap junction Intermediate filament Hemidesmosome Basal surface

Basal lamina

v FIGURE 15-24 Micrograph of the microvilli that form the

lumenal surface of intestinal epithelial cells, obtained by the deep-etching technique. The surface of each microvillus is covered with a series of bumps believed to be integral membrane proteins. The glycocalyx, which covers the apices (tips) of the microvilli, is composed of a network of glycoproteins and digestive enzymes. [From N. Hirokawa and J. E. Heuser, 1981,
J. Cell Biol. 91:399; courtesy of N. Hirokawa and J. E. Heuser.]

across the epithelial cells lining the intestine and kidney. Similar proteins are used to transport amino acids across these epithelia. Figure 15-25 depicts the transport of glucose from the intestinal lumen to the blood. Glucose is imported against its concentration gradient from the intestinal lumen across the apical surface of the epithelial cells by a two-Na/oneglucose symporter located in the microvillar membranes. As noted above, this symporter couples the energetically unfavorable inward movement of one glucose molecule to the energetically favorable inward transport of two Na ions (see Figure 15-19). In the steady state, all the Na ions transported from the intestinal lumen into the cell during Na/glucose symport, or the similar process of Na/amino acid symport, are pumped out across the basolateral membrane, often called the serosal (blood-facing) membrane. Thus the low intracellular Na concentration is maintained. The Na/K ATPase that accomplishes this is found in these cells exclusively on the basolateral surface of the plasma membrane. The coordinated operation of these transporters allows uphill movement of glucose and amino acids from the intestine into the cell, and ultimately is powered by ATP hydrolysis by the Na/K ATPase. Glucose and amino acids concentrated inside intestinal cells by symporters are exported down their concentration gradients into the blood via uniport proteins in the basolateral membrane. In the case of glucose, this movement is mediated by GLUT2, a glucose transporter that is localized in the basal and lateral membranes of intestinal cells (see

Microvilli

Glycocalyx

FIGURE 15-23 Schematic diagram of epithelial cells lining the small intestine and the principal types of cell junctions that connect them. As in all epithelia, the basal surface of the cells rests on the basal lamina, a fibrous network of collagen and proteoglycans that supports the epithelial cell layer. The apical surface faces the intestinal lumen. Tight junctions, lying just under the microvilli, prevent diffusion of substances between the intestinal lumen and the blood via the extracellular space between cells. Gap junctions allow movement of small molecules and ions between the cytosol of adjacent cells. The remaining three types of junctions, adherens junctions, spot desmosomes, and hemidesmosomes are critical to cell-cell and cell-matrix adhesion.

604

CHAPTER 15

Transport across Cell Membranes

Intestinal lumen Dietary glucose High (dietary) Na+

Blood High Na+ Low K +

Epithelial cells Low Na+ High K +

Parietal Cells Acidify the Stomach Contents While Maintaining a Neutral Cytosolic pH
The mammalian stomach contains a 0.1 M solution of hydrochloric acid (HCl). This strongly acidic medium denatures many ingested proteins before they are degraded by proteolytic enzymes in the stomach (e.g., pepsin) that function at acidic pH. Hydrochloric acid is secreted into the stomach by parietal cells (also known as oxyntic cells) in the gastric lining. These cells contain a H/K ATPase in their apical membrane, which faces the stomach lumen and generates a concentration of H ions 106 times greater in the stomach lumen than in the cell cytosol (pH 1.0 versus pH 7.0). This enzyme is a P-class ATPase, similar in structure and function to the Na/K ATPase discussed earlier. Operation of the Na/K ATPase results in a net outward movement of one charged ion per ATP (see Figure 15-13). In contrast, the action of the H/K ATPase, which exports one H ion and imports one K ion for each ATP hydrolyzed, produces no net movement of electric charge. The numerous mitochondria in parietal cells produce abundant ATP for use by the H/K ATPase. If parietal cells simply exported H ions in exchange for K ions, a rise in the concentration of OH ions and thus a marked rise in cytosolic pH would occur, since in the cytosol, as in all aqueous solutions, the product of the H and OH concentrations is a constant (1014 M2). However, during acidification of the stomach lumen, the pH of the parietal-cell cytosol remains neutral. Parietal cells accomplish this feat by means of a Cl/HCO3 antiporter in the basolateral membrane (Figure 15-26). The excess cytosolic OH, generated by exporting protons, combines with CO2 that diffuses into the cell from the blood, forming HCO3 in a reaction catalyzed by cytosolic carbonic anhydrase. The HCO3 ion is transported across the basolateral membrane of the cell into the blood in exchange for an incoming Cl ion by means of an anion antiporter that is similar in structure and function to the erythrocyte AE1. The Cl ions thus imported into the cell exit through Cl channels in the apical membrane, entering the stomach lumen. To preserve electroneutrality, each Cl ion that moves into the stomach lumen across the apical membrane is accompanied by a K ion that moves outward through a separate K channel. In this way, the excess K ions pumped inward by the H/K ATPase are returned to the stomach lumen, thus maintaining the intracellular K concentration. The net result is accumulation of both H and Cl ions (i.e., HCl) in the stomach lumen, while the pH of the cytosol remains neutral and the excess OH ions, as HCO3, are transported into the blood.

GLUT 2 Glucose Na+ Na+/K + ATPase K+ Basolateral membrane Na+ ATP K+ Glucose 2 Na+ Glucose 2 Na+ Na+/glucose symport protein Apical membrane

ADP + Pi

Tight junction

v FIGURE 15-25 Transport of glucose from the intestinal lumen into the blood. Activity of the Na/K ATPase (green) in the basolateral surface membrane generates Na and K concentration gradients, and the K gradient generates an insidenegative membrane potential. Both the Na concentration gradient and the membrane potential are used to drive the uptake of glucose from the intestinal lumen by the two-Na/one-glucose symporter (blue) located in the apical surface membrane. Glucose leaves the cell via facilitated diffusion catalyzed by GLUT2 (orange), a glucose uniporter located in the basolateral membrane.

Figure 5-1c). (GLUT2 is a homolog of GLUT1; as discussed earlier; however, GLUT1 generally functions to import glucose into many body cells.) The net result of the operation of these various transport proteins is movement of Na ions, amino acids, and glucose from the intestinal lumen across the intestinal epithelium into the interstitial spaces surrounding the cells, and eventually into the blood. Tight junctions between the epithelial cells prevent these molecules from diffusing back into the intestinal lumen. The epithelial cells lining kidney tubules, which have an architecture similar to that of intestinal epithelial cells, reabsorb glucose from the blood filtrate that is the forming urine and return it to the blood. In the first part of a kidney tubule, the epithelial cells transport glucose against a relatively small glucose concentration gradient. These cells utilize a second type of Na/glucose symport proteina one-Na/one-glucose symporter, which has a high transport rate but cannot transport glucose against a steep concentration gradient. At the intracellular Na concentration and membrane potential depicted in Figure 15-9, this symporter can generate an intracellular glucose concentration 100 times that of the extracellular medium (here the forming urine). In the latter part of a kidney tubule, however, the epithelial cells take up the remaining glucose against a more than 100-fold glucose concentration gradient. To accomplish this, these cells contain in their apical membrane the same two-Na/one-glucose symporter found in intestinal epithelial cells. The two types of Na/glucose symport proteins are similar in amino acid sequence, predicted structure, and mechanism but have evolved to transport glucose under different conditions.

Tight Junctions Seal Off Body Cavities and Restrict Diffusion of Membrane Components
For polarized epithelial cells to carry out their transport functions, extracellular fluids surrounding their apical and basolateral membranes must be kept separate. This is

Transport across Epithelia Central ductule

605

Basolateral membrane

Apical membrane Cl channel protein

Apical membrane

Anion Cl antiport protein HCO3

Cl
+ HCO3 H

Cl H+ H+/K + ATPase K+ K+ K+ channel protein Secretory vesicles Tight junction

ATP ADP + Pi K+

Carbonic anhydrase

H2O
OH

CO2

CO2 +

Tight junction Stomach lumen

Basolateral membrane

v FIGURE 15-26 Acidification of the stomach lumen by parietal cells in the gastric lining. The apical membrane of parietal cells contains a H/K ATPase (a P-class pump) as well as Cl and K channel proteins. Note the cyclic K transport across the apical membrane: K ions are pumped inward by the H/K ATPase and exit via a K channel. The basolateral membrane contains an anion antiporter that exchanges HCO3 and Cl ions. The combined operation of these four different transport proteins acidifies the stomach lumen while maintaining the neutral pH and electroneutrality of the cytosol. See the text for more details.

Single acinar cell

v FIGURE 15-27 Diagram of pancreatic acinar cells. An

acinus is a spherical aggregate of about a dozen cells; the lumen of an acinus is connected to a ductule that merges with other ductules and eventually leads into a main pancreatic duct, which empties into the lumen of the small intestine (Figure 5-48). Acinar cells synthesize degradative enzymes and store them as inactive precursors (zymogens) in secretory vesicles, which cluster under the apical region of the plasma membrane adjacent to the ductule. The basolateral membrane covers the sides of an acinar cell below the apical (lumen-facing) surface and extends along the base of the cell; nutrients in the blood in the surrounding vessels are transported through this region of the plasma membrane into the cell. Note the tight junctions (orange) just below the apical region between adjacent cells; they prevent movement of substances between the central ductule and the blood.

accomplished by tight junctions, which connect adjacent epithelial cells and usually are located just below the apical surface (see Figure 15-23). These specialized regions of the plasma membrane form a barrier that seals off body cavities such as the intestine, the stomach lumen, ductules in pancreatic acini, and the bile duct in the liver. For example, tight junctions prevent diffusion of small molecules directly from the intestinal lumen into the interstitial spaces that surround the basolateral plasma membrane and that lead to the blood. Thus intestinal epithelial cells must transport nutrients through the cells as previously described. In the pancreas, tight junctions between acinar cells likewise prevent leakage of secreted proteins, including digestive enzymes, from the central ductules into the blood (Figure 15-27). Tight junctions also prevent diffusion of membrane proteins and glycolipids between the apical and basolateral regions of the plasma membrane, ensuring that these regions contain different membrane components.
Structure of Tight Junctions Tight junctions are composed of thin bands of plasma-membrane proteins that completely encircle a polarized cell and are in contact with similar thin bands on adjacent cells. When thin sections of cells are viewed in an electron microscope, the plasma membranes of adjacent cells appear to touch each other at intervals and

even to fuse (Figure 15-28a). Freeze-fracture electron microscopy affords a striking view of the tight junction. The microvillar tight junction shown in Figure 15-28b appears to comprise an interlocking network of ridges in the plasma membrane. More specifically, there appear to be ridges on the cytosolic face of the plasma membrane of each of the two contacting cells. (Corresponding grooves not shown here are found on the exoplasmic face.) High magnification reveals that these ridges are made up of protein particles 34 nm in diameter. In the model shown in Figure 15-28c, the tight junction is formed by a double row of these particles, one row donated by each cell. The two principal integral membrane proteins found in tight junctions are occludin and claudin. Each of these proteins has four membrane-spanning helices. Although the molecular structure of the junction is not known, the extracellular domains of rows of occludin and claudin proteins

606
(a)

CHAPTER 15

Transport across Cell Membranes

v FIGURE 15-28 Tight junctions. (a) Thin-section electron

micrograph of the apical region of two liver epithelial cells, illustrating the tight junction just below the microvilli and the adherens junction. From the apical region of these liver cells, which faces the lumen of the bile duct, phospholipids and other components of bile are secreted into the duct. (b) Freeze-fracture electron micrograph of a tight junction between two intestinal epithelial cells. The fracture plane passes through the plasma membrane of one of the two adjacent cells. The honeycomblike

network of ridges of particles below the microvilli forms the tight junction. (c) A model showing how a tight junction might be formed by linkage of rows of protein particles in adjacent cells (see also Figure 15-23). [Part (a) from P. A. Cross and K. L.
Mercer, 1993, Cell and Tissue Ultrastructure, A Functional Perspective, W. H. Freeman and Company, p. 50; part (b) courtesy of L. A. Staehelin; part (c) adapted from L. A. Staehelin and B. E. Hull, 1978, Sci. Am. 238(5):140, and D. Goodenough, 1999, Proc. Natl. Acad. Sci. USA 96:319.]

in the plasma membrane of one cell probably form extremely tight links with similar rows of claudin and occludin in the adjacent cell, essentially fusing two adjacent cells and creating an impenetrable seal. Treatment of an epithelium with the protease trypsin destroys the tight junctions, supporting the proposal that proteins are essential structural components of these junctions. The long C-terminal cytosolic-facing domain of occludin is bound to one of a group of large cytosolic proteins (ZO-1,

ZO-2, and ZO-3) that, in turn, are bound to other cytoskeletal proteins and to actin fibers. These interactions appear to stabilize the linkage between occludin molecules that is essential for integrity of the tight junction (Chapter 22).
Impermeability of Tight Junctions to Aqueous Solutions That tight junctions are impermeable to most water-

soluble substances can be demonstrated in an experiment in which lanthanum hydroxide (an electron-dense colloid of high molecular weight) is injected into the pancreatic blood

Transport across Epithelia

607

Lipids in the cytosolic leaflets of the apical and basolateral membranes of epithelial cells have the same composition and apparently can diffuse from one region of the membrane to the other. In contrast, the lipid compositions of the exoplasmic leaflets of the apical and basolateral membrane regions are very different, and membrane lipids in the exoplasmic leaflets cannot diffuse through tight junctions. All the glycolipid in MDCK cells, for instance, is present in the exoplasmic face of the apical membrane, as are all proteins anchored to the membrane by fatty acids linked to a glycosylphosphatidylinositol group (see Figure 3-36a). In fact, the only lipids in the exoplasmic leaflet of the apical plasma membrane are glycolipids, fatty acid components of glycosylphosphatidylinositol anchors, and cholesterol. Phosphatidylcholine, conversely, is present almost exclusively in the exoplasmic face of the basolateral plasma membrane.
v FIGURE 15-29 Experimental demonstration that tight junctions prevent passage of water-soluble substances. Pancreatic acinar tissue is fixed and prepared for microscopy a few minutes after electron-opaque lanthanum hydroxide is injected into the blood of an experimental animal. As shown in this electron micrograph of adjacent acinar cells, the lanthanum hydroxide can penetrate between the cells but is arrested at the level of the tight junction. [Courtesy of D. Friend.]

Other Junctions Interconnect Epithelial Cells and Control Passage of Molecules between Them
In order to function in an integrated manner, the individual cells composing epithelia and other organized tissues must adhere to one another and to the surrounding extracellular matrix and also control the movement of ions and small molecules between them. Several specialized cell junctions are critical to adhesion and passage of molecules between cells in tissues (see Figure 15-23). Three types of cell junctions, called desmosomes, function in cell-cell and cell-matrix adhesion. Epithelial and some other types of cells, such as smooth muscle, are bound tightly together by spot desmosomes. These are buttonlike points of contact between cells, often thought of as a spot-weld between adjacent plasma membranes, that confer mechanical strength on these tissues. Hemidesmosomes, similar in structure to spot desmosomes, anchor the plasma membrane to regions of the extracellular matrix. Bundles of intermediate filaments course through the cell, interconnecting spot desmosomes and hemidesmosomes. Finally, adherens junctions (also known as belt desmosomes), which are found primarily in epithelial cells, form a belt of cell-cell adhesion just under the tight junctions. The lateral surfaces of adjacent cells contain numerous gap junctions. These junctions help to integrate the metabolic activities of all cells in a tissue by allowing the direct passage of ions and small molecules from the cytosol of one cell to that of another (see the chapter opening figure). Among these are intracellular signaling molecules (e.g., cyclic AMP and Ca2) and precursors of DNA and RNA. Electron micrographs of animal tissue sections have shown that a space of about 20 nm ordinarily is present between the nonjunctional regions of plasma membranes of adjacent cells. This space contains integral membrane and extracellular surface glycoproteins that assist junctions in intercellular adhesion.

vessel of an experimental animal; a few minutes later the pancreatic acinar cells are fixed and prepared for microscopy. As shown in Figure 15-29, the lanthanum hydroxide diffuses from the blood into the space that separates the lateral surfaces of adjacent acinar cells, but cannot penetrate past the outermost tight junction. Other studies have shown that tight junctions also are impermeable to salts. For instance, when MDCK cells are grown in a medium containing very low concentrations of Ca2, they form a monolayer in which the cells are not connected by tight junctions; as a result, fluids and salts flow freely across the cell layer. When Ca2 is added to such a monolayer, tight junctions form within an hour, and the cell layer becomes impermeable to fluids and salts (see Figure 6-7).
Ability of Tight Junctions to Block Diffusion of Proteins and Lipids in the Plane of the Plasma Membrane

When liposomes containing a fluorescent-tagged glycoprotein are added to the medium in contact with the apical surface of a monolayer of MDCK cells, some spontaneously fuse with the plasma membrane. Fluorescent glycoprotein is detectable in the apical but not in the basolateral surface of the cells so long as the tight junctions between adjacent cells are intact. However, if the tight junctions are destroyed by removing Ca2 from the medium, the fluorescent protein is soon detectable in the basolateral surface, indicating that it can diffuse from the apical to the basolateral regions of the plasma membrane. These results indicate that plasma membrane proteins cannot diffuse through tight junctions.

608

CHAPTER 15

Transport across Cell Membranes

An understanding of the structure and function of desmosomes requires knowledge about actin microfilaments and intermediate filaments. Likewise, an understanding of gap junctions and their equivalent in plant cells (plasmodesmata) depends on knowledge of cellular metabolism and signaling. Therefore, we defer detailed discussion of these junctions until later chapters when these related topics are examined.

15.8

Osmosis, Water Channels, and the Regulation of Cell Volume

S U M M A R Y Transport across Epithelia

The apical and basolateral plasma membrane domains of epithelial cells contain different transport proteins and carry out quite different transport processes. In the intestinal epithelial cell, Na/glucose and Na/amino acid symporters are in the apical membrane region facing the intestinal lumen, while Na/K ATPases and glucose and amino acid uniporters are in the basolateral membrane region facing the blood capillaries. The coordinated operation of these membrane transport proteins allows the uphill transepithelial movement of amino acids and glucose from the lumen to the blood, powered by ATP hydrolysis by the Na/K ATPase (see Figure 15-25). Parietal cells in the stomach lining, which secrete HCl into the lumen, have ATP-powered H/K pumps, K channels, and Cl channels on the apical membrane and pH-sensitive Cl/HCO3 antiporters on the basolateral membrane. The combined action of these proteins allows the cytosolic pH to be maintained near neutrality, despite the active export of protons from the cells into the stomach lumen, causing its acidification (see Figure 15-26). The plasma membrane contains specialized regions that form various types of cell junctions between adjacent cells (see Figure 15-23). Tight junctions interconnecting epithelial and other polarized cells seal off body cavities and restrict diffusion of plasma-membrane proteins from the apical to the basolateral surfaces. Tight junctions also prevent diffusion of lipids in the exoplasmic (but not the cytosolic leaflet) from the apical to the basolateral domains of the plasma membrane. Adherens junctions and spot desmosomes bind the plasma membranes of adjacent cells in a way that gives strength and rigidity to the entire tissue. Hemidesmosomes help connect cells to the extracellular matrix. Gap junctions in animal cells and plasmodesmata in plant cells interconnect the cytosol of two adjacent cells, allowing small molecules and ions to pass between them.

In this section, we examine two types of transport phenomena that, at first glance, may seem unrelated: the regulation of cell volume in both plant and animal cells, and the bulk flow of water (the movement of water containing dissolved solutes) across one or more layers of cells. In humans, for example, water moves from the blood filtrate that will form urine across a layer of epithelial cells lining the kidney tubules and into the blood, thus concentrating the urine. (If this did not happen, one would excrete several liters of urine a day!) In higher plants, water and minerals are absorbed by the roots and move up the plant through conducting tubes (the xylem); water is lost from the plant mainly by evaporation from the leaves. What these processes have in common is osmosis the movement of water from a region of lower solute concentration to a region of higher solute concentration. We begin with a consideration of some basic facts about osmosis, and then show how they explain several physiological properties of animals and plants.

Osmotic Pressure Causes Water to Move across Membranes

As noted early in this chapter, most biological membranes are relatively impermeable to ions and other solutes, but like all phospholipid bilayers, they are somewhat permeable to water (see Figure 15-1). Permeability to water is increased by water-channel proteins discussed below. Water tends to move across a membrane from a solution of low solute concentration to one of high. Or, in other words, since solutions with a high amount of dissolved solute have a lower concentration of water, water will move from a solution of high water concentration to one of lower. This process is known as osmotic flow. Osmotic pressure is defined as the hydrostatic pressure required to stop the net flow of water across a membrane separating solutions of different compositions (Figure 15-30). In this context, the membrane may be a layer of cells or a plasma membrane. If the membrane is permeable to water but not to solutes, the osmotic pressure across the membrane is given by
(15-11)

where is the osmotic pressure in atmospheres (atm) or millimeters of mercury (mmHg); R is the gas constant; T is the absolute temperature; and C is the difference in total solute concentrations, CA and CB, on each side of the membrane. It is the total number of solute molecules that is important. For example, a 0.5 M NaCl solution is actually 0.5 M Na ions and 0.5 M Cl ions and has approximately the same osmotic pressure as a 1 M solution of glucose or lactose.

Osmosis, Water Channels, and the Regulation of Cell Volume

609

v FIGURE 15-30 Experimental system for demonstrating osmotic pressure. Solutions A and B are separated by a membrane that is permeable to water but impermeable to all solutes. If CB (the total concentration of solutes in solution B) is greater than CA, water will tend to flow across the membrane from solution A to solution B. The osmotic pressure between the solutions is the hydrostatic pressure that would have to be applied to solution B to prevent this water flow. From the vant Hoff equation, RT (CB CA).

Consequently, it is essential that animal cells be maintained in an isotonic medium, which has a solute concentration close to that of the cell cytosol (see Figure 5-22). Even in an isotonic environment, all animal cells face a problem in maintaining their cell volume. Cells contain a large number of charged macromolecules and small metabolites that attract ions of opposite charge (e.g., K, Ca2, PO43). Also recall that there is a slow leakage of extracellular ions, particularly Na and Cl, into cells down their concentration gradient. As a result of these factors, in the absence of some countervailing mechanism, the cytosolic solute concentration would increase, causing an osmotic influx of water and eventually cell lysis. To prevent this, animal cells actively export inorganic ions as rapidly as they leak in. The export of Na by the ATP-powered Na/K pump plays the major role in this mechanism for preventing cell swelling. If cultured cells are treated with an inhibitor that prevents production of ATP, they swell and eventually burst, demonstrating the importance of active transport in maintaining cell volume. Unlike animal cells, plant, algal, fungal, and bacterial cells are surrounded by a rigid cell wall. Because of the cell wall, the osmotic influx of water that occurs when such cells are placed in a hypotonic solution (even pure water) leads to an increase in intracellular pressure but not in cell volume. In plant cells, the concentration of solutes (e.g., sugars and salts) usually is higher in the vacuole than in the cytosol, which in turn has a higher solute concentration than the extracellular space. The osmotic pressure, called turgor pressure, generated from the entry of water into the cytosol and then into the vacuole pushes the cytosol and the plasma membrane against the resistant cell wall. Cell elongation during growth occurs by a hormone-induced localized loosening of a region of the cell wall, followed by influx of water into the vacuole, increasing its size (see Figure 22-33). Although most protozoans (like animal cells) do not have a rigid cell wall, many contain a contractile vacuole that permits them to avoid osmotic lysis. A contractile vacuole takes up water from the cytosol and, unlike a plant vacuole, periodically discharges its contents through fusion with the plasma membrane (Figure 15-31). Thus,
v
FIGURE 15-31 The contractile vacuole in Paramecium caudatum, a typical ciliated protozoan, as revealed by Nomarski microscopy of a live organism. The vacuole is filled by radiating canals that collect fluid from the cytosol. When the vacuole is full, it fuses for a brief period with the plasma membrane and expels its contents. (a) A full vacuole and system of radiating canals. (b) A nearly empty vacuole; the radiating canals are collecting more fluid from the cytosol to refill it.

From Equation 15-11 we can calculate that a hydrostatic pressure of 0.22 atm (167 mmHg) would just balance the water flow across a semipermeable membrane produced by a concentration gradient of 10 mM sucrose or 5 mM NaCl.

Different Cells Have Various Mechanisms for Controlling Cell Volume

Animal cells will swell when they are placed in a hypotonic solution (i.e., one in which the concentration of solutes is lower than it is in the cytosol). Some cells, such as erythrocytes, will actually burst as water enters them by osmotic flow. Rupture of the plasma membrane by a flow of water into the cytosol is termed osmotic lysis. Immersion of all animal cells in a hypertonic solution (i.e., one in which the concentration of solutes is higher than it is in the cytosol) causes them to shrink as water leaves them by osmotic flow.

610

CHAPTER 15

Transport across Cell Membranes

even though water continuously enters the protozoan cell by osmotic flow, the contractile vacuole prevents too much water from accumulating in the cell and swelling it to the bursting point.

(e.g., the kidney cells that resorb water from the urine) that exhibit high permeability for water.

Water Channels Are Necessary for Bulk Flow of Water across Cell Membranes
Even though a pure phospholipid bilayer is only slightly permeable to water, small changes in extracellular osmotic strength cause most animal cells to swell or shrink rapidly. In contrast, frog oocytes and eggs, which have an internal salt concentration comparable to other cells (150 mM), do not swell when placed in pond water of very low osmotic strength. These observations led investigators to suspect that the plasma membranes of erythrocytes and other cell types contain water-channel proteins that accelerate the osmotic flow of water. The absence of these water channels in frog oocytes and eggs protects them from osmotic lysis. Microinjection experiments with mRNA encoding aquaporin, an erythrocyte membrane protein, provided convincing evidence that this protein increases the permeability of cells to water (Figure 15-32). In its functional form, aquaporin is a tetramer of identical 28-kDa subunits, each of which contains six transmembrane helices that form three pairs of homologs in an unusual orientation (Figure 15-33a). The channel through which water moves is thought to be lined by eight transmembrane helices, two from each subunit (Figure 15-33b). Aquaporin or homologous proteins are expressed in abundance in erythrocytes and in other cells

Simple Rehydration Therapy Depends on Osmotic Gradient Created by Absorption of Glucose and Na
An understanding of osmosis and the intestinal absorption of glucose forms the basis for a simple therapy that has saved millions of lives, particularly in less-developed countries. In these countries, diarrhea caused by cholera and other intestinal pathogens is a major cause of death of young children. A cure demands not only killing the bacteria with antibiotics, but also rehydration replacement of the water that is lost from the blood and other tissues. Simply drinking water does not help, because it is excreted from the gastrointestinal tract almost as soon as it enters. To understand the simple therapy that is used, recall that absorption of glucose by the small intestine involves the coordinated movement of Na; one cannot be transported without the other (see Figure 15-25). The movement of NaCl and glucose from the intestinal lumen, across the epithelial cells, and into the blood creates a transepithelial osmotic gradient, forcing movement of water from the intestinal lumen into the blood. Thus, giving affected children a solution of sugar and salt to drink (but not sugar or salt alone) causes the bulk flow of water into the blood from the intestinal lumen and leads to rehydration.

v FIGURE 15-32 Experimental demonstration that aquaporin is a water-channel protein. Frog oocytes, which normally do not express aquaporin, were microinjected with erythrocyte mRNA encoding aquaporin. These photographs show control oocytes (bottom image in each panel) and microinjected oocytes (top image in each panel) at the indicated times after transfer from an isotonic salt solution (0.1 mM) to a hypotonic salt

solution (0.035 M). The volume of the control oocytes remained unchanged, because they are poorly permeable to water. In contrast, the microinjected oocytes expressing aquaporin swelled because of an osmotic influx of water, indicating that aquaporin increases their permeability to water. [Courtesy of
Gregory M. Preston and Peter Agre, Johns Hopkins University School of Medicine.]

Osmosis, Water Channels, and the Regulation of Cell Volume

611

Changes in Intracellular Osmotic Pressure Cause Leaf Stomata to Open

Although most plants cells do not change their volume or shape because of the osmotic movement of water, the opening and closing of stomata the pores through which CO2 enters a leafprovides an important exception. The external epidermal cells of a leaf are covered by a waxy cuticle that is largely impenetrable to water and to CO2, a gas required for photosynthesis by the

FIGURE 15-34 The opening and closing of stomata. (a) Light micrograph of a leaf of a wandering Jew (Tradescantia sp) plant shows two stomata, each surrounded by a pair of guard cells. (b) Opening of K and Cl channels in the plasma membrane of the guard cells is followed by an influx of K and Cl into the cytosol and then into the vacuole. This triggers the osmotic influx of water, causing the cells to bulge and opening the stomatal pore. [See D. J. Cosgrove and
R. Hedrich, 1991, Planta 186:143. Part (a) courtesy Runk /Schoenberger, from Grant Heilman.]

FIGURE 15-33 The structure of aquaporin, a waterchannel protein in the erythrocyte plasma membrane. This tetrameric protein has four identical subunits. (a) Schematic model of an aquaporin subunit showing the three pairs of homologous transmembrane helices, A and A, B and B, and C and C. As indicated by the arrows showing the N-terminal n C-terminal directionality of the helices, the homologous segments are oriented in the opposite direction. (b) Head-on view of tetrameric aquaporin showing the packing of the transmembrane helices in the plane of the membrane, as determined by x-ray crystallography. The helices (represented as circles) in each of the four subunits are shown in different colors. Although the identity of the two helices from each subunit that line the central channel is not known, they probably are a pair of homologous segments. The opposite orientation of the two helices in a pair within the membrane would account for the ability of the channel to transport water equally in both directions across the membrane. [Adapted from A. Chang et al., 1997, Nature 387:627.]

chlorophyll-laden mesophyll cells in the leaf interior. As CO2 enters a leaf, water vapor is simultaneously losta process that can be injurious to the plant. Thus it is essential that the stomata open only during periods of light, when photosynthesis occurs; even then, they must close if too much water vapor is lost. Two guard cells surround each stomate (Figure 15-34a). Changes in turgor pressure lead to changes in the shape of these guard cells, thereby opening or closing the pores. Stomatal opening is caused by an increase in the concentration of ions or other solutes within the guard cells because of (1) opening of K and Cl channels and the subsequent influx of K and Cl ions from the environment, (2) the metabolism of stored sucrose to smaller compounds, or (3) a combination of these two processes. The resulting increase in the intracellular solute concentration causes water to enter the guard cells osmotically, increasing their

612

CHAPTER 15

Transport across Cell Membranes

turgor pressure (Figure 15-34b). Since the guard cells are connected to each other only at their ends, the turgor pressure causes the cells to bulge outward, opening the stomatal pore between them. Stomatal closing is caused by the reverse processa decrease in solute concentration and turgor pressure within the guard cells. Stomatal opening is under tight physiological control by at least two mechanisms. A drop in CO2 within the leaf, resulting from active photosynthesis, causes the stomata to open, permitting additional CO2 to enter the leaf interior so that photosynthesis can continue. When more water exits the leaf than enters it from the roots, the mesophyll cells produce the hormone abscissic acid, which causes K efflux from the guard cells; water then exits the cells osmotically, and the stomata close, protecting the leaf from further dehydration.

S U M M A R Y Osmosis, Water Channels, and the Regulation of Cell Volume

Most biological membranes are more permeable to water than to ions or other solutes, and water moves across them by osmosis from a solution of lower solute concentration to one of higher solute concentration. Animal cells swell or shrink when placed in hypotonic or hypertonic solutions, respectively. To maintain their normal cytosolic osmolarity and hence cell volume, animal cells must export Na and other ions that leak or are transported from the extracellular space into the cytosol. The rigid cell wall surrounding plant cells prevents their swelling and leads to generation of turgor pressure in response to the osmotic influx of water. In response to the entry of water, protozoans maintain their normal cell volume by extruding water from contractile vacuoles. Aquaporin in the erythrocyte plasma membrane and other water-channel proteins increase the water permeability of biomembranes (see Figure 15-33). Opening and closing of K and Cl channels and the resulting changes in cytosolic solute concentrations of guard cells cause stomata in leaves to open and close (see Figure 15-34).

ATPase that acidifies the stomach is the most widely used drug for treating stomach ulcers. As we discuss in Chapter 21, the plasma membrane of nerve cells contains Na-symport proteins that are specifically inhibited by many drugs of abuse (e.g., cocaine) and antidepression medications (e.g., Prozac). Inhibitors of kidney channel proteins are widely used to control hypertension (high blood pressure); by blocking resorption of water from the forming urine into the blood, these drugs reduce blood volume and thus blood pressure. Calcium-channel blockers are widely employed to control the intensity of contraction of the heart. Within the next years, the human genome project will generate the sequences of all human membrane transport proteins. Soon after, researchers will discover in which types of cells and tissues these proteins are expressed. Using recombinant DNA techniques, scientists will be able to generate lines of cultured cells that express these in abundance, so that their molecular properties can be studied. Gene-knockout studies in mice will provide clues to their role in human physiology and disease. This basic knowledge will enable drug company researchers to identify new types of compounds that inhibit or activate just one of these transport proteins and not its homologs expressed in other types of cells. In this way, new and highly specific drugs will be developed to treat a variety of diseases. Physicians will also be able to identify individuals who may be at risk for certain types of diseases (e.g., hypertension or diabetes) because they have mutations in certain membrane transport proteins. And at the level of basic biology, we will all learn precisely how the human body digests and metabolizes all kinds of food and controls the levels of sugars, salts, fats, and other essential molecules in the blood and tissues.

PERSPECTIVES

in the Literature

PERSPECTIVES

for the Future

In this chapter, we have explained certain aspects of human physiology in terms of the action of specific membrane transport proteins. Such a molecular physiology approach has many medical applications. Even today, specific inhibitors or activators of channels, pumps, and transporters constitute the largest single class of drugs. For instance, an inhibitor of the gastric H/K

The Escherichia coli lactose permease is one of the most extensively studied membrane transport proteins. Encoded by the y gene of the lac operon (see Figure 10-1), this proton lactose symporter is essential for transport of lactose into the bacterial cell against its concentration gradient. Despite extensive efforts, the lactose permease has not been crystallized, and thus no three-dimensional structure is available. Nonetheless, Cysteine-scanning mutagenesis, in which every amino acid, in turn, is changed to cysteine, has been particularly revealing. The new cysteine residue can be subjected to a number of chemical modifications to determine its location relative to other amino acids and its involvement in lactose binding and transport. As you read the recent papers listed below, and earlier ones referenced in Frillingos et al., focus on the following questions: 1. What types of chemical, biochemical, and biophysical techniques were applied to the mutant proteins? What types of data do these different techniques produce?

MCAT/GRE-Style Questions

613

2. How did such studies lead to a detailed three-dimensional model of the arrangements of the twelve membrane-spanning -helices? 3. How did these studies identify amino acid residues crucial for binding of lactose at the exoplasmic surface and residues thought to conduct protons through the protein? 4. How did the authors determine that binding of the substrate induced tilting of several membrane-spanning -helices that might accompany transport of the proton and sugar? 5. What other experimental techniques might be applied to shed additional light on the structure and function of this protein? Frillingos, S., et al. 1998. Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins. FASEB J. 12:12811299. (A review.) Venkatesan, P., and H. R. Kaback. 1998. The substrate-binding site in the lactose permease of Escherichia coli. Proc. Natl Acad. Sci. USA 95:98029807. Wang, Q., et al. 1998. Proximity of helicesVIII (Ala 273) and IX (Met 299) in the lactose permease of Escherichia coli. Biochemistry 37:49104915. Wu, J., D. Hardy, and H. R. Kaback. 1998. Transmembrane helix tilting and ligand-induced conformational changes in the lactose permease determined by site-directed chemical crosslinking in situ. J. Mol. Biol. 282:959967.

MCAT/GRE-Style Questions
Key Concept Please read the section titled Na Entry

into Mammalian Cells Has a Negative G (p. 587) and answer the following questions (assume a membrane potential of 70 mV and the Na and K concentrations shown in Figure 15-8): 1. The Na/K ATPase pumps 2 moles of Na out of the cell for every 3 moles of K pumped into the cell. What is the G for pumping 1 mole of Na out of the cell? a. 3.03 kcal/mol. b. 1.41 kcal/mol. c. 1.61 kcal/mol. d. 3.03 kcal/mol. 2. What is the G for pumping 1 mole of K into the cell? a. 0.19 kcal/mol. b. 1.41 kcal/mol. c. 1.61 kcal/mol. d. 3.03 kcal/mol. 3. What is the overall energetics of one complete round of transport of 2 moles of Na and 3 moles of K? a. 6.63 kcal. b. 5.49 kcal. c. 6.03 kcal. d. 6.63 kcal. 4. How many ATPs are minimally consumed during one complete round of transport? a. 1. b. 2. c. 3. d. 5. 5. You treat the cell with a drug, a K ionophore, that selectively equilibrates K concentrations across the membrane. What now is the G for K transport by the ATPase? Assume that the membrane potential stays the same. a. 1.61 kcal/mol. b. 0.19 kcal/mol. c. 1.42 kcal/mol. d. 3.03 kcal/mol.
Key Experiment Please read the section titled Tight Junctions Seal Off Body Cavities and Restrict Diffusion of Membrane Components (p. 604) and answer the following questions:

Testing Yourself on the Concepts

1. The concentration of glucose in the mammalian bloodstream is about 5 mM and varies within the range of 3 mM after a few days of fasting to 7 mM after a feast. Considering these to be typical glucose levels, do you expect the Km for a glucose transporter to be 10 6 M or 10 3 M. Why? 2. Chemically, steroid hormones are lipids. On the basis of their expected permeability properties in biological membranes, predict whether receptor proteins for steroid hormones would be expected to be cell-surface or intracellular proteins. 3. Membrane transport proteins, be they uniports, symports, antiports, or ATPases, all have several transmembrane helices. How does this contribute to the function of the transport protein? 4. A Na/glucose symport can transport glucose against a concentration gradient. How is this energetically unfavorable process linked to ATP consumption, be it direct or indirect? 5. How might water channels be important in the opening of leaf stomata?

6. Functions of tight junctions include all the following except: a. Separation of extracellular fluids. b. Sealing of body cavities.

614

CHAPTER 15

Transport across Cell Membranes

c. Prevention of diffusion of membrane proteins and lipids between apical and basolateral regions. d. Tight communication and exchange of small molecules between neighboring cells. 7. Molecules present in or associated with tight junctions include all the following except: a. Connexin. b. Occludin and claudin. c. ZO-1, ZO-2, and ZO-3. d. Cytoskeletal linking proteins and actin. 8. Tight junctions may be reversibly dissociated by a. Mg2 removal and addition. b. Ca2 removal and addition. c. Glycosidase treatment. d. Trypsin treatment. 9. What is the expected effect on the distribution of plasmamembrane proteins between the apical and basolateral regions if tight junctions are dissociated? a. The distribution stays the same. b. Apical and basolateral proteins intermix. c. The distribution becomes even more distinct. d. The proteins are degraded. 10. Fluorescent lipids may be selectively introduced into the cytosolic leaflet of the apical membrane of epithelial cells in a two-step procedure. After completing the procedure, what is the expected distribution of the fluorescent lipids? a. Restricted to the apical surface. b. Restricted to the basolateral surface. c. Distributed in equal concentrations in the cytosolic and exoplasmic leaflets of the membrane. d. Distributed in equal concentrations in the apical and basolateral regions of the cell.
Key Medical Application Please read the section ti-

b. The ability of recombinant CFTR expressed in test COS cells to alter the Cl transport properties of these cells. c. The ability of recombinant CFTR expressed to confer cAMP sensitivity to C transport. d. The ability of recombinant CFTR inserted into liposomes to form Cl transport channels. 13. Introduction of CFTR into the lung is an attractive route for genetic correction of at least some of the symptoms of cystic fibrosis. This is true for all the following reasons except: a. DNA introduced into the lung rather than the bloodstream easily crosses the blood-brain barrier. b. The lung is easily accessible to DNA-containing aerosols. c. The lung is one of the major organs affected in CF. d. The introduction of DNA into lung cells does not alter the DNA of germ cells.

Key Terms
active transport 580 antiport 598 basal lamina 602 cell junctions 602 cotransport 598 epithelium 602 hypertonic 609 hypotonic 609 isotonic 609 Nernst equation 587 osmosis 608 passive diffusion 579 symport 598 tight junctions 605 uniport 582

References
Uniporter-Catalyzed Transport of Specific Molecules

tled Cystic Fibrosis Transmembrane Regulator (CFTR) Protein (p. 597) and answer the following questions: 11. Given the genetic and phenotypic traits of CF patients, the likely molecular defect in CF is a. A multigene trait. b. A defect in cAMP regulation of CFTR. c. Due to a mutation in the Q domain of CFTR. d. Due to a mutation in the protein with which CFTR interacts and which it regulates. 12. The name CFTR implies that as originally described the protein was thought not to be a Cl channel itself but rather a regulator of the Cl channel. What experimental observation shows that the CFTR protein itself is a Cl channel? a. The ability of recombinant CFTR expressed in the epithelial cells of cystic fibrosis patients to restore the Cl transport properties of these cells.

Bell, G., et al. 1993. Structure and function of mammalian facilitative sugar transporters. J. Biol. Chem. 268:1916119164. Henderson, P. J. 1993. The 12-transmembrane helix transporters. Curr. Opin. Cell Biol. 5:708721. Mueckler, M. 1994. Facilitative glucose transporters. Eur. J. Biochem. 219:713725. Malandro, M., and M. Kilberg. 1996. Molecular biology of mammalian amino acid transporters. Ann. Rev. Biochem. 66:305 336.
Ion Channels, Intracellular Ion Environment, and Membrane Electric Potential

Clapham, D., and B. Ehrlich, eds. 1996. Organellar Ion Channels and Transporters. Society of General Physiologists. Marine Biological Laboratory and Rockefeller University Press, New York. Choe, S., and R. Robinson. 1998. An ingenious filter: the structural basis for ion selectivity. Neuron 20:821823. Dawson, D., and R. Frizzell, eds. 1995. Ion Channels and Genetic Diseases. Society of General Physiologists. Marine Biological Laboratory and Rockefeller University Press, New York. Doyle, D. A., et al. 1998. The structure of the potassium channel: molecular basis of K conduction and selectivity. Science 280:69 77.

References

615

Hille, B. 1991. Ion Channels of Excitable Membranes. 2d ed. Sinauer Associates (Sunderland, Mass.). Racker, E. 1985. Reconstitutions of Transporters, Receptors, and Pathological States. Academic Press. Stein, W. D., and W. R. Leib. 1986. Transport and Diffusion across Cell Membranes. Academic Press.
Active Transport and ATP Hydrolysis

Boyer, P. D. 1997. The ATP synthasea splendid molecular machine. Ann. Rev. Biochem. 66:717749. Carafoli, E. 1992. The Ca 2 pump of the plasma membrane. J. Biol. Chem. 267:21152118. Doige, C. A., and G. F. Ames. 1993. ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance. Ann. Rev. Microbiol. 47:291319. Gottesman, M. M., I. Pastan, and S. V. Ambudkar. 1996. P-glycoprotein and multidrug resistance. Curr. Opin. Genet. Devel. 6:610617. Glynn, I. M. 1993. All hands to the sodium pump. J. Physiol. (London) 462:130. Higgins, C. F. 1995. The ABC of channel regulation. Cell 82:693 696. Jencks, W. P. 1995. The mechanism of coupling chemical and physical reactions by the calcium ATPase of sarcoplasmic reticulum and other coupled vectorial systems. Biosci. Rep. 15:283287. Lingrel, J. B., and T. Kuntzweiler. 1994. Na,K-ATPase. J. Biol. Chem. 269:1965919662. Linton, K. J., and C. F. Higgins. 1998. The Escherichia coli ATPbinding cassette (ABC) proteins. Mol. Microbiol. 28:513. Lutsenko, S., and J. Kaplan. 1995. Organization of P-type ATPases: significance of structural diversity. Biochemistry 34:15607 15613. MacLennan, D. H., W. J. Rice, and N. M. Green. 1997. The mechanism of Ca 2 transport by sarco(endo)plasmic reticulum Ca2ATPases. J. Biol. Chem. 272:2881528818. Nelson, N., and D. J. Klionsky. 1996. Vacuolar H-ATPase: from mammals to yeast and back. Experientia 52:11011110. Rea, P. A., et al. 1992. Vacuolar H-translocating pyrophosphatases: a new category of ion translocase. Trends Biochem. Sci. 17:348 353. Sachs, G. 1997. Proton pump inhibitors and acid-related diseases. Pharmacotherapy 17:2237. Shapiro, A. B., and V. Ling. 1995. Using purified P-glycoprotein to understand multidrug resistance. J. Bioenerg. Biomemb. 27:713. Stevens, T., and M. Forgac. 1997. Structure, function, and regulation of the vacuolar (H) ATPase. Ann. Rev. Cell Devel. Biol. 13:779808. Sze, H., J. M. Ward, and S. Lai. 1992. Vacuolar H-translocating ATPases from plants: structure, function, and isoforms. J. Bioenerg. Biomemb. 24:371381. Welsh, M. J., and A. E. Smith. 1993. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73:1251 1254. Zhang, P., et al. 1998. Structure of the calcium pump from sarcoplasmic reticulum at 8 resolution. Nature 392:835.
Cotransport Catalyzed by Symporters and Antiporters

Barkla, B., and O. Pantoja. 1996. Physiology of ion transport across the tonoplast of higher plants. Ann. Rev. Plant Physiol. and Plant Mol. Biol. 47:159184. Jay, D. G. 1996. Role of band 3 in homeostasis and cell shape. Cell 86:853854. Maathuis, F. J., and D. Sanders. 1992. Plant membrane transport. Curr. Opin. Cell Biol. 4:661669. Orlowski, J., and S. Grinstein. 1997. Na/H exchangers of mammalian cells. J. Biol. Chem. 272:2237322376. Ruetz, S., A. E. Lindsey, and R. R. Kopito. 1993. Function and biosynthesis of erythroid and nonerythroid anion exchangers. Soc. Gen. Physiol. Ser. 48:193200. Shrode, L. D., H. Tapper, and S. Grinstein. 1997. Role of intracellular pH in proliferation, transformation, and apoptosis. J. Bioenerg. Biomemb. 29:393399. Turk, E., and E. Wright. 1996. Membrane topology motifs in the SGLT cotransporter family. J. Memb. Biol. 159:120. Wakabayashi, S., M. Shigekawa, and J. Pouyssegur. 1997. Molecular physiology of vertebrate Na/H exchangers. Physiol. Rev. 77:5174. Wright, E. M., D. D. Loo, E. Turk, and B. A. Hirayama. 1996. Sodium cotransporters. Curr. Opin. Cell Biol. 8:468473.
Transport across Epithelia

Anderson, J. M., and C. M. Van Itallie. 1995. Tight junctions and the molecular basis for regulation of paracellular permeability. Am. J. Physiol. 269:G467G475. Cereijido, M., J. Valds, L. Shoshani, and R. Conteras. 1998. Role of tight junctions in establishing and maintaining cell polarity. Ann. Rev. Physiol. 60:161177. Goodenough, D. A. 1999. Plugging the leaks. Proc. Natl. Acad. Sci. USA 96:319. Gumbiner, B. M. 1996. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345357. Le Gall, A. H., C. Yeaman, A. Muesch, and E. Rodriquez-Boulan. 1995. Epithelial cell polarity: new perspectives. Semin. Nephrol. 15:272284. Mitic, L., and J. Anderson. 1998. Molecular architecture of tight junctions. Ann. Rev. Physiol. 60:121142. Nelson, W. J. 1992. Regulation of cell surface polarity from bacteria to mammals. Science 258:948955. Rubin, L. L. 1992. Endothelial cells: adhesion and tight junctions. Curr. Opin. Cell Biol. 4:830833. Schultz, S., et al., eds. 1997. Molecular Biology of Membrane Transport Disorders. Plenum Press.
Osmosis, Water Channels, and the Regulation of Cell V olume

Alper, S. L. 1991. The band 3-related anion exchanger (AE) gene family. Ann. Rev. Physiol. 53:549564. Baldwin, S. A. 1993. Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins. Biochim. Biophys. Acta 1154:1749.

Agre, P., M. Bonhivers, and M. Borgina. 1998. The aquaporins, blueprints for cellular plumbing systems. J. Biol. Chem. 273:14659. Chrispeels, M. J., and P. Agre. 1994. Aquaporins: water channel proteins of plant and animal cells. Trends Biochem. Sci. 19:421 425. Maathuis, F. J., A. M. Ichida, D. Sanders, and J. I. Schroeder. 1997. Roles of higher plant K channels. Plant Physiol. 114:1141 1149. Maurel, C. 1997. Aquaporins and water permeability of plant membranes. Ann. Rev. Plant Physiol. and Plant Mol. Biol. 48:399 430. Sarkadi, B., and J. C. Parker. 1991. Activation of ion transport pathways by changes in cell volume. Biochim. Biophys. Acta 1071:407427. Verkman, A. S., et al. 1996. Water transport across mammalian cell membranes. Am. J. Physiol. 270:C12C30.