Transport Across Cell Membrane

The plasma membrane is a selectively permeable barrier between thecell and the extracellular environment. Its permeability propertiesensure that essential molecules such as glucose, amino acids, and

lipids readily enter the cell, metabolic intermediates remain in the cell, andwaste compounds leave the cell. In short, the selectivepermeability of the plasma membrane allows the cell tomaintain a constant internal environment. In severalearlier chapters, we examined the components andstructural organization of cell membranes (see Figures 3-32 and 5-30). The phospholipid bilayer—the basicstructural unit of biomembranes—is essentiallyimpermeable to most water-soluble molecules, such asglucose and amino acids, and to ions. Transport of suchmolecules and ions across all cellular membranes ismediated by transport proteins associated with theunderlying bilayer. Because different cell types requiredifferent mixtures of low-molecular-weight compounds,the plasma membrane of each cell type contains a specificset of transport proteins that allow only certain ions ormolecules to cross. Similarly, organelles within the celloften have a different internal environment from that of the surrounding cytosol, and organelle membranescontain specific transport proteins that maintain thisdifference.

In animals, sheets of epithelial cells line all the bodycavities (e.g., the stomach, intestines, urinary bladder)

and the skin (see Figure 6-4). Epithelial cells frequently transport ions orsmall molecules from one side to the other. Those lining the small intestine,for instance, transport products of digestion (e.g., glucose and amino acids)

Transport across Cell Membranes

15

OUTLINE

15.1 Diffusion of Small Moleculesacross 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 585

15.5 Active Transport by ATP-Powered Pumps 588

15.6 Cotransport by Symporters and Antiporters 597

15.7 Transport across Epithelia 602

15.8 Osmosis, Water Channels, and the Regulation of Cell Volume 608

MEDIA CONNECTIONS

Overview: Biological Energy Interconversions

Classic Experiment 15.1: Stumbling uponActive Transport

MOHRIG: EXPER. ORGANIC CHEM. Figure: CD ICON 100% of size

Fine Line Illustrations (516) 781-7200 5/29/97�6/9/97

Three-dimensional structure of a gapjunction membrane channel

connecting two adjacent cells.

into the blood, and those lining the stomach secretehydrochloric acid into the stomach lumen. In order forepithelial cells to carry out these transport functions, theirplasma membrane must be organized into at least twodiscrete regions, each with different sets of transportproteins. In addition, specialized regions of the plasmamembrane interconnect epithelial cells, imparting strengthand rigidity to the sheet and preventing material on oneside from moving between the cells to the other.

In the first two sections of this chapter, we discuss the protein-independent movement of small hydrophobicmolecules across phospholipid bilayers and present anoverview of the various types of transport proteins presentin cell membranes. We then describe each of the maintypes of transport proteins. We also explain how specificcombinations of transport proteins in different subcellularmembranes enable cells to carry out essential physiologicalprocesses, including the maintenance of cytosolic pH, the transport of glucose across the absorptive intestinalepithelium, the accumulation of sucrose and salts in plant-cell vacuoles, and the directed flow of water in both plantsand animals. Often the same type of transport protein isinvolved in quite different physiological processes.

15.1 Diffusion of SmallMolecules acrossPhospholipid Bilayers

An artificial membrane composed of pure phospholipid orof phospholipid and cholesterol is permeable to gases, suchas O2 and CO2, and small, uncharged polar molecules, suchas urea and ethanol (Figure 15-1). Such molecules also cancross cellular membranes by passive diffusion unaided bytransport proteins. No metabolic energy is expended becausemovement is from a high to a low concentration of the mole-cule, down its chemical concentration gradient. As noted inChapter 2, such transport reactions have a positive DS value(increase in entropy) and a negative DG (decrease in free en-ergy). The relative diffusion rate of a substance across thebilayer is proportional to its concentration gradient acrossthe layer and to its hydrophobicity. There is little specificityto the process, in that any small hydrophobic molecule willbe transported.

The first step in transport by passive diffusion is move-ment of a molecule from the aqueous solution into thehydrophobic interior of the phospholipid bilayer. The hydro-phobicity of a substance is measured by its partition coeffi-cient, K, the equilibrium constant for its partition betweenoil and water. Since the composition of the interior of thephospholipid bilayer resembles that of oil, the partition co-efficient of a substance moving across a bilayer equals theratio of its concentration just inside the hydrophobic coreof the bilayer Cm to its concentration in the aqueous solu-tion Caq:

(15-1)

The partition coefficient is a measure of the relative affinityof a substance for lipid versus water: the higher a substance’spartition 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 40.0002) more hydrophobic than urea, will diffuse through

Diffusion of Small Molecules across Phospholipid Bilayers 579

Amino acidsATPGlucose 6-phosphate

H2O

CO2N2O2

Ethanol

Smallunchargedpolarmolecules

Largeunchargedpolarmolecules

Ions

Chargedpolarmolecules

Gases

WaterUrea

Glucose

K+, Mg2+, Ca2+,Cl−, HCO3

−,HPO4

2−

O

NH2NH2 C

▲ FIGURE 15-1 A pure artificial phospholipid bilayer ispermeable to small hydrophobic molecules and smalluncharged polar molecules. It is slightly permeable to waterand urea and impermeable to ions and to large uncharged polarmolecules. When a small phospholipid bilayer separates twoaqueous compartments, membrane permeability can be easilydetermined by adding a small amount of radioactive material toone compartment and measuring its rate of appearance in theother compartment.

phospholipid bilayer membranes about 50 times faster thanurea. Diethylurea also enters cells about 50 times fasterthan urea.

Once a molecule moves into the hydrophobic interior ofa bilayer, it diffuses across it; finally, the molecule movesfrom the bilayer into the aqueous medium on the other sideof the membrane. Because the hydrophobic core of a typi-cal cell membrane is 100–1000 times more viscous than wa-ter, the diffusion rate of all substances across a phospholipidmembrane is very much slower than the diffusion rate ofthe same molecule in water. Thus, movement across the hy-drophobic portion of a membrane is the rate-limiting stepin the passive diffusion of molecules across cell membranes.

Now let’s consider the passive diffusion of small mole-cules through a membrane more quantitatively. Suppose amembrane of surface area A and thickness x separates twosolutions of concentrations C1

aq and C2aq, where C1

aq . C2aq

(Figure 15-2). In this case, the diffusion rate dn/dt (in mol/s)is given by a modification of Fick’s law, which states thatthe diffusion rate across the membrane is directly propor-tional to the permeability coefficient P, to the difference insolution concentrations C1

aq 2 C2aq, and to the area A, or

(15-2)

For any molecule, the value of P, and thus its rate of pas-sive diffusion, is proportional to its partition coefficient K:

(15-3)

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

Thus we can see that the rate of diffusion is proportional toboth the partition coefficient and the diffusion constant andis inversely proportional to the membrane thickness. How-ever, the thickness of the hydrophobic interior of all phos-pholipid bilayer membranes is approximately the same,about 2.5 to 3 nm, and the diffusion coefficient D is thesame for most substances. Thus differences in the rate atwhich molecules passively diffuse across membranes de-pends 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 asethanol and urea, enter and leave cells by passive diffusionacross the plasma membrane. This transport is described byFick’s law. In the following sections, we will see how move-ment of other molecules and ions across cell membranes dif-fers from simple diffusion.

15.2 Overview of MembraneTransport Proteins

Very few molecules enter or leave cells, or cross organellarmembranes, unaided by proteins. Even transport of mole-cules, such as water and urea, that can diffuse across purephospholipid bilayers is frequently accelerated by transportproteins. The three major classes of membrane transportproteins are depicted in Figure 15-3a. All are integral trans-membrane proteins and exhibit a high degree of specificityfor the substance transported. The rate of transport by thethree types differs considerably owing to differences in theirmechanism of action.

ATP-powered pumps (or simply pumps) are ATPases thatuse the energy of ATP hydrolysis to move ions or small mole-cules across a membrane against a chemical concentrationgradient or electric potential. This process, referred to as ac-tive transport, is an example of a coupled chemical reaction(Chapter 2). In this case, transport of ions or small mole-cules “uphill” against a concentration gradient or electricpotential across a membrane, which requires energy, is cou-pled to the hydrolysis of ATP to ADP and Pi, which releasesenergy. The overall reaction—ATP hydrolysis and the “up-hill” movement of ions or small molecules—is energeticallyfavorable. Such pumps maintain the low calcium (Ca21) andsodium (Na1) ion concentrations inside virtually all animalcells relative to that in the medium, and generate the lowpH inside animal-cell lysosomes, plant-cell vacuoles, and thelumen of the stomach.

580 CHAPTER 15 Transport across Cell Membranes

x

C2aqC1

aq C1m C2

m

x

A

▲ FIGURE 15-2 A simple model for passive diffusionof small hydrophobic molecules directly across thehydrocarbon core of a pure phospholipid bilayer of thicknessx in centimeters and area A in square centimeters. C1

aq andC2

aq are the concentrations of two solutions on sides 1 and 2 ofthe membrane; C1

m and C2m are the corresponding con-

centrations just within the hydrocarbon core of the bilayer.Movement of a solute molecule is indicated by the blue arrow.

Channel proteins transport water or specific types of ionsdown their concentration or electric potential gradients, anenergetically favorable reaction. They form a protein-linedpassageway across the membrane through which multiplewater molecules or ions move simultaneously, single file ata very rapid rate—up to 108 per second. As discussed in alater section, the plasma membrane of all animal cells con-tains potassium-specific channel proteins that are generallyopen and are critical to generating the normal, resting elec-tric potential across the plasma membrane. Many othertypes of channel proteins are usually closed, and open onlyin response to specific signals. Because these types of ionchannels play a fundamental role in the functioning of nervecells, they will be discussed in detail in Chapter 21.

Transporters, a third class of membrane transport pro-teins, move a wide variety of ions and molecules across cellmembranes. In contrast to channel proteins, transporters

bind only one (or a few) substrate molecules at a time; af-ter binding substrate molecules, the transporter undergoesa conformational change such that the bound substrate mol-ecules, and only these molecules, are transported across themembrane. Because movement of each substrate molecule(or small number of molecules) requires a conformationalchange in the transporter, transporters move only about102–104 molecules per second, a lower rate than that asso-ciated with channel proteins.

Three types of transporters have been identified (Figure15-3b). Uniporters transport one molecule at a time downa concentration gradient. This type of transporter, for ex-ample, moves glucose or amino acids across the plasmamembrane into mammalian cells. In contrast, antiportersand symporters couple the movement of one type of ion ormolecule against its concentration gradient to the movementof a different ion or molecule down its concentration gra-dient. Like ATP pumps, antiporters and symporters medi-ate coupled reactions in which an energetically unfavorablereaction is coupled to an energetically favorable reaction.Because symporters and antiporters catalyze “uphill” move-ment of certain molecules, they are often referred to as “ac-tive transporters,” but unlike pumps, they do not hydrolyzeATP (or any other molecule) during transport. A better termfor these proteins is cotransporters, referring to their abil-ity to transport two different solutes simultaneously.

To study the functional properties of the different kindsof membrane-transport proteins, researchers need experi-mental systems in which a particular transport protein pre-dominates. In one common approach, a specific transportprotein is extracted and purified; the purified protein thenis reincorporated into pure phospholipid bilayer membranes,such as liposomes (Figure 15-4). Alternatively, the gene en-coding a transport protein can be expressed at high levelsin a cell normally not expressing it; the difference in trans-port of a substance by the transfected and nontransfectedcells will be due to the expressed transport protein. In thesesystems, the functional properties of the various membraneproteins can be examined without ambiguity.

S U M M A R Y Overview of Membrane TransportProteins

• The plasma membrane regulates the traffic of mole-cules into and out of the cell.

• Gases and small hydrophobic molecules diffuse directlyacross the phospholipid bilayer at a rate proportional totheir ability to dissolve in a liquid hydrocarbon.

• Ions, sugars, amino acids, and sometimes water can-not diffuse across the phospholipid bilayer at sufficientrates to meet the cell’s needs and must be transportedby a group of integral membrane proteins includingchannels, transporters, and ATP-powered ion pumps(see Figure 15-3).

Overview of Membrane Transport Proteins 581

ATP

(b)

ATP-powered pump(100 − 103 ions/s)

Ion channel(107 − 108 ions/s)

Transporter(102 − 104 molecules/s)

ClosedOpen

Uniporter Symporter Antiporter

Exterior

Cytosol

ADP + Pi

(a)

▲ FIGURE 15-3 Schematic diagrams illustrating action ofmembrane transport proteins. Gradients are indicated bytriangles with the tip pointing toward lower concentration,electrical potential, or both. (a) The three major types of transportproteins. Pumps utilize the energy released by ATP hydrolysis topower movement of specific ions (red circles) or small moleculesagainst their electrochemical gradient. Channels catalyze movementof specific ions (or water) down their electrochemical gradient.Transporters, which fall into three groups, facilitate movement ofspecific small molecules or ions (black circles). (b) The threegroups of transporters. Uniporters, also shown in part (a), transporta single type of molecule down its concentration gradient.Cotransport proteins (symporters and antiporters) catalyze themovement 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 ofcotransporters differ in the relative direction of movement of thetransported molecule and cotransported ion.

• Two common experimental systems for studying thefunctions of transport proteins are liposomes containinga purified transport protein (see Figure 15-4) and cellstransfected with the gene encoding a particular trans-port protein.

15.3 Uniporter-CatalyzedTransport

We begin our discussion of membrane transport proteinswith the simplest type, which catalyze uniport transport. Theplasma membrane of most cells contains several uniportersthat enable amino acids, nucleosides, sugars, and other smallmolecules to enter and leave cells down their concentrationgradients. Similar to enzymes, uniporters accelerate a reac-tion that is already thermodynamically favored, and the

movement of a substance across a membrane down its con-centration gradient will have the same negative DG valuewhether or not a protein transporter is involved. This typeof movement sometimes is referred to as facilitated trans-port (or facilitated diffusion). As we stressed in Chapter 2,many chemical reactions that are thermodynamically favoredwill not occur unless an appropriate enzyme is present; suchis also the case with movement of hydrophilic moleculesacross biological membranes. Unlike the substrates of enzy-matic reactions, however, transported substances undergono chemical change during movement across a membrane.

Three Main Features Distinguish UniportTransport from Passive DiffusionThree properties of uniporter-catalyzed movement of glucoseand other small hydrophilic molecules across a membranedistinguish this type of transport from passive diffusion:

1. The rate of facilitated transport by uniporters is far higherthan predicted by Fick’s equation describing passive diffu-sion (Figure 15-5). Because the transported molecules neverenter the hydrophobic core of the phospholipid bilayer, thepartition coefficient K is irrelevant.

2. Transport is specific. Each uniporter transports only a single species of molecule or a single group of closely relatedmolecules.

3. Transport occurs via a limited number of uniporter mol-ecules, rather than throughout the phospholipid bilayer.Consequently, there is a maximum transport rate Vmax thatis achieved when the concentration gradient across the mem-brane is very large and each uniporter is working at its max-imal rate.

Figure 15-5 shows the initial rate of glucose uptake byerythrocytes at different external glucose concentrations.Since the concentration of glucose is usually higher in theextracellular medium than in the cell, the plasma-membraneglucose transporters usually catalyze net movement of glu-cose in one direction: from the medium into the cell. Underthis condition, Vmax is achieved at high external glucose con-centrations. However, if the concentration gradient is re-versed, the glucose transporter, like all uniporters, is equallyable to catalyze net movement in the reverse direction: fromthe cell into the medium. Such a situation occurs in livercells during periods of starvation, when these cells synthe-


Intacterythrocytemembrane

Detergentmolecules

Dialyze or diluteto remove detergent

Othertransport protein

Glucosetransport protein

Disrupt membrane,solubilize protein withdetergents, and purify

Phospholipids

Mix withphospholipids

Glucose

Liposome withglucose transport protein

Glucose

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

▲

size glucose (from fatty acids, amino acids, and other smallmolecules) and release it into the blood, and in intestinal ep-ithelial cells during transport of glucose from the intestineto the blood.

GLUT1 Transports Glucose into Most Mammalian CellsVirtually all mammalian cells use blood glucose as the majorsource of cellular energy, and most express GLUT1, a plasma-membrane uniporter that catalyzes movement of glucosedown its concentration gradient. The properties of GLUT1,as well as of many other transport proteins, have been ex-tensively studied in the mammalian erythrocyte, since thiscell has no nucleus and no internal membranes; it is essen-tially a “bag” of hemoglobin containing relatively few otherintracellular proteins (Figure 15-6). We discuss GLUT1 insome detail as an example of the uniport type of transportprotein.

The glucose transporter GLUT1 alternates between twoconformational states: in one, a glucose-binding site facesthe outside of the membrane; in the other, a glucose-bind-

ing site faces the inside. Figure 15-7 depicts the sequence ofevents occurring during the unidirectional transport of glu-cose from the cell exterior inward to the cytosol. GLUT1also can catalyze the net movement of glucose from the cyto-sol outward by reversal of steps 1–4 shown in Figure 15-7.Experimental support for this model, which is thought toapply to other uniport proteins as well, has come from kine-tic experiments discussed below.

Kinetics of GLUT1-Catalyzed Movement of Glucose Asnoted previously, a plot of the entry rate of glucose into ery-throcytes versus external glucose concentration is not linear;rather, it is a curve that levels off at Vmax at high externalglucose concentrations (see Figure 15-5). The kinetics of theunidirectional transport of glucose (and other small mole-cules) from the outside of a cell inward via a uniporter canbe described by the same type of equation used to describea simple enzyme-catalyzed chemical reaction.

For simplicity, let’s assume that a substance S (say, glucose)is present initially only on the outside of the membrane. Inthis case, we can write

where Km is the substance-transporter binding constant andVmax is the maximum transport rate of S into the cell. By asimilar derivation used to arrive at the Michaelis-Mentenequation in Chapter 3, we can derive the following expres-sion for v, the transport rate for S into the cell:

(15-4)

where C is the concentration of Sout (initially, the concentra-tion of Sin 5 0); Vmax is the rate of transport if all moleculesof the transporter contain a bound S, which occurs at high

Uniporter-Catalyzed Transport 583

500

250

Rat

e o

f g

luco

se u

pta

ke (

v)

External concentration of glucose (mM)1 2 3 4 5 6 7 8 9 10 11 12 13 140

1/2Vmax

Vmax

Facilitated transport

Km

Passive diffusion

▲ FIGURE 15-5 Comparison of the observed uptake rate ofglucose by erythrocytes (red curve) with the calculated rateif glucose were to enter solely by passive diffusion throughthe phospholipid bilayer (blue curve). The rate of glucoseuptake (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 theinitial concentration of glucose in the erythrocyte is zero, so thatthe concentration gradient of glucose across the membrane isthe same as the external concentration. The glucose transporterin the erythrocyte membrane clearly increases the rate ofglucose transport, compared with that associated with passivediffusion, at all glucose concentrations. Like enzymes, thetransporter-catalyzed uptake of glucose exhibits a maximumtransport rate Vmax and is said to be saturable. The Km is theconcentration at which the rate of glucose uptake is half-maximal.

▲ FIGURE 15-6 Normal human erythrocytes, viewed bydifferential interference light microscopy, are disk shapedand contain no internal membranes. The opposite surfacealso is concave. [Courtesy of M. Murayama, Biological Photo Service.]

ds ds dsds ds

dsdsdsds

dsdsds

ds ds

ds

Sout concentrations; and Km is the substrate concentrationat which half-maximal transport occurs across the mem-brane. The lower the value of Km, the more tightly the sub-strate binds to the transporter, and the greater the transportrate. Equation 15-4 describes the curve for glucose uptakeshown in Figure 15-5.

For GLUT1 in the erythrocyte membrane, the Km forglucose transport is 1.5 millimolar (mM); at this concen-tration roughly half the transporters with outward-facingbinding sites would have a bound glucose. Blood glucose isnormally 5 mM, or 0.9 g/L. At this concentration, the ery-throcyte glucose transporter is functioning at 77 percent ofthe maximal rate Vmax, as can be seen from Figure 15-5.

The kinetics of glucose transport are more complex andmore revealing than this simple analysis suggests. For instance,if [14C]glucose is added to a suspension of erythrocyteswhose intracellular glucose concentration is zero, the labeledglucose is transported inward at a particular initial rate proportional to the concentration of labeled glucose, as de-scribed by Equation 15-4. This initial rate of [14C]glucosetransport is accelerated severalfold if unlabeled glucose ispresent inside the cells before addition of the labeled glucose.This unexpected experimental observation indicates that theslow (rate-determining) step in the inward transport of glu-cose is the change in GLUT1 from a conformation with anunoccupied inward-facing glucose-binding site to a confor-mation with an unoccupied outward-facing binding site (step4 n step 5 in Figure 15-7). This conformational change isaccelerated severalfold when an unlabeled glucose moleculebinds to the inward-facing site and is transported outward.This result adds strong support to the conformational-change model of GLUT1 depicted in Figure 15-7.

Specificity and Structure of GLUT1 As noted above, theKm for glucose transport by GLUT1 is 1.5 mM. The Km forthe nonbiological L-isomer of glucose is .3000 mM; thusat concentrations at which D-glucose is readily transported


Plasmamembrane

Outward-facingglucose-bindingsite

Inward-facing glucose-binding siteBound glucose

GlucoseExterior

Cytosol

1 2 3 4 5

Glucose

▲ FIGURE 15-7 Model of the mechanism of uniport transportby GLUT1, which is believed to shuttle between twoconformational states. In one conformation ( 1 , 2 , and 5 ), theglucose-binding site faces outward; in the other ( 3 , 4 ), thebinding site faces inward. Binding of glucose to the outward-facingbinding site ( 1 n 2 ) triggers a conformational change in thetransporter ( 2 n 3 ), moving the bound glucose through theprotein 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 reverseconformational change ( 4 n 5 ), inactivating the inward-facingglucose binding site and regenerating the outward-facing one. Ifthe concentration of glucose is higher inside the cell than outside,the cycle will work in reverse ( 4 n 1 ), catalyzing net movementof glucose from inside to out.

into the erythrocyte, L-glucose does not enter at a measur-able rate. The isomeric sugars D-mannose and D-galactose,which differ from D-glucose in the configuration at only onecarbon atom (see Figure 2-8), also are transported by GLUT1at measurable rates. However, the Km for D-mannose is20 mM and for D-galactose is 30 mM, so that considerablyhigher concentrations of these substrates than of D-glucoseare needed to half-saturate the transport reaction. ThusGLUT1 is quite specific, having a much higher affinity (in-dicated by a lower Km) for the normal substrate D-glucosethan for other substrates.

After glucose is transported into the erythrocyte, it is rap-idly phosphorylated, forming glucose 6-phosphate, which can-not leave the cell (see Figure 16-3). Because this reaction isthe first step in the metabolism of glucose, the intracellularconcentration of free glucose does not increase as glucose istaken up by the cell. Consequently, the glucose concentra-tion gradient across the membrane is maintained, as is therate of glucose entry into the cell.

GLUT1 is an integral, transmembrane protein with a mole-cular weight of 45,000. It accounts for 2 percent of the protein in the plasma membrane of erythrocytes. Insertionof purified GLUT1 into artificial liposomes dramatically increases their permeability to D-glucose (see Figure 15-4).This artificial system exhibits all the properties of glucoseentry 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 glu-cose transporter indicate that it contains 12 a helices that spanthe phospholipid bilayer. Although the amino acid residues inthe transmembrane a helices are predominantly hydrophobic,several helices bear amino acid residues (e.g., serine, threo-nine, asparagine, and glutamine) whose side chains can formhydrogen bonds with the hydroxyl groups on glucose. Theseresidues are thought to form the inward-facing and outward-facing glucose-binding sites in the interior of the protein.

S U M M A R Y Uniporter-Catalyzed Transport

• Uniport-type membrane transport proteins operate toimport many types of molecules into the cell driven onlyby a concentration gradient, a process termed facilitatedtransport or facilitated diffusion.

• Three main features distinguish uniport transportfrom passive diffusion: the rate of transport is far higherthan predicted by Fick’s equation, transport is specific,and transport occurs via a limited number of trans-porter proteins rather than throughout the phospholipidbilayer.

• 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 inthe plasma membrane of most mammalian cells, allowsonly glucose and closely related sugars to cross the bi-layer 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 thoughtto involve a similar conformational-change mechanism.

15.4 Intracellular IonEnvironment andMembrane ElectricPotential

The movement of ions across the plasma membrane and or-ganelle membranes is mediated by several types of transportproteins: all symporters and certain antiporters cotransportions simultaneously along with specific small molecules,whereas ion channels, ion pumps, and some antiporterstransport only ions. In all cases, the rate and extent of iontransport across membranes is influenced not only by theion concentrations on the two sides of the membrane butalso by the voltage (i.e., the electric potential) that existsacross the membrane. Here we discuss the origin of the elec-tric potential across the plasma membrane and its relation-ship to ion channels within the membrane.

Ionic Gradients and an Electric Potential AreMaintained across the Plasma MembraneThe specific ionic composition of the cytosol usually differsgreatly from that of the surrounding fluid. In virtually allcells—including microbial, plant, and animal cells—the cy-tosolic pH is kept near 7.2 and the cytosolic concentrationof K1 is much higher than that of Na1. In addition, in bothinvertebrates and vertebrates, the concentration of K1 is

20–40 times higher in cells than in the blood, while the con-centration of Na1 is 8–12 times lower in cells than in theblood (Table 15-1). The concentration of Ca21 free in thecytosol is generally less than 0.2 micromolar (2 3 1027 M),a thousand or more times lower than that in the blood. Plantcells and many microorganisms maintain similarly high cy-tosolic concentrations of K1 and low concentrations of Ca21

and Na1 even if the cells are cultured in very dilute salt so-lutions. The ATP-driven ion pumps that generate and main-tain these ionic gradients are discussed later.

In addition to ion pumps, which transport ions againsttheir concentration gradients, the plasma membrane containschannel proteins that allow the principal cellular ions (Na1,K1, Ca21, and Cl2) to move through it at different ratesdown their concentration gradients. Ion concentration gra-dients 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 respectto the outside. This value does not seem like much until werealize that the plasma membrane is only about 3.5 nm thick.Thus the voltage gradient across the plasma membrane is0.07 V per 3.5 3 1027 cm, or 200,000 volts per centimeter!(To appreciate what this means, consider that high-voltagetransmission lines for electricity utilize gradients of about200,000 volts per kilometer!) As explained below, the plasmamembrane, like all biological membranes, acts like a capac-itor—a device consisting of a thin sheet of nonconductingmaterial (the hydrophobic interior) surrounded on bothsides by electrically conducting material (the polar head

Intracellular Ion Environment and Membrane Electric Potential 585

TABLE 15-1 Typical Ion Concentrations in Invertebrates and Vertebrates

Cell BloodIon (mM) (mM)

SQUID AXON*K1 400 20Na1 50 440Cl2 40–150 560Ca21 0.0003 10X2† 300–400 5–10

MAMMALIAN CELL

K1 139 4Na1 12 145Cl2 4 116HCO3

2 12 29X2 138 9Mg21 0.8 1.5Ca21 ,0.0002 1.8

*The large nerve axon of the squid, an invertebrate cell, has been widelyused in studies of the mechanism of conduction of electric impulses.†X2represents proteins, which have a net negative charge at the neutralpH of blood and cells.

groups and the ions in the surrounding aqueous medium)—that can store positive charges on one side and negativecharges on the other.

The ionic gradients and electric potential across the plasmamembrane drive many biological processes. Opening andclosing of Na1, K1, and Ca21 channels are essential to theconduction of an electric impulse down the axon of a nervecell (Chapter 21). In many animal cells, the Na1 concen-tration gradient and the membrane electric potential powerthe uptake of amino acids and other molecules against theirconcentration gradient; this transport is catalyzed by ion-linked symport and antiport proteins. In most cells, a risein the cytosolic Ca21 concentration is an important regula-tory signal, initiating contraction in muscle cells and trig-gering secretion of digestive enzymes in the exocrine pan-creatic cells.

Here we discuss the role of ion channels in generatingthe membrane electric potential. Later we examine the ATP-powered ion pumps that generate ion concentration gradi-ents, and ion-linked cotransport proteins.

The Membrane Potential in Animal CellsDepends Largely on Resting K1 ChannelsIn the experimental system outlined in Figure 15-8a, the dis-tribution of K1, Na1, and Cl2 ions is similar to that betweenan animal cell and its aqueous environment. A membraneseparates a 15 mM KCl/150 mM NaCl solution on the rightside (representing the “outside” of the cell) from a 150 mMKCl/15 mM NaCl solution on the left side (the “inside”).A potentiometer (voltmeter) is connected to the solution oneach side to measure any difference in electric potentialacross the membrane. If the membrane is impermeable toall ions, no ions will flow across it; there will be no electricpotential across it.


15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

+ –+ –+ –

Na+

Na+

Na+ Na+ channel

Net charge

(b) Membrane permeable to Na+ only

At equilibrium, potential is –59 mV,with the right side negative with respect to the left

K+ channel

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

– +– +– +

K+

K+

K+

(c) Membrane permeable to K+ only

Net charge

At equilibrium, potential is +59 mV,with the right side positivewith respect to the left

−60 +60

0

−60 +60

0

(a) Membrane impermeable to Na+, K+, and Cl−

−60 +60

0

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

Zero potentialdifference

Cell cytosol Extracellularmedium

Potentiometer

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

+ –+ –+ –

Na+

Na+

Na+ Na+ channel

Net charge



K+ channel

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

– +– +– +

K+

K+

K+


Net charge


−60 +60

0

−60 +60

0


−60 +60

0

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−



Potentiometer

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

+ –+ –+ –

Na+

Na+

Na+ Na+ channel

Net charge



K+ channel

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−

– +– +– +

K+

K+

K+


Net charge


−60 +60

0

−60 +60

0


−60 +60

0

15 mMNa+Cl−

150 mMK+Cl−

150 mMNa+Cl−

15 mMK+Cl−



Potentiometer

FIGURE 15-8 Experimental system for generating atransmembrane voltage potential across a membraneseparating a 150 mM KCl/15 mM NaCl solution (a similarcomposition to that of the cell cytosol) from a 15 mMKCl/150 mM NaCl solution (concentrations similar to thosein blood). (a) An impermeable membrane prevents ion movementacross the membrane, and thus no difference in electric potentialis registered on the potentiometer connecting the two solutions.(b) If the membrane is selectively permeable only to Na1, thenNa1 ions diffuse from right to left, through Na1 channels. As aconsequence, a net positive charge builds up on the left side anda net negative charge builds up on the right side of the membrane.At equilibrium, the membrane potential caused by the chargeseparation becomes equal to the Nernst potential ENa registeredon the potentiometer, and the movement of Na1 ions in the twodirections becomes equal. (c) If the membrane is selectivelypermeable only to K1, diffusion of K1 ions from left to rightthrough K1 channels causes accumulation of a net negativecharge on the left side and a net positive charge on the right side.At equilibrium, the membrane electric potential is equal to EK.

▲

Now suppose that the membrane contains Na1-channelproteins that accommodate Na1 ions but exclude K1 andCl2 ions. Na1 ions then tend to move down their concen-tration gradient from the right side to the left, leaving anexcess of negative Cl2 ions compared with Na1 ions on theright side and generating an excess of positive Na1 ions com-pared with Cl2 ions on the left side. The excess Na1 on theleft and Cl2 on the right remain near the respective surfacesof the membrane, since, as in a capacitor, the excess posi-tive charges on one side of the membrane are attracted tothe excess negative charges on the other side. The resultingseparation of charge across the membrane can be measuredby a potentiometer as an electric potential, or voltage, withthe right side of the membrane negative (having excess neg-ative charge) with respect to the left (Figure 15-8b).

As more and more Na1 ions move through channelsacross the membrane, the magnitude of this charge differ-ence (i.e., voltage) increases. However, continued right-to-leftmovement of the Na1 ions eventually is inhibited by the mutual repulsion between the excess positive (Na1) chargesaccumulated on the left side of the membrane and by the attraction of Na1 ions to the excess negative charges builtup on the right side. The system soon reaches an equilibriumpoint at which the two opposing factors that determine themovement of Na1 ions—the membrane electric potential andthe ion concentration gradient—balance each other out. Atequilibrium, no net movement of Na1 ions occurs across themembrane. Thus the excess negative (Cl2) charges bound tothe right surface of the membrane are separated from andattracted to the excess positive (Na1) ones on the left. In thisway, the phospholipid membrane, with its nonconducting hy-drophobic interior bounded by the conducting polar headgroups and adjacent aqueous medium, stores the chargeacross it exactly as does a capacitor in an electric circuit.

If a membrane is permeable only to Na1 ions, then themeasured electric potential across the membrane equals thesodium equilibrium potential in volts, ENa. The magnitudeof ENa is given by the Nernst equation, which is derivedfrom basic principles of physical chemistry:

(15-5)

where R (the gas constant) 5 1.987 cal/(degree ? mol), or8.28 joules/(degree ? mol); T (the absolute temperature) 5293 K at 20 °C, Z (the valency) 5 11, F (the Faraday con-stant) 5 23,062 cal/(mol ? V), or 96,000 coulombs/(mol ? V), and [Nal] and [Nar] are the Na1 concentrations on the leftand right sides, respectively, at equilibrium. The Nernstequation is similar to the equations used to calculate thevoltage change associated with oxidation or reduction re-actions (Chapter 2), which also involve movement of elec-tric charges. At 20 °C, Equation 15-5 reduces to

(15-6)

If [Nal]/[Nar] 5 0.1, as in Figure 15-8b, then ENa 520.059 V (259 mV), with the right side negative with respectto the left.

If the membrane is permeable only to K1 ions and notto Na1 or Cl2 ions, then a similar equation describes thepotassium equilibrium potential EK:

(15-7)

The magnitude of the membrane electric potential is thesame (59 mV), except that the right side is now positive withrespect to the left (Figure 15-8c), opposite to the polarityobtained with selective Na1 permeability.

As noted earlier, the membrane potential across the plasmamembrane of animal cells is about 270 mV; that is, the cyto-solic face is negative with respect to the exoplasmic (outside)face. These membranes contain many open K1 channels butfew open Na1 or Ca21 channels. As a result, the major ionicmovement across the plasma membrane is that of K1 fromthe inside outward, leaving an excess of negative charge onthe inside and creating an excess of positive charge on theoutside. Thus the flow of K1 ions through these open chan-nels, called K1 leak channels or resting K1 channels, is themajor determinant of the inside-negative membrane potential.Quantitatively, the usual resting membrane potential of 270mV is close to but less than that of the potassium equilibriumpotential calculated from the Nernst equation. The K1 con-centration gradient that drives the flow of ions through rest-ing K1 channels is generated by an ion pump that transportsK1 ions into the cytosol from the extracellular medium andNa1 ions out. In the absence of this pump, which is discussedlater, the K1 concentration gradient could not be maintainedand eventually the membrane potential would fall.

Recent cloning and molecular characterization of restingK1 channels show that the channel protein is built of fouridentical subunits. Each subunit contains two membrane-spanning a helices, which partially line the ion-conductingpore in the middle of the protein, and a shorter looped Psegment, which acts as a filter to allow K1 but not otherions to enter the pore and cross the membrane. As we dis-cuss in Chapter 21, the structure of resting K1 channels isgenerally similar to the structures of other ion channels thatare critical to the function of nerve cells.

Although resting K1 channels play the dominant role ingenerating the electric potential across the plasma membraneof animal cells, this is not the case in plant and fungal cells.The inside-negative membrane potential in these cells is gen-erated by transport of H1 ions out of the cell by an ATP-powered proton pump.

Na1 Entry into Mammalian Cells Has a Negative DGAs we’ve seen, two forces govern the movement of such ionsas K1, Cl2, and Na1 across selectively permeable membranes:

Intracellular Ion Environment and Membrane Electric Potential 587

the voltage and the ion concentration gradient across themembrane. These forces may act in the same direction or inopposite directions. To calculate the free-energy change DGcorresponding to the transport of any ion across a mem-brane, we need to consider the contribution from each ofthese forces independent of the other.

For example, in a reaction where Na1 moves from out-side to inside the cell, the free-energy change generated fromthe Na1 concentration gradient is given by

(15-8)

At the concentrations of Nain and Naout shown in Figure15-9, which are typical for many mammalian cells, DGc

would be 21.45 kcal/mol, the change in free energy for thethermodynamically favored transport of 1 mol of Na1 ionsfrom outside to inside the cell if there were no membraneelectric potential. The free-energy change generated from themembrane electric potential is given by

(15-9)

where F is the Faraday constant and E is the membraneelectric potential. If E 5 270 mV, then DGm would be21.6 kcal/mol, the change in free energy for the thermody-

namically favored transport of 1 mol of Na1 ions from out-side to inside the cell if there were no Na1 concentrationgradient. Given both forces acting on Na1 ions, the totalDG will be the sum of the two partial values:

In this typical example, the Na1 concentration gradient andthe membrane electric potential contribute almost equally tothe total DG for transport of Na1 ions. Since DG is ,0, theinward movement of Na1 ions is thermodynamically favored.As discussed later, certain cotransport proteins use the in-ward movement of Na1 to power the uphill movement ofseveral ions and small molecules into or out of animal cells.

S U M M A R Y Intracellular Ion Environmentand Membrane Electric Potential

• ATP-driven ion pumps generate and maintain ionicgradients across the plasma membrane. As a result, theionic composition of the cytosol usually differs greatlyfrom that of the surrounding fluid (see Table 15-1).

• In both invertebrates and vertebrates, the K1 concen-tration is higher and the Na1 concentration is lower incells than in the blood. The cytosolic Ca21 concentrationis maintained at less than 0.2 mM.

• An inside-negative electric potential (voltage) of50–70 mV exists across the plasma membrane of allcells; this is equivalent to a voltage gradient of200,000 volts per centimeter.

• In animal cells, the electric potential across theplasma membrane is generated primarily by movementof cytosolic K1 ions through resting K1 channels to theexternal medium. Unlike most other ion channels,which open only in response to various signals, theseK1 channels are usually open.

• In plants and fungi, the membrane potential is main-tained by the ATP-driven pumping of protons from thecytosol across the membrane.

• Two forces govern the movement of ions across selec-tively permeable membranes: the membrane electric poten-tial and the ion concentration gradient, which may act inthe same or opposite directions. For the thermodynami-cally favored inward movement of Na1 into animal cells,these forces act in the same direction (see Figure 15-9).

15.5 Active Transport by ATP-Powered Pumps

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


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OutsideInsideOutsideInside––––

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OutsideInside–

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12 mMNa+

145 mMNa+

∆Gc = −1.45 kcal/mol

∆G = ∆Gc + ∆Gm = −3.06 kcal/mol

∆Gm = −1.61 kcal/mol

Free-energy change duringtransport of Na+ from outsideto inside

Na+

Na+

Ion concentrationgradient

Membraneelectrical potential

–

▲ FIGURE 15-9 Transmembrane forces acting on Na1 ions.As with all ions, the movement of Na1 ions across the plasmamembrane is governed by the sum of two separate forces—themembrane electric potential and the ion concentration gradient. Inthe case of Na1 ions, these forces usually act in the same direction.

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of these transport proteins are depicted in Figure 15-10, andtheir properties are summarized in Table 15-2. Note that theP, F, and V classes transport ions only, whereas the ABC super-family class transports small molecules as well as ions.

P-class ion pumps contain a transmembrane catalytic asubunit, which contains an ATP-binding site, and usually asmaller b subunit, which may have regulatory functions.Many of these pumps are tetramers composed of two a andtwo b subunits. During the transport process, at least oneof the a subunits is phosphorylated (hence the label “P”),and the transported ions are thought to move through thephosphorylated subunit. This class includes the Na1/K1

ATPase in the plasma membrane, which maintains the Na1

and K1 gradients typical of animal cells, and several Ca21

ATPases, which pump Ca21 ions out of the cytosol into theexternal medium or into the lumen of the sarcoplasmic retic-ulum (SR) of muscle cells. Another member of the P class,found in acid-secreting cells of the mammalian stomach,transports protons (H1 ions) out of and K1 ions into thecell. The H1 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 sim-ilar to each other but unrelated to and more complicatedthan P-class pumps. F- and V-class pumps contain at leastthree kinds of transmembrane proteins and five kinds of ex-trinsic polypeptides that form the cytosolic domain. Severalof the transmembrane and extrinsic subunits in F-class and

V-class pumps exhibit sequence homology, and each pair ofhomologous subunits is thought to have evolved from a com-mon polypeptide.

All known V and F pumps transport only protons in aprocess that does not involve a phosphoprotein intermediate.V-class pumps generally function to maintain the low pH ofplant vacuoles and of lysosomes and other acidic vesicles inanimal cells by using the energy released by ATP hydrolysisto pump protons from the cytosolic to the exoplasmic faceof the membrane against the proton electrochemical gradient.F-class pumps are found in bacterial plasma membranes andin mitochondria and chloroplasts. In contrast to V pumps,they generally function to power the synthesis of ATP fromADP and Pi by movement of protons from the exoplasmicto the cytosolic face of the membrane down the proton elec-trochemical gradient. Because of their importance in ATPsynthesis in chloroplasts and mitochondria, F-class protonpumps are treated separately in the next chapter.

The final class of ATP-powered transport proteins islarger and more diverse than the other classes. Referred toas the ABC (ATP-binding cassette) superfamily, this class in-cludes more than 100 different transport proteins found inorganisms ranging from bacteria to humans. Each ABC pro-tein is specific for a single substrate or group of related sub-strates including ions, sugars, peptides, polysaccharides, andeven proteins. All ABC transport proteins share a commonorganization consisting of four “core” domains: two trans-membrane (T) domains, forming the passageway through

Active Transport by ATP-Powered Pumps 589

c

b A

T

A

T

a

Exterior

Cytosol

ATP-binding region

P-class pump F- and V-class pump ABC superfamily

c c

α α

β

βα

ε

δ

γ

ATP-binding region

▲ FIGURE 15-10 The four classes of ATP-powered transportproteins. P-class pumps are composed of two differentpolypeptides, a and b, and become phosphorylated as part ofthe transport cycle. The sequence around the phosphorylatedresidue, located in the larger a subunits, is homologous amongdifferent pumps. F-class and V-class pumps do not formphosphoprotein intermediates. Their structures are similar andcontain similar proteins, but none of their subunits are relatedto those of P-class pumps. All members of the large ABC

superfamily of proteins contain four core domains: twotransmembrane (T) domains and two cytosolic ATP-binding (A)domains that couple ATP hydrolysis to solute movement. Thesecore domains are present as separate subunits in some ABCproteins (depicted here), but are fused into a single polypeptidein 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. Nat’l. Acad. Sci. USA 94:10583; and T. Elston, H. Wang,

and G. Oster, 1998, Nature 391:510.]

which transported molecules cross the membrane, and twocytosolic 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 multi-domain polypeptides.

All classes of ATP-powered pumps have one or more bind-ing sites for ATP, and these are always on the cytosolic face ofthe membrane (see Figure 15-10). Although these proteins areoften called ATPases, they normally do not hydrolyze ATP intoADP and Pi unless ions or other molecules are simultaneously

transported. Because of the tight coupling between ATP hydro-lysis and transport, the energy stored in the phosphoanhy-dride bond is not dissipated. Thus ATP-powered transportproteins are able to collect the free energy released duringATP hydrolysis and use it to move ions or other moleculesuphill against a potential or concentration gradient.

The energy expended by cells to maintain the concentra-tion gradients of Na1, K1, H1, and Ca21 across the plasmaand intracellular membranes is considerable. In nerve and kid-ney cells, for example, up to 25 percent of the ATP produced


TABLE 15-2 Comparison of Major Classes of ATP-Powered Ion and Small-Molecule Pumps

P Class F Class V Class ABC Class

Substances Transported

H1, Na1, K1, Ca21 H1 only H1 only Ions and various smallmolecules

Structural and Functional Features

Large catalytic a Multiple transmembrane Multiple transmembrane Two transmembranesubunits (often two) and cytosolic subunits and cytosolic subunits domains form the pathwaybecome phosphorylated generally function generally use energy for solute; two cytosolicduring solute transport; to synthesize ATP released by ATP hydrolysis ATP-binding domainssmaller b subunits may on b cytosolic subunits to pump H1 ions from couple ATP hydrolysisregulate transport. powered by movement cytosol to organelle to solute movement.

of H1 down an lumens, acidifying them. Domains may be in oneelectrochemical gradient. or separate subunits.

Location of Specific Pumps

Plasma membrane of Bacterial plasma Vacuolar membranes in Bacterial plasma plants, fungi, bacteria membranes plants, yeast, other fungi membranes (amino acid,(H1 pump) sugar, and peptide

transporters)

Plasma membrane of Inner mitochondrial Endosomal and lysosomal Mammalian endoplasmichigher eukaryotes membrane membrane in animal cells reticulum (transporters of(Na1/K1 pump) peptides associated with

antigen presentation byMHC proteins)

Apical plasma membrane Thylakoid membrane of Plasma membrane ofof mammalian stomach chloroplast certain acid-secreting animalcells (H1/K1 pump) cells (e.g., osteoclasts and

some kidney tubule cells)

Plasma membrane of Mammalian plasmaall eukaryotic cells membranes (transporters(Ca21 pump) of small molecules,

phospholipids, small lipidlike drugs)

Sarcoplasmic reticulummembrane in muscle cells (Ca21 pump)

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 pro-duction of ATP (e.g., 2,4-dinitrophenol), the ion concentra-tion inside the cell gradually approaches that of the exteriorenvironment as the ions move through plasma membranechannels down their electric and concentration gradients.Eventually treated cells die: partly because protein synthesisrequires a high concentration of K1 ions and partly becausein the absence of a Na1 gradient across the cell membrane,a cell cannot import certain nutrients such as amino acids.Studies on the effects of such poisons provided early evidencefor the existence of ion pumps. In this section, we discussin some detail examples of the P, V, and ABC classes of ATP-powered pumps.

Plasma-Membrane Ca21 ATPase Exports Ca21 Ions from CellsAs discussed in Chapter 20, small increases in the concen-tration of free Ca21 ions in the cytosol trigger a variety ofcellular responses. In order for Ca21 to function in intra-cellular signaling, its cytosolic concentration usually mustbe kept below 0.1–0.2 mM. (Although some cytosolic Ca21

is bound to negatively charged groups, it is the concentra-tion of free, unbound Ca21 that is critical to its signalingfunction.) The plasma membranes of animal, yeast, andprobably plant cells contain Ca21 ATPases that transportCa21 out of the cell against its electrochemical gradient.These P-class ion pumps help maintain the concentration offree Ca21 ions in the cytosol at a low level.

In addition to a catalytic a subunit containing an ATP-binding site, as found in other P-class pumps, plasma-membrane Ca21 ATPases also contain the Ca21-binding reg-ulatory protein calmodulin. A rise in cytosolic Ca21 inducesthe binding of Ca21 ions to calmodulin, which triggers anallosteric activation of the Ca21 ATPase; as a result, the ex-port of Ca21 ions from the cell accelerates, and the originallow cytosolic concentration of free Ca21 is restored rapidly.

Muscle Ca21 ATPase Pumps Ca21 Ions from the Cytosol into the Sarcoplasmic ReticulumBesides the plasma-membrane Ca21 ATPase, muscle cells con-tain a second, different Ca21 ATPase that transports Ca21

from the cytosol into the lumen of the sarcoplasmic reticu-lum (SR), an internal organelle that concentrates and storesCa21 ions. As discussed in Chapter 18, the SR and its cal-cium pump (referred to as the muscle calcium pump) arecritical in muscle contraction and relaxation: release of Ca21

ions from the SR into the muscle cytosol causes contraction,and the rapid removal of Ca21 ions from the cytosol by themuscle calcium pump induces relaxation.

Because the muscle calcium pump constitutes more than80 percent of the integral protein in SR membranes, it is eas-ily purified and characterized. Each transmembrane catalytic

a subunit has a molecular weight of 100,000 and transportstwo Ca21 ions per ATP hydrolyzed. In the cytosol of musclecells, the free Ca21 concentration ranges from 1027 M (rest-ing cells) to more than 1026 M (contracting cells), whereas thetotal Ca21 concentration in the SR lumen can be as high as1022 M. Sites on the cytosolic surface of the muscle calciumpump have a very high affinity for Ca21 (Km 5 1027 M),allowing the pump to transport Ca21 efficiently from thecytosol into the SR against the steep concentration gradient.

The concentration of free Ca21 within the sarcoplasmicreticulum is actually much less than the total concentrationof 1022 M. Two soluble proteins in the lumen of SR vesi-cles bind Ca21 and serve as a reservoir for intracellular Ca21,thereby reducing the concentration of free Ca21 ions in theSR vesicles, and consequently decreasing the energy neededto pump Ca21 ions into them from the cytosol. The activityof the muscle Ca21 ATPase is so regulated that if the freeCa21 concentration in the cytosol becomes too high, the rateof calcium pumping increases until the cytosolic Ca21 con-centration is reduced to less than 1 µM. Thus in muscle cells,the calcium pump in the SR membrane can supplement theactivity of the plasma-membrane pump, assuring that thecytosolic concentration of free Ca21 remains below 1 mM.

The current model of the mechanism of action of the Ca21

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 theprotein is in one conformation, termed E1, two Ca21 ionsbind in sequence to high-affinity sites on the cytosolic sur-face (step 1). Then an ATP binds to its site on the cytosolicsurface; in a reaction requiring that a Mg21 ion be tightlycomplexed to the ATP, the bound ATP is hydrolyzed to ADPand the liberated phosphate is transferred to a specific aspar-tate residue in the protein, forming a high-energy acyl phos-phate bond, denoted by E1,P (step 2). The protein thenchanges its conformation to E2–P, generating two low-affinity Ca21-binding sites on the exoplasmic surface, whichfaces the SR lumen; this conformational change simultane-ously propels the two Ca21 ions through the protein to thesesites (step 3) and inactivates the high-affinity Ca21-bindingsites on the cytosolic face. The Ca21 ions then dissociate fromthe exoplasmic surface of the protein (step 4). Following this,the aspartyl-phosphate bond in E2–P is hydrolyzed, causingE2 to revert to E1, a change that inactivates the exoplasmic-facing Ca21-binding sites and regenerates the cytosolic-facing Ca21-binding sites (step 5).

Thus phosphorylation of the muscle calcium pump by ATPfavors conversion of E1 to E2, and dephosphorylation favorsthe conversion of E2 to E1. While only E2–P, not E1,P, isactually hydrolyzed, the free energy of hydrolysis of the as-partyl-phosphate bond in E1,P is greater than that for E2–P. The reduction in free energy of the aspartyl-phosphatebond in E2–P, relative to E1,P, can be said to power theE1 n E2 conformational change. The affinity of Ca21 forthe cytosolic-facing binding sites in E1 is a thousandfoldgreater than the affinity of Ca21 for the exoplasmic-facing


sites in E2; this difference enables the protein to transportCa21 unidirectionally from the cytosol, where it binds tightlyto the pump, to the exoplasm, where it is released.

Much evidence supports the model depicted in Figure15-11. For instance, the muscle calcium pump has been iso-lated with phosphate linked to an aspartate residue, andspectroscopic studies have detected slight alterations in pro-tein conformation during the E1 n E2 conversion. On thebasis of the protein’s amino acid sequence and various bio-chemical studies, investigators proposed the structural model


Ca2+

E1

2 Ca2+

E1 E1

ATP ADP

P

1 2

3

Low-affinity Ca+-binding sites

High-affinity Ca+-binding sites

ATP siteCytosol

4

PP

E2 E2

▲ FIGURE 15-11 Model of the mechanism of action of muscleCa21 ATPase, which is located in the sarcoplasmic reticulum(SR) membrane. Only one of the two a subunits of this P-classpump is depicted. E1 and E2 are alternate conformational formsof the protein in which the Ca21-binding sites are on the cytosolicand exoplasmic faces, respectively. An ordered sequence of steps,

as diagrammed here, is essential for coupling ATP hydrolysis andthe transport of Ca21 ions (red circles) across the membrane. ,Pindicates a high-energy acyl phosphate bond; —P indicates alow-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.]

SR lumen

1 2 3 4 5 6 7 8 9 10

Cytosol

Ca2+-binding residue

Transmembraneα helix

COO−12

nm

Energytransduction

Phosphorylationof aspartate

ATPbinding

H3+N

Globulardomains

FIGURE 15-12 Schematic structuralmodel for the catalytic (a) subunit ofmuscle Ca21 ATPase. The 10transmembrane a helices are thought toform a channel through which Ca21 ionsmove. Site-specific mutagenesis studieshave identified four residues (red dots),located in four of the transmembranehelices, that participate in Ca21 binding.Trypsin digestion releases three cytosolicglobular domains, which constitute thebulk of the protein. One cytosolic domainfunctions in ATP binding; a second bearsthe 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.]

▲

for the catalytic a subunit shown in Figure 15-12. The mem-brane-spanning a helices are thought to form the passage-way through which Ca21 ions move. The bulk of the sub-unit consists of cytosolic globular domains that are involvedin ATP binding, phosphorylation of aspartate, and energytransduction. These domains are connected by “stalks” tothe membrane-embedded domain.

As noted previously, all P-class ion pumps, regardless ofwhich ion they transport, are phosphorylated during thetransport process. The amino acid sequences around the

phosphorylated aspartate in the catalytic a subunit arehighly conserved in all proteins of this type. Thus the mech-anistic model in Figure 15-11 probably is generally applicableto all these ATP-powered ion pumps. In addition, the a sub-units of all the P pumps examined to date have a similar mole-cular weight and, as deduced from their amino acid sequencesderived from cDNA clones, have a similar arrangement oftransmembrane a helices (see Figure 15-12). These findingsstrongly suggest that all these proteins evolved from a com-mon precursor, although they now transport different ions.

Na1/K1 ATPase Maintains the IntracellularNa1 and K1 Concentrations in Animal CellsA second P-class ion pump that has been studied in consid-erable detail is the Na1/K1 ATPase present in the plasmamembrane of all animal cells. This ion pump is a tetramerof subunit composition a2b2. (Classic Experiment 15.1 de-scribes the discovery of this enzyme.) The b polypeptide isrequired for newly synthesized a subunits to fold properlyin the endoplasmic reticulum but apparently is not involved

directly in ion pumping. The a subunit is a 120,000-MWnonglycosylated polypeptide whose amino acid sequenceand predicted membrane structure are very similar to thoseof the muscle Ca21 ATPase. In particular, the Na1/K1 AT-Pase has a stalk on the cytosolic face that links domains con-taining the ATP-binding site and the phosphorylated aspar-tate to the membrane-embedded domain. The overallprocess of transport moves three Na1 ions out of and twoK1 ions into the cell per ATP molecule split (Figure 15-13a).

Several lines of evidence indicate that the Na1/K1 ATPaseis responsible for the coupled movement of K1 and Na1 intoand out of the cell, respectively. For example, the drug ouabain,which binds to a specific region on the exoplasmic surface ofthe protein and specifically inhibits its ATPase activity, alsoprevents cells from maintaining their Na1/K1 balance. Anydoubt that the Na1/K1 ATPase is responsible for ion move-ment was dispelled by the demonstration that the enzyme,when purified from the membrane and inserted into lipo-somes, propels K1 and Na1 transport in the presence of ATP.

The mechanism of action of the Na1/K1 ATPase, outlinedin Figure 15-13b, is similar to that of the muscle calcium


P

2 K+

3 Na+

K+

Na+Exterior

Cytosol

ATP

Oligosaccharide

ADP + Pi

Low-affinityNa+-binding sites

(a)

(b) High-affinityK+-binding site

ATP

ATP site

ADP

PP

E2E1E1

Binding of ATP,phosphorylationof aspartate

High-affinityNa+-bindingsite

Low-affinityK+-bindingsites

E1E2E2 E1

Pi

E2 E1conformationalchange, inwardtransport of K+

Hydrolysisof aspartylphosphate

Binding of 3 Na+ ions

Dissociationof Na+ ,binding of K+

Dissociationof K+ ions

E1 E2conformationalchange, outwardtransport of Na+

α β

FIGURE 15-13 Models for thestructure and function of the Na1/K1

ATPase in the plasma membrane.(a) This P-class pump comprises twocopies each of a small glycosylated bsubunit and a large a subunit, whichperforms ion transport. Hydrolysis of one molecule of ATP to ADP and Pi iscoupled to export of three Na1 ions (bluecircles) and import of two K1 ions (darkred triangles) against their concentrationgradients (large triangles). It is not knownwhether only one a subunit, or both, in asingle ATPase molecule transports ions.(b) Ion pumping by the Na1/K1 ATPase involves a high-energy acyl phosphateintermediate (E1,P) and conformationalchanges, similar to transport by themuscle Ca21 ATPase. In this case,hydrolysis of the E2–P intermediatepowers transport of a second ion (K1)inward. Na1 ions are indicated by bluecircles; K1 ions, by red triangles. Seetext for details. [Adapted from P. Läuger,

1991, Electrogenic Ion Pumps, Sinauer

Associates, p. 178.]

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pump, except that ions are pumped in both directions acrossthe membrane. In its E1 conformation, the Na1/K1 ATPasehas three high-affinity Na1-binding sites and two low-affinityK1-binding sites on the cytosolic-facing surface of the pro-tein. The Km for binding of Na1 to these cytosolic sites is0.6 mM, a value considerably lower than the intracellularNa1 concentration of <12 mM; as a result, Na1 ions nor-mally will fill these sites. Conversely, the affinity of the cyto-solic K1-binding sites is low enough that K1 ions, trans-ported inward through the protein, dissociate from E1 intothe cytosol despite the high intracellular K1 concentration.During the E1 n E2 transition, the three bound Na1 ionsmove outward through the protein. Transition to the E2 con-formation also generates two high-affinity K1 sites and threelow-affinity Na1 sites on the exoplasmic face. Because theKm for K1 binding to these sites (0.2 mM) is considerablylower than the extracellular K1 concentration (4 mM), thesesites will fill quickly with K1 ions. In contrast, the threeNa1 ions, transported outward through the protein, will dis-sociate into the extracellular medium from the low-affinityNa1 sites on the exoplasmic surface despite the high extra-cellular Na1 concentration. Similarly, during the E2 n E1transition, the two bound K1 ions are transported inward.

V-Class H1 ATPases Pump Protonsacross Lysosomal and Vacuolar MembranesAll V-class ATPases transport H1 ions only. These protonpumps, present in the membranes of lysosomes, endosomes,and plant vacuoles, function to acidify the lumen of these organ-elles. The acidity of the lysosomal lumen, usually <4.5–5.0,can be measured precisely in living cells by use of particleslabeled with a pH-sensitive fluorescent dye. Cells phagocy-tose these particles (see Figure 5-44a) and transfer them tothe lysosomes. The ability of different wavelengths of visiblelight to excite fluorescence is highly dependent on pH, andthe lysosomal pH can be calculated from the spectrum of thefluorescence emitted. Maintenance of the 100-fold or moreproton gradient between the lysosomal lumen (pH <4.5–5.0)and the cytosol (pH <7.0) depends on ATP production bythe cell.

The ATP-powered proton pumps in lysosomal and vac-uolar membranes have been isolated, purified, and incor-porated into liposomes. As illustrated in Figure 15-10, theseV-class proton pumps contain two discrete domains: a cyto-solic-facing hydrophilic domain (V1) composed of five dif-ferent polypeptides and a transmembrane domain (V0) con-taining 9–12 copies of proteolipid c, one copy of protein b,and one copy of protein a. The subunit composition of thecytosolic domain is a3b3gde; the a and b subunits containthe sites where ATP binding and hydrolysis occur. Eachtransmembrane c subunit is thought to span the membranetwo times; the c and a subunits together form the proton-conducting channel. Unlike P-class ion pumps, the V-classH1 ATPases are not phosphorylated and dephosphorylatedduring proton transport.

Similar V-class ATPases are found in the plasma mem-brane of certain acid-secreting cells. These include osteoclasts,bone-resorbing macrophagelike cells, which bind to a boneand seal off a small segment of extracellular space betweenthe plasma membrane and the surface of the bone. HCl secreted into this space by osteoclasts dissolves the calciumphosphate crystals that give bone its rigidity and strength.

Another example is the mitochondria-rich epithelial cellslining the toad bladder; the apical plasma membrane of thesecells contain many V-class H1 ATPases, which function toacidify the urine (Figure 15-14). As we discuss later, the mem-brane of plant vacuoles contains two proton pumps: a typicalV-class H1 ATPase and another one that utilizes the energyreleased by hydrolysis of inorganic pyrophosphate (PPi) topump protons into the vacuole. This PPi-hydrolyzing protonpump, believed to be unique to plants, has an amino acid se-quence different from any other ion-transporting proteins.

ATP-powered proton pumps cannot acidify the lumen ofan organelle (or the extracellular space) by themselves. Thereason for this is that pumping of protons would rapidlycause a buildup of positive charge on the exoplasmic faceof the membrane on the inside of the vesicle membrane anda corresponding buildup of negative charges on the cytoso-lic face. In other words, the pump would generate a voltageacross the membrane, exoplasmic face positive, which wouldprevent movement of protons into the vesicle before a sig-nificant H1 concentration gradient had been established. Infact, this is the way that H1 pumps generate an inside-negative potential across plant and yeast plasma membranes.In order for an organelle lumen or an extracellular space


▲ FIGURE 15-14 The plasma membrane of certain acid-secreting cells contains an almost crystalline array of V-classH1 ATPases. This electron micrograph is of a platinum replica ofthe cytosolic surface of the apical plasma membrane of a toadbladder epithelial cell. Each stud is a single V-class H1 ATPase(<600,000 MW) composed of several polypeptide subunitssurrounding a central channel. [From D. Brown, S. Gluck, and

J. Hartwig, 1987, J. Cell Biol. 105:1637.]

(e.g., the outside of an osteoclast) to become acidic, move-ment of H1 up its concentration gradient must be accom-panied by (1) movement of an equal number of anions inthe same direction or (2) movement of equal numbers of adifferent cation in the opposite direction. The first processoccurs in lysosomes and plant vacuoles whose membranescontain V-class H1 ATPases and ion channels through whichaccompanying anions (e.g., Cl2) move. The second occursin the lining of the stomach, which contains a P-class H1/K1

ATPase that pumps one H1 outward and one K1 inward.

The ABC Superfamily Transports a Wide Variety of SubstratesAs noted earlier, all members of the very large and diverseABC superfamily of transport proteins contain two trans-membrane (T) domains and two cytosolic ATP-binding (A)domains (see Figure 15-10). The T domains, each built ofsix membrane-spanning a helices, form the pathway throughwhich the transported substance (substrate) crosses themembrane and determine the substrate specificity of eachABC protein. The sequence of the A domains is <30 to 40percent homologous in all members of this superfamily, indicating a common evolutionary origin. Some ABC pro-teins also contain a substrate-binding subunit or regulatorysubunit.

Bacterial Plasma-Membrane Permeases The plasmamembrane of many bacteria contain numerous permeasesthat belong to the ABC superfamily. These proteins use theenergy released by hydrolysis of ATP to transport specificamino acids, sugars, vitamins, or even peptides into the cell.Since bacteria frequently grow in soil or pond water wherethe concentration of nutrients is low, these ABC transportproteins allow the cells to concentrate amino acids and othernutrients in the cell against a substantial concentration

gradient. Bacterial permeases generally are inducible; that is,the quantity of a transport protein in the cell membrane isregulated by both the concentration of the nutrient in themedium and the metabolic needs of the cell.

In E. coli histidine permease, a typical bacterial ABC pro-tein, the two transmembrane domains and two cytosolicATP-binding domains are formed by four separate subunits.In gram-negative bacteria such as E. coli, which have anouter membrane, a soluble histidine-binding protein in theperiplasmic space assists in transport (Figure 15-15). Thissoluble protein binds histidine tightly and directs it to theT subunits, through which histidine crosses the membranepowered by ATP hydrolysis. Mutant E. coli cells that aredefective in any of the histidine-permease subunits or thesoluble binding protein are unable to transport histidine intothe cell, but are able to transport other amino acids whoseuptake is facilitated by other transport proteins. Such ge-netic analyses provide strong evidence that histidine perme-ase and similar ABC proteins function to transport solutesinto the cell.

Mammalian MDR Transport Proteins A seriesof rather unexpected observations led to discoveryof the first eukaryotic ABC protein. Oncologists

noted that tumor cells often became simultaneously resist-ant to several chemotherapeutic drugs with unrelated chem-ical structures; similarly, cell biologists observed that cul-tured cells selected for resistance to one toxic substance (e.g.,colchicine, a microtubule inhibitor) frequently became resis-tant to several other drugs, including the anticancer drugadriamycin. Subsequent studies showed that this resistanceis due to enhanced expression of a multidrug-resistance(MDR) transport protein known as MDR1. In this memberof the ABC superfamily, all four domains are “fused” intoa single 170,000-MW protein (Figure 15-16). This protein


ADP + Pi

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FIGURE 15-15 Gram-negativebacteria import many solutes by meansof ABC proteins (permeases) that utilizea soluble substrate-binding proteinpresent in the periplasmic space.Depicted here is the import of the aminoacid histidine. After diffusing throughporins in the outer membrane, histidine is bound by a specific periplasmichistidine-binding protein, which undergoesa conformational change. The histidine-protein complex binds to the exoplasmicsurface of a T subunit in histidine permeaselocated in the plasma membrane.Hydrolysis of ATP bound to the A subunitthen powers movement of histidinethrough the protein into the cytosol. Thetransport process does not appear toinvolve a phosphoprotein intermediate.

▲

ds

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uses the energy derived from ATP hydrolysis to export alarge variety of drugs from the cytosol to the extracellularmedium. The Mdr1 gene is frequently amplified in multidrug-resistant cells, resulting in a large overproduction of theMDR1 protein.

Most drugs transported by MDR1 are small hydrophobicmolecules, which diffuse from the culture medium across theplasma membrane into the cell. The ATP-powered export ofsuch drugs from the cytosol by MDR1 means a much higherextracellular drug concentration is required to kill cells. ThatMDR1 is an ATP-powered small-molecule pump has beendemonstrated with liposomes containing the purified protein(see Figure 15-4). The ATPase activity of these liposomes isenhanced by different drugs in a dose-dependent mannercorresponding to their ability to be transported by MDR1.

Not only does MDR1 transport a varied group of mol-ecules, but all these substrates compete with one another fortransport by MDR1. Although the mechanism of action ofMDR1-assisted transport has not been definitively demon-strated, 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 thecytosolic-facing leaflet of the plasma membrane. The hydro-phobic portion of a substrate molecule is oriented towardthe hydrophobic core of the membrane, and the chargedportion toward the polar cytosolic face of the membraneand is still in the cytosol. The substrate diffuses laterally un-til encountering and binding to a site on the MDR1 proteinthat is within the bilayer. The protein then “flips” the chargedsubstrate molecule into the exoplasmic leaflet, an energeti-cally unfavorable reaction powered by the coupled ATPaseactivity of MDR1. Once in the exoplasmic face, the sub-strate diffuses into the aqueous phase on the outside of thecell. Support for the flippase model of transport by MDR1comes from MDR2, a homologous protein present in the region of the liver cell plasma membrane that faces the bileduct. MDR2 has been shown to flip phospholipids from thecytosolic-facing leaflet of the plasma membrane to the exo-plasmic leaflet, thereby generating an excess of phospholipids


Transmembraneα helix

Exterior

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H3+N COO−

ATP binding ATP binding

FIGURE 15-16 Schematic structuralmodel for mammalian MDR1 protein. Inthis member of the ABC superfamily, thetwo transmembrane domains and twocytosolic ATP-binding domains are part ofa single polypeptide. Each transmembranedomain contains six a helices. The two halvesof this 1280-aa protein have similar aminoacid sequences. A variety of lipid-solublemolecules that diffuse across the plasmamembrane into the cell are transportedoutward by MDR1.

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Chargedend

HydrophobicendSubstrate

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3

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2

▲ FIGURE 15-17 Possible mechanisms of action of the MDR1protein. (a) The flippase model proposes that a lipid-solublemolecule first dissolves in the cytosolic-facing leaflet of theplasma membrane ( 1 ) and then diffuses in the membrane untilbinding to a site on the MDR1 protein that is within the bilayer( 2 ). Powered by ATP hydrolysis, the substrate molecule flips intothe exoplasmic leaflet ( 3 ), from which it can move directly into

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

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.]

in the exoplasmic leaflet; these phospholipids peel off intothe bile duct and form an essential part of the bile. An alter-native pump model also has been proposed for MDR1 (Figure 15-17b). According to this model, drug molecules inthe cytosol bind directly to a single small-molecule bindingsite on the cytosolic face of the MDR1 protein; subsequentATP hydrolysis powers movement of the bound drugthrough the protein to the aqueous phase on the outside ofthe cell by a mechanism similar to that of other ATP-pow-ered pumps.

MDR1 protein is expressed in abundance in the liver, intestines, and kidney—sites from which natural toxic prod-ucts are removed from the body. Thus the natural functionof MDR1 may be to transport a variety of natural and meta-bolic toxins into the bile, intestinal lumen, or forming urine.During the course of its evolution, MDR1 appears to havecoincidentally acquired the ability to transport drugs whosestructures are similar to those of these toxins. Tumors de-rived from these cell types, such as hepatomas (liver cancers),frequently are resistant to virtually all chemotherapeuticagents and thus difficult to treat, presumably because thetumors exhibit increased expression of the MDR1 or MDR2proteins.

Cystic Fibrosis Transmembrane Regulator(CFTR) Protein Discovery of another ABC trans-port protein came from studies of cystic fibrosis

(CF), the most common lethal autosomal recessive geneticdisease of Caucasians. This disease is caused by a mutationin the CFTR gene, which encodes a chloride-channel proteinthat is regulated by cyclic AMP (cAMP), an intracellular second messenger. These Cl2 channels are present in the apical plasma membranes of epithelial cells in the lung, sweatglands, pancreas, and other tissues. An increase in cAMPstimulates Cl2 transport by such cells from normal individ-uals, but not from CF individuals who have a defectiveCFTR protein.

The sequence and predicted structure of the encodedCFTR protein, based on analysis of the cloned gene, are verysimilar to those of MDR1 protein except for the presenceof an additional domain, the regulatory (R) domain, on thecytosolic face. The Cl2-channel activity of CFTR proteinclearly is enhanced by binding of ATP. Moreover, as detailedin Chapter 20, cAMP activates a protein kinase that phos-phorylates, and thereby activates, CFTR. When purifiedCFTR protein is incorporated into liposomes, it forms Cl2

channels with properties similar to those in normal epithe-lial cells. And when the wild-type CFTR protein is expressedby recombinant techniques in cultured epithelial cells fromCF patients, the cells recover normal Cl2-channel activity.This latter result raises the possibility that gene therapymight reverse the course of cystic fibrosis.

Since CFTR protein is similar to MDR1 in structure, itmay also function as an ATP-powered pump of some as-yetunidentified molecule. In any case, much remains to be learnedabout this fascinating class of ABC transport proteins.

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

• Four types of membrane transport proteins couplethe energy-releasing hydrolysis of ATP with the energy-requiring transport of substances against their concen-tration gradient (see Figure 15-10 and Table 15-2).

• In P-class pumps, phosphorylation of the a subunitand a change in conformational states are essential forcoupled transport of H1, Na1, K1, or Ca21 ions (seeFigures 15-11 and 15-13).

• The P-class Na1/K1 ATPase pumps three Na1 ions outof and two K1 ions into the cell per ATP hydrolyzed. Ahomolog, the Ca21 ATPase, pumps two Ca21 ions out ofthe cell or, in muscle, into the sarcoplasmic reticulum perATP hydrolyzed. The combined action of these pumps inanimal cells creates an intracellular ion milieu of high K1,low Ca21, and low Na1 very different from the extra-cellular fluid milieu of high Na1, high Ca21, and low K1.

• In the multisubunit V-and F-class ATPases, whichpump protons exclusively, a phosphorylated protein isnot an intermediate in transport.

• A V-class H1 pump in animal lysosomal and endo-somal 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 super-family of transport proteins contain four core domains:two transmembrane domains, which form a pathwayfor solute movement and determine substrate specificity,and two cytosolic ATP-binding domains.

• The ABC superfamily includes bacterial amino acidand sugar permeases (see Figure 15-15); the mammalianMDR1 protein, which exports a wide array of drugsfrom cells; and CFTR protein, a Cl2 channel that is defective in cystic fibrosis.

• According to the flippase model of MDR1 activity, asubstrate molecule diffuses into the cytosolic leaflet ofthe plasma membrane, then is flipped to the exoplasmicleaflet in an ATP-powered process, and finally diffusesfrom the membrane into the extracellular space (see Figure 15-17a).

15.6 Cotransport by Symporters and Antiporters

Besides ATP-powered pumps, cells have a second, discreteclass of proteins that import or export ions and small mole-cules, such as glucose and amino acids, against a concen-tration gradient. These proteins use the energy stored in theelectrochemical gradient of Na1 or H1 ions to power the

Cotransport by Symporters and Antiporters 597

uphill movement of another substance, a process called co-transport. For instance, the energetically favored movementof a Na1 ion (the “cotransported” ion) into a cell across theplasma membrane, driven both by its concentration gradi-ent and by the transmembrane voltage gradient (see Figure15-9), can be coupled obligatorily to movement of the“transported” molecule (e.g., glucose) against its concen-tration gradient. When the transported molecule and co-transported ion move in the same direction, the process iscalled symport; when they move in opposite directions, theprocess is called antiport (see Figure 15-2b).

Na1-Linked Symporters Import Amino Acidsand Glucose into Many Animal CellsMany cells, such as those lining the small intestine and thekidney tubules, need to concentrate glucose against a verylarge concentration gradient. Such cells utilize a twoNa1/one-glucose symporter; a protein that couples trans-membrane movement of one glucose molecule to the trans-port of two Na1 ions:

As discussed earlier, movement of Na1 from the externalmedium into the cell is driven by two forces: by the Na1

concentration gradient (the Na1 concentration is lower in-side the cell than in the medium) and by the inside-negativemembrane electric potential (see Figure 15-9). Quantita-tively, the free-energy change for the symport transport oftwo Na1 ions and one glucose molecule can be written

where F is the Faraday constant, E is the electric potentialacross the plasma membrane, and the other parameters are

as defined previously. According to our previous calcula-tions, the membrane electric potential and the Na1 concen-tration gradient each contribute about 21.5 kcal per moleof Na1 transported inward, or a total of about 3 kcal /mol(see Equations 15-8 and 15-9). Thus the change in free energy DG for transport of two moles of Na1 inward isabout 26 kcal. By substituting this value into the partialequation for glucose transport

we can calculate that a DG of 26 kcal /mol can generate anequilibrium concentration of glucose inside the cell that is<30,000 times greater than the exterior concentration. Bytransporting two Na1 ions per glucose, this Na1/glucosesymport protein can accumulate glucose against a muchsteeper concentration gradient than if only one Na1 ion weretransported per glucose.

The Na1/glucose symporter contains 14 transmembranea helices (Figure 15-18). A recombinant protein consistingof only the five C-terminal transmembrane a helices has beenshown to transport glucose across the plasma membrane,down its concentration gradient, independently of Na1. Thisportion of the molecule thus functions as a glucose-permeation pathway. The N-terminal portion of the protein,including helices 1–9, is required to couple Na1 binding and glucose transport against a concentration gradient. Figure 15-19 depicts the current model of transport byNa1/glucose symporters. This model, which has not yet beenexperimentally supported, entails conformational changesanalogous to those that occur in uniport transporters suchas GLUT1, which do not require a cotransported ion (seeFigure 15-7).

Na1-Linked Antiporter Exports Ca21 from Cardiac Muscle CellsThe plasma membrane of most cells contains one or moretypes of antiporters, which couple movement of a cotrans-


FIGURE 15-18 Structural model for thetwo-Na1/one-glucose symporter. This662-aa protein forms 14 transmembrane a helices with the N-and C-termini facing the cytosol. The five C-terminal helices formthe sugar-permeation pathway; the rest ofthe protein may be required to couple Na1

binding and glucose transport. Theexoplasmic surface of the protein hasbinding sites for two Na1 ions and oneglucose <3.5 nm apart, but the location ofthese sites has not yet been determined.[Adapted from M. Panayotova-Heiermann et al.,

1997, J. Biol. Chem. 272:20324.]

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ported ion (often Na1) down its electrochemical gradient tomovement of a different molecule in the opposite directionagainst a concentration gradient (see Figure 15-3b). In car-diac muscle cells, for example, a Na1/Ca21 antiporter, ratherthan a plasma membrane Ca21 ATPase, plays the principalrole in maintaining a low concentration of Ca21 in the cyto-sol. The reaction of this cation antiporter can be written

Note that the movement of three Na1 ions is required topower the export of one Ca21 ion against the greater than10,000-fold concentration gradient between the cell interior(2 3 1027 M) and cell exterior (2 3 1023 M). As in othermuscle cells, a rise in the intracellular Ca21 concentrationin cardiac muscle triggers contraction. Thus the operationof the Na1/Ca21 antiporter lowers the cytosolic concentra-tion of Ca21 and reduces the strength of heart muscle con-traction. The Na1/K1 ATPase in the plasma membrane ofcardiac cells, as in other body cells, creates the Na1 con-centration gradient used to power export of Ca21 ions.

The drugs ouabain and digoxin increase the forceof heart muscle contraction and are widely used inthe treatment of congestive heart failure. The pri-

mary effect of these drugs is to inhibit the Na1/K1 ATPase,thereby raising the intracellular Na1 concentration (andlowering intracellular K1). Because the Na1/Ca21 antiporterfunctions less efficiently with a lower Na1 concentrationgradient, fewer Ca21 ions are exported and the intracellularCa21 concentration increases. This increase causes the mus-cle to contract more strongly.

AE1 Protein, a Cl2/HCO32 Antiporter,

Is Crucial to CO2 Transport by ErythrocytesIn addition to cation antiporters, which transport only pos-itive ions, many cells also contain anion transporters, which

transport only negative ions. An important example is AE1protein, the predominant integral protein of the mammalianerythrocyte. This anion antiporter catalyzes the one-for-oneexchange of Cl2 and HCO3

2 across the plasma membrane.Since one singly charged negative ion is exchanged for an-other, there is no net movement of electric charge and thereaction is not affected by the membrane potential. Thus,the direction of the reaction is dependent only on the con-centration gradients of the transported ions.

Transmembrane anion exchange is essential to an im-portant function of the erythrocyte—the transport of wastecarbon dioxide (CO2), which is generated in peripheral tis-sues, to the lungs for excretion by respiratory exhalation(Figure 15-20). Waste CO2 released from cells into the cap-illary blood diffuses across the erythrocyte membrane. In itsgaseous form, CO2 dissolves poorly in aqueous solutions,such as the cytosol or blood plasma, but the potent enzymecarbonic anhydrase inside the erythrocyte converts CO2 tothe water-soluble bicarbonate (HCO3

2) anion:

Since

we can write the overall reaction for carbonic anhydrase as

The release of oxygen from hemoglobin into the peri-pheral capillaries induces a conformational change in theglobin polypeptide that enables a histidine side chain to bindthe proton produced by the carbonic anhydrase reaction.Meanwhile, the HCO3

2 formed by carbonic anhydrase istransported out of the erythrocyte in exchange for an enter-ing Cl2 via AE1 protein (see Figure 15-20, top).


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▲ FIGURE 15-19 Proposed model for operation of thetwo-Na1/one-glucose symporter. The simultaneous bindingof Na1 and glucose to sites on the exoplasmic surfaceinduces a conformational change, generating atransmembrane pore or tunnel that allows both bound Na1

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ionsand glucose to move through the protein to binding sites on the

cytosolic domain and then to pass into the cytosol. After thispassage, 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.]

The entire anion-exchange process in peripheral-blooderythrocytes is completed within 50 milliseconds (ms), dur-ing which time 5 3 109 HCO3

2 ions are exported from thecell down its concentration gradient. If anion exchange didnot occur, HCO3

2 would accumulate inside the erythrocyteto toxic levels during periods of exercise, when much CO2

is generated. About 80 percent of the CO2 in blood is trans-ported as HCO3

2 generated inside erythrocytes; anion ex-change allows about two-thirds of this HCO3

2 to be trans-ported by blood plasma external to the cells, increasing theamount of CO2 that can be transported from tissues to thelungs. Also, without anion exchange, the increased HCO3

2

concentration in the erythrocyte would cause the cytosol tobecome alkaline. The exchange of HCO3

2 (which we can

think of as equal to OH2 1 CO2) for Cl2 causes the cyto-solic pH to remain near neutrality.

The overall direction of this anion-exchange process isreversed in the lungs. CO2 diffuses out of the erythrocyteand is eventually expelled in breathing. The lowered concen-tration of CO2 within the cytosol drives the carbonic anhy-drase reaction, as written above, from right to left: HCO3

2

reacts to yield CO2 and OH2. At the same time, oxygenbinding to hemoglobin causes a proton to be released fromhemoglobin; the proton combines with the OH2 to formH2O. The lowered intracellular HCO3

2 concentrationcauses HCO3

2 to enter the erythrocyte in exchange for Cl2

(see Figure 15-20, bottom).AE1, which has been studied extensively, carries out the

precise one-for-one sequential exchange of anions on oppo-site sides of the membrane required to preserve electroneu-trality in the cell; only once every 10,000 or so transportcycles does an anion move unidirectionally from one side ofthe membrane to the other. AE1 has a large membrane-embedded domain, folded into at least 12 transmembranea helices, which carry out anion transport, and a cytosolic-facing domain, which anchors certain cytoskeletal proteinsto the membrane. Although the precise transport mechanismis not known, conformational changes most likely have akey role as in other membrane transport proteins.

Several Cotransporters Regulate Cytosolic pHThe anaerobic metabolism of glucose yields lactic acid, andaerobic metabolism yields CO2, which is hydrated by car-bonic anhydrase to carbonic acid (H2CO3). These weak acidsdissociate, yielding H1 ions (protons); if these protons werenot exported from cells, the cytosolic pH would drop pre-cipitously, endangering cellular functioning. Two types of co-transport proteins are employed to remove some of the “excess” protons generated during metabolism of animalcells. One is a Na1HCO3

2/Cl2 cotransporter, which importsone Na1 ion down its concentration gradient, together withone HCO3

2, in exchange for export of one Cl2 ion againstits concentration gradient. The imported HCO3

2 ions combine with protons generated by metabolism to produceCO2, which diffuses out of the cell. Thus the overall actionof this transporter raises the cytosolic pH (reduces the H1

concentration). Also important in removing excess protonsis a Na1/H1 antiporter, which couples entry of one Na1 ioninto the cell down its concentration gradient to export oneH1 ion.

The plasma membranes of most animal cells also con-tain a Na1-independent Cl2/HCO3

2 antiporter similar to theerythrocyte AE1 protein discussed previously. This anion-exchange protein functions to lower the cytosolic pH, in ef-fect removing “excess” OH2 ions. Recall that a HCO3

2 ioncan be viewed as a complex of OH2 and CO2, so export ofHCO3

2 lowers the cytosolic pH. Exchange of cytosolicHCO3

2 for extracellular Cl2 is powered by the import ofCl2 down its concentration gradient (Cl2out . Cl2in).


CO2 + OH−

H2OH+

HCO3−

O2CO2

AE1 protein

Hemoglobin

HCO3−

Erythrocyteplasma membrane

In systemic capillariesHigh CO2 pressureLow O2 pressure

NC

H

N CC

NC

H

N CC

Cl−

CO2 + OH−

H2OH+

HCO3−

CO2

HCO3−

Carbonicanhydrase

In pulmonary capillariesLow CO2 pressureHigh O2 pressure

Cl−

Carbonicanhydrase

O2

▲ FIGURE 15-20 Schematic drawings showing aniontransport across the erythrocyte membrane in systemic and pulmonary capillaries. AE1 protein (purple)—an anionantiporter—catalyzes the reversible exchange of Cl2 and HCO3

2

ions across the membrane and works in conjunction withcarbonic anhydrase. In systemic capillaries, the overall reactioncauses HCO3

2 to be released from the cell, which is essentialfor CO2 transport from the tissues to the lungs. In the lungs, theoverall reaction is reversed. See the text for further discussion.

The activity of all three of these antiport proteins dependson pH, providing cells with a fine-tuned mechanism for con-trolling the cytosolic pH (Figure 15-21). The proton-exporting transporters, which are activated when the pH ofthe cytosol falls, act to raise the cytosolic pH. Similarly, arise in pH above 7 stimulates the Cl2/HCO3

2 antiporter,leading to a more rapid export of HCO3

2 and decrease inthe cytosolic pH. In this manner the cytosolic pH of grow-ing cells is maintained very close to pH 7.4.

Small changes in the cytosolic pH may have profoundeffects on the overall cellular metabolic rate. For instance,primary fibroblast cells grown to maximal density (conflu-ence) in tissue culture generally become quiescent: DNA syn-thesis stops; the rates of RNA synthesis, glucose catabolism,and protein synthesis are reduced; and the cytosolic pHdrops from the characteristic 7.4 of growing cells to <7.2.Treatment of quiescent cells with a mixture of serum growthfactors restimulates cell growth and DNA synthesis. An earlyeffect of these growth factors is a marked increase in thecytosolic pH to 7.4; this dramatic change is caused in partby stimulation of the Na1/H1 antiport, which expels pro-tons into the medium. The rise in cytosolic pH is believedto help activate certain metabolic pathways required for cellgrowth and division.

Numerous Transport Proteins Enable PlantVacuoles 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 notedearlier, the vacuolar membrane contains a V-class

ATP-powered pump and a unique PPi-powered pump, bothof which function to pump H1 ions into the vacuolar lumenagainst a concentration gradient. As illustrated in Figure 15-22, the vacuolar membrane also contains Cl2 and NO3

2

channels that transport these anions from the cytosol intothe vacuole. Entry of these anions against their concentra-tion gradients is driven by the inside-positive potential gener-ated by the H1 pumps. Operation of both types of protonpumps in conjunction with these anion channels producesan inside-positive electric potential of about 20 mV acrossthe vacuolar membrane and also a substantial pH gradient.

The proton gradient and electric potential across the plantvacuole membrane are used in much the same way as theNa1 gradient and electric potential across the animal-cellplasma membrane: to power the selective uptake or extrusionof ions and small molecules. In the leaf, for example, excesssucrose generated during photosynthesis in the day is storedin the vacuole; during the night the stored sucrose movesinto the cytoplasm and is metabolized to CO2 and H2O withconcomitant generation of ATP from ADP and Pi. A proton-sucrose antiporter in the vacuolar membrane operates to ac-cumulate sucrose in plant vacuoles. The inward movementof sucrose is powered by the outward movement of H1, whichis favored by its concentration gradient (lumen . cytosol)and by the outward-negative potential across the vacuolarmembrane (see Figure 15-22). Uptake of Ca21 and Na1 intothe vacuole from the cytosol against their concentration gradients is similarly mediated by proton antiporters.


100

50

Per

cen

t m

axim

al r

ate

6.8 7.2 7.6

Na+/H+

antiporter

Cl−/HCO3−

antiporter

Na+ HCO3−/Cl−

cotransporter

Intracellular pH

▲ FIGURE 15-21 Effect of intracellular pH on activity ofmembrane 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.]

2H+

Cl−Ionchannelproteins

Na+ Ca2+ SucrosePlantvacuolemembrane

(pH = 3−6)

(pH = 7.5)

20 mV

NO3−

Proton antiport proteins

H+

H+-pumping proteins

ATPADP + Pi

PPi2Pi

+++

–––

H+ H+ H+

▲ FIGURE 15-22 Concentration of ions and sucrose by theplant vacuole. The vacuolar membrane contains two types ofproton pumps: a V-class H1 ATPase (light green) and a uniquepyrophosphate-hydrolyzing proton pump (dark green). Thesepumps generate a lowered luminal pH as well as an inside-positive electric potential across the vacuolar membrane owingto the inward pumping of H1 ions. The inside-positive potentialpowers the movement of Cl2 and NO3

2 from the cytosol throughseparate channel proteins (dark purple). Proton antiporters (lightpurple), powered by the H1 gradient, accumulate Na1, Ca21, andsucrose 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.]

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

• A small molecule or ion may be imported or exportedagainst its concentration gradient by coupling its move-ment to that of another molecule or ion, usually H1 orNa1, down its electrochemical gradient.

• Two forces power the movement of H1 or Na1

across a membrane: the electric potential and the ionconcentration gradient.

• Entry of glucose and amino acids into certain cellsagainst their concentration gradient is coupled by sym-port proteins to the energetically favorable entry of Na1

(see Figure 15-19).

• In cardiac muscle cells, the export of Ca21 is coupled to the import of Na1 by a cation antiporter,which transports 3 Na1 ions inward for each Ca21

ion exported.

• The erythrocyte membrane contains a Cl2/HCO32

anion antiporter (AE1 protein) that facilitates transportof CO2 by the blood (see Figure 15-20).

• Two proton-exporting transporters—aNa1Cl2/HCO3

2 cotransporter and a Na1/H1 an-tiporter—maintain the cytosolic pH in animal cells veryclose to 7.4 despite metabolic production of carbonicand lactic acids. A Na1-independent Cl2/HCO3

2

antiporter, similar to AE1 protein, functions to exportHCO3

2 when the cytosolic pH rises above normal,causing a decrease in pH.

• Uptake of sucrose, Na1, Ca21, and other substancesinto plant vacuoles is carried out by proton antiportersin the vacuolar membrane. Ion channels in the mem-brane are critical in generating a proton concentrationgradient large enough to power accumulation of ionsand metabolites in vacuoles by these proton antiporters(see Figure 15-22).

15.7 Transport across EpitheliaWith few exceptions, all the internal and external body sur-faces of animals, such as the skin, stomach, and intestines,are covered with a layer of epithelial cells called an epithe-lium (see Figure 6-4). Many epithelial cells transport ions orsmall molecules from one side to the other of the epithe-lium. Those lining the stomach, for instance, secrete hydro-chloric acid into the stomach lumen, which after a meal be-comes pH 1, while those lining the small intestine transportproducts of digestion (e.g., glucose and amino acids) intothe blood. All epithelial cells in a sheet are interconnectedby several types of specialized regions of the plasma mem-brane called cell junctions. These impart strength and rigid-ity to the tissue and prevent water-soluble material on oneside of the sheet (as in the intestinal lumen) from moving

across to the other side. In this section we first describe thepolarized nature of epithelia and how different combina-tions of membrane proteins enable epithelial cells to carryout their transport or secretory functions. Then we discussthe structure and function of the junctions that interconnectepithelial cells.

The Intestinal Epithelium Is Highly PolarizedAn epithelial cell is said to be polarized because one side dif-fers in structure and function from the other. In particular,its plasma membrane is organized into at least two discreteregions, each with different sets of transport proteins. In theepithelial cells that line the intestine, for example, that por-tion of the plasma membrane facing the intestine, the apicalsurface, is specialized for absorption; the rest of the plasmamembrane, the lateral and basal surfaces, often referred toas the basolateral surface, mediates transport of nutrients fromthe cell to the surrounding fluids which lead to the bloodand forms junctions with adjacent cells and the underlyingextracellular matrix called the basal lamina (Figure 15-23).

Extending from the lumenal (apical) surface of intestinalepithelial cells are numerous fingerlike projections (100 nmin diameter) called microvilli (singular, microvillus). Oftenreferred to collectively as the brush border because of theirappearance, microvilli greatly increase the area of the api-cal surface and thus the number of transport proteins it cancontain, enhancing the absorptive capacity of the intestinalepithelium. A bundle of actin filaments that runs down thecenter of each microvillus gives rigidity to the projection.Overlying the brush border is the glycocalyx, a loose net-work composed of the oligosaccharide side chains of inte-gral membrane glycoproteins, glycolipids, and enzymes thatcatalyze the final stages in the digestion of ingested carbo-hydrates and proteins (Figure 15-24). The action of theseenzymes produces monosaccharides and amino acids, whichare transported across the intestinal epithelium and eventu-ally into the bloodstream.

Transepithelial Movement of Glucose and Amino Acids Requires Multiple Transport ProteinsMovement of monosaccharides and amino acids from theintestinal lumen into the blood is a two-stage transcellularprocess. The first stage, import of substances from the lumeninto intestinal epithelial cells, is carried out by membranetransport proteins in the microvilli on the apical surface ofintestinal cells. The second stage, export of substances fromthe cells into the fluid surrounding the basolateral surface,is carried out by other transport proteins on the basolateralplasma membrane. In order for such transepithelial trans-port to occur, the epithelial cell must be polarized, with dif-ferent sets of transport proteins localized in the basolateraland apical surfaces. To illustrate this process, we examinethe membrane transport proteins required to move glucose


across the epithelial cells lining the intestine and kidney. Sim-ilar proteins are used to transport amino acids across theseepithelia.

Figure 15-25 depicts the transport of glucose from theintestinal lumen to the blood. Glucose is imported againstits concentration gradient from the intestinal lumen acrossthe apical surface of the epithelial cells by a two-Na1/one-glucose symporter located in the microvillar membranes. Asnoted above, this symporter couples the energetically unfavor-able inward movement of one glucose molecule to the ener-getically favorable inward transport of two Na1 ions (seeFigure 15-19). In the steady state, all the Na1 ions trans-ported from the intestinal lumen into the cell duringNa1/glucose symport, or the similar process of Na1/aminoacid symport, are pumped out across the basolateral mem-brane, often called the serosal (blood-facing) membrane.Thus the low intracellular Na1 concentration is maintained.The Na1/K1 ATPase that accomplishes this is found in thesecells exclusively on the basolateral surface of the plasmamembrane. The coordinated operation of these transportersallows uphill movement of glucose and amino acids fromthe intestine into the cell, and ultimately is powered by ATPhydrolysis by the Na1/K1 ATPase.

Glucose and amino acids concentrated inside intestinalcells by symporters are exported down their concentrationgradients into the blood via uniport proteins in the baso-lateral membrane. In the case of glucose, this movement ismediated by GLUT2, a glucose transporter that is localizedin the basal and lateral membranes of intestinal cells (see

Transport across Epithelia 603

Microvillus

Tight junctions

Adherens junction

Spot desmosome

Gap junction

Intermediatefilament

Hemidesmosome

Basal lamina

Basal surface

Lateral surface

Apical surface

FIGURE 15-23 Schematic diagram ofepithelial cells lining the small intestineand the principal types of cell junctionsthat connect them. As in all epithelia, thebasal surface of the cells rests on the basallamina, a fibrous network of collagen andproteoglycans that supports the epithelial celllayer. The apical surface faces the intestinallumen. Tight junctions, lying just under themicrovilli, prevent diffusion of substancesbetween the intestinal lumen and the bloodvia the extracellular space between cells. Gapjunctions allow movement of small moleculesand ions between the cytosol of adjacentcells. The remaining three types of junctions,adherens junctions, spot desmosomes, andhemidesmosomes are critical to cell-cell andcell-matrix adhesion.

▲

Gly

coca

lyx

Mic

rovi

lli

▲ FIGURE 15-24 Micrograph of the microvilli that form thelumenal 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 integralmembrane proteins. The glycocalyx, which covers the apices(tips) of the microvilli, is composed of a network of glycoproteinsand digestive enzymes. [From N. Hirokawa and J. E. Heuser, 1981,

J. Cell Biol. 91:399; courtesy of N. Hirokawa and J. E. Heuser.]

Figure 5-1c). (GLUT2 is a homolog of GLUT1; as discussedearlier; however, GLUT1 generally functions to import glu-cose into many body cells.) The net result of the operationof these various transport proteins is movement of Na1 ions,amino acids, and glucose from the intestinal lumen acrossthe intestinal epithelium into the interstitial spaces surround-ing the cells, and eventually into the blood. Tight junctionsbetween the epithelial cells prevent these molecules from dif-fusing back into the intestinal lumen.

The epithelial cells lining kidney tubules, which have anarchitecture similar to that of intestinal epithelial cells, re-absorb glucose from the blood filtrate that is the formingurine and return it to the blood. In the first part of a kid-ney tubule, the epithelial cells transport glucose against arelatively small glucose concentration gradient. These cellsutilize a second type of Na1/glucose symport protein—aone-Na1/one-glucose symporter, which has a high transportrate but cannot transport glucose against a steep concen-tration gradient. At the intracellular Na1 concentration andmembrane potential depicted in Figure 15-9, this symportercan generate an intracellular glucose concentration <100times that of the extracellular medium (here the formingurine). In the latter part of a kidney tubule, however, the ep-ithelial cells take up the remaining glucose against a morethan 100-fold glucose concentration gradient. To accomplishthis, these cells contain in their apical membrane the sametwo-Na1/one-glucose symporter found in intestinal epithe-lial cells. The two types of Na1/glucose symport proteinsare similar in amino acid sequence, predicted structure, andmechanism but have evolved to transport glucose under dif-ferent conditions.

Parietal Cells Acidify the Stomach ContentsWhile Maintaining a Neutral Cytosolic pH

The mammalian stomach contains a 0.1 M solution of hydro-chloric acid (H1Cl2). This strongly acidic medium denaturesmany ingested proteins before they are degraded by prote-olytic enzymes in the stomach (e.g., pepsin) that function atacidic pH. Hydrochloric acid is secreted into the stomachby parietal cells (also known as oxyntic cells) in the gastriclining. These cells contain a H1/K1 ATPase in their apicalmembrane, which faces the stomach lumen and generates aconcentration of H1 ions 106 times greater in the stomachlumen than in the cell cytosol (pH 5 1.0 versus pH 5 7.0).This enzyme is a P-class ATPase, similar in structure andfunction to the Na1/K1 ATPase discussed earlier. Operationof the Na1/K1 ATPase results in a net outward movementof one charged ion per ATP (see Figure 15-13). In contrast,the action of the H1/K1 ATPase, which exports one H1 ionand imports one K1 ion for each ATP hydrolyzed, producesno net movement of electric charge. The numerous mitochon-dria in parietal cells produce abundant ATP for use by theH1/K1 ATPase.

If parietal cells simply exported H1 ions in exchange forK1 ions, a rise in the concentration of OH2 ions and thusa marked rise in cytosolic pH would occur, since in the cyto-sol, as in all aqueous solutions, the product of the H1 andOH2 concentrations is a constant (10214 M2). However,during acidification of the stomach lumen, the pH of theparietal-cell cytosol remains neutral. Parietal cells accom-plish this feat by means of a Cl2/HCO3

2 antiporter in thebasolateral membrane (Figure 15-26). The “excess” cytosolicOH2, generated by exporting protons, combines with CO2

that diffuses into the cell from the blood, forming HCO32

in a reaction catalyzed by cytosolic carbonic anhydrase. TheHCO3

2 ion is transported across the basolateral membraneof the cell into the blood in exchange for an incoming Cl2

ion by means of an anion antiporter that is similar in struc-ture and function to the erythrocyte AE1. The Cl2 ions thusimported into the cell exit through Cl2 channels in the api-cal membrane, entering the stomach lumen. To preserve elec-troneutrality, each Cl2 ion that moves into the stomach lumenacross the apical membrane is accompanied by a K1 ion thatmoves outward through a separate K1 channel. In this way,the excess K1 ions pumped inward by the H1/K1 ATPaseare returned to the stomach lumen, thus maintaining the in-tracellular K1 concentration. The net result is accumulationof both H1 and Cl2 ions (i.e., HCl) in the stomach lumen,while the pH of the cytosol remains neutral and the excessOH2 ions, as HCO3

2, are transported into the blood.

Tight Junctions Seal Off Body Cavities andRestrict Diffusion of Membrane Components

For polarized epithelial cells to carry out their transportfunctions, extracellular fluids surrounding their apical and basolateral membranes must be kept separate. This is


K+K+

GLUT 2

ADP + Pi

ATPNa+

Na+/K+ ATPase

Na+GlucoseGlucose Glucose

Apicalmembrane

2 Na+ 2 Na+

Na+/glucosesymportprotein

Dietary glucoseHigh (dietary) Na+

Tight junctionBasolateralmembrane

High Na+ Low K+

Low Na+

High K+

Blood Epithelial cells Intestinal lumen

▲ FIGURE 15-25 Transport of glucose from the intestinallumen into the blood. Activity of the Na1/K1 ATPase (green) in the basolateral surface membrane generates Na1 and K1

concentration gradients, and the K1 gradient generates an inside-negative membrane potential. Both the Na1 concentration gradientand the membrane potential are used to drive the uptake ofglucose from the intestinal lumen by the two-Na1/one-glucosesymporter (blue) located in the apical surface membrane. Glucoseleaves the cell via facilitated diffusion catalyzed by GLUT2(orange), a glucose uniporter located in the basolateral membrane.

accomplished by tight junctions, which connect adjacent epi-thelial cells and usually are located just below the apical sur-face (see Figure 15-23). These specialized regions of the plasmamembrane form a barrier that seals off body cavities suchas the intestine, the stomach lumen, ductules in pancreaticacini, and the bile duct in the liver. For example, tight junc-tions prevent diffusion of small molecules directly from theintestinal lumen into the interstitial spaces that surround thebasolateral plasma membrane and that lead to the blood.Thus intestinal epithelial cells must transport nutrientsthrough the cells as previously described. In the pancreas,tight junctions between acinar cells likewise prevent leakageof secreted proteins, including digestive enzymes, from thecentral ductules into the blood (Figure 15-27). Tight junc-tions also prevent diffusion of membrane proteins and gly-colipids between the apical and basolateral regions of theplasma membrane, ensuring that these regions contain dif-ferent membrane components.

Structure of Tight Junctions Tight junctions are com-posed of thin bands of plasma-membrane proteins that com-pletely encircle a polarized cell and are in contact with sim-ilar thin bands on adjacent cells. When thin sections of cellsare viewed in an electron microscope, the plasma membranesof adjacent cells appear to touch each other at intervals and

even to fuse (Figure 15-28a). Freeze-fracture electron micro-scopy affords a striking view of the tight junction. Themicrovillar tight junction shown in Figure 15-28b appearsto comprise an interlocking network of ridges in the plasmamembrane. More specifically, there appear to be ridges onthe cytosolic face of the plasma membrane of each of thetwo contacting cells. (Corresponding grooves not shownhere are found on the exoplasmic face.) High magnificationreveals that these ridges are made up of protein particles3–4 nm in diameter. In the model shown in Figure 15-28c,the tight junction is formed by a double row of these parti-cles, one row donated by each cell.

The two principal integral membrane proteins found intight junctions are occludin and claudin. Each of these pro-teins has four membrane-spanning a helices. Although themolecular structure of the junction is not known, the extra-cellular domains of rows of occludin and claudin proteins


Basolateralmembrane

Anionantiportprotein

HCO3− HCO3

−

CO2 CO2 + −OH

H2OADP + Pi

ATP

Cl− Cl−

H+H+

Cl−

K+K+K+H+/K+ ATPase

K+ channelprotein

Cl− channelprotein

Apicalmembrane

Tightjunction

Stomach lumen

Carbonicanhydrase

▲ FIGURE 15-26 Acidification of the stomach lumen byparietal cells in the gastric lining. The apical membrane ofparietal cells contains a H1/K1 ATPase (a P-class pump) as wellas Cl2 and K1 channel proteins. Note the cyclic K1 transportacross the apical membrane: K1 ions are pumped inward by the H1/K1 ATPase and exit via a K1 channel. The basolateralmembrane contains an anion antiporter that exchanges HCO3

2

and Cl2 ions. The combined operation of these four differenttransport proteins acidifies the stomach lumen while maintainingthe neutral pH and electroneutrality of the cytosol. See the textfor more details.

Basolateralmembrane

Single acinar cell

Central ductule

Apicalmembrane

Secretoryvesicles

Tight junction

▲ FIGURE 15-27 Diagram of pancreatic acinar cells. Anacinus is a spherical aggregate of about a dozen cells; the lumenof an acinus is connected to a ductule that merges with otherductules and eventually leads into a main pancreatic duct, whichempties into the lumen of the small intestine (Figure 5-48). Acinarcells synthesize degradative enzymes and store them as inactiveprecursors (zymogens) in secretory vesicles, which cluster underthe apical region of the plasma membrane adjacent to the ductule.The basolateral membrane covers the sides of an acinar cellbelow the apical (lumen-facing) surface and extends along thebase of the cell; nutrients in the blood in the surrounding vesselsare transported through this region of the plasma membrane intothe cell. Note the tight junctions (orange) just below the apicalregion between adjacent cells; they prevent movement ofsubstances between the central ductule and the blood.

ZO-2, and ZO-3) that, in turn, are bound to other cyto-skeletal proteins and to actin fibers. These interactions ap-pear to stabilize the linkage between occludin molecules thatis essential for integrity of the tight junction (Chapter 22).

Impermeability of Tight Junctions to Aqueous Solu-tions That tight junctions are impermeable to most water-soluble substances can be demonstrated in an experiment inwhich lanthanum hydroxide (an electron-dense colloid ofhigh molecular weight) is injected into the pancreatic blood


(a)

▲ FIGURE 15-28 Tight junctions. (a) Thin-section electronmicrograph of the apical region of two liver epithelial cells,illustrating the tight junction just below the microvilli and theadherens junction. From the apical region of these liver cells,which faces the lumen of the bile duct, phospholipids and othercomponents of bile are secreted into the duct. (b) Freeze-fractureelectron micrograph of a tight junction between two intestinalepithelial cells. The fracture plane passes through the plasmamembrane 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 adjacentcells (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 extremelytight links with similar rows of claudin and occludin in theadjacent cell, essentially fusing two adjacent cells and cre-ating an impenetrable seal. Treatment of an epithelium withthe protease trypsin destroys the tight junctions, supportingthe proposal that proteins are essential structural compo-nents of these junctions.

The long C-terminal cytosolic-facing domain of occludinis bound to one of a group of large cytosolic proteins (ZO-1,

vessel of an experimental animal; a few minutes later thepancreatic acinar cells are fixed and prepared for micro-scopy. As shown in Figure 15-29, the lanthanum hydroxidediffuses from the blood into the space that separates the lat-eral surfaces of adjacent acinar cells, but cannot penetratepast the outermost tight junction.

Other studies have shown that tight junctions also are im-permeable to salts. For instance, when MDCK cells are grownin a medium containing very low concentrations of Ca21,they form a monolayer in which the cells are not connectedby tight junctions; as a result, fluids and salts flow freelyacross the cell layer. When Ca21 is added to such a mono-layer, tight junctions form within an hour, and the cell layerbecomes impermeable to fluids and salts (see Figure 6-7).

Ability of Tight Junctions to Block Diffusion of Pro-teins and Lipids in the Plane of the Plasma MembraneWhen liposomes containing a fluorescent-tagged glycopro-tein are added to the medium in contact with the apical sur-face of a monolayer of MDCK cells, some spontaneously fusewith the plasma membrane. Fluorescent glycoprotein is detect-able in the apical but not in the basolateral surface of thecells so long as the tight junctions between adjacent cells areintact. However, if the tight junctions are destroyed by remov-ing Ca21 from the medium, the fluorescent protein is soondetectable in the basolateral surface, indicating that it candiffuse from the apical to the basolateral regions of the plasmamembrane. These results indicate that plasma membraneproteins cannot diffuse through tight junctions.

Lipids in the cytosolic leaflets of the apical and baso-lateral membranes of epithelial cells have the same compo-sition and apparently can diffuse from one region of themembrane to the other. In contrast, the lipid compositionsof the exoplasmic leaflets of the apical and basolateral mem-brane regions are very different, and membrane lipids in theexoplasmic leaflets cannot diffuse through tight junctions.All the glycolipid in MDCK cells, for instance, is present inthe 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 compo-nents of glycosylphosphatidylinositol anchors, and choles-terol. Phosphatidylcholine, conversely, is present almost exclusively in the exoplasmic face of the basolateral plasmamembrane.

Other Junctions Interconnect Epithelial Cells and Control Passage of Molecules between ThemIn order to function in an integrated manner, the individualcells composing epithelia and other organized tissues mustadhere to one another and to the surrounding extracellularmatrix and also control the movement of ions and smallmolecules between them. Several specialized cell junctionsare critical to adhesion and passage of molecules betweencells in tissues (see Figure 15-23).

Three types of cell junctions, called desmosomes, func-tion in cell-cell and cell-matrix adhesion. Epithelial and someother types of cells, such as smooth muscle, are bound tightlytogether by spot desmosomes. These are buttonlike pointsof contact between cells, often thought of as a “spot-weld”between adjacent plasma membranes, that confer mechani-cal strength on these tissues. Hemidesmosomes, similar instructure to spot desmosomes, anchor the plasma membraneto regions of the extracellular matrix. Bundles of interme-diate filaments course through the cell, interconnecting spotdesmosomes and hemidesmosomes. Finally, adherens junc-tions (also known as belt desmosomes), which are found pri-marily in epithelial cells, form a belt of cell-cell adhesionjust under the tight junctions.

The lateral surfaces of adjacent cells contain numerousgap junctions. These junctions help to integrate the meta-bolic activities of all cells in a tissue by allowing the directpassage of ions and small molecules from the cytosol of onecell to that of another (see the chapter opening figure).Among these are intracellular signaling molecules (e.g.,cyclic AMP and Ca21) and precursors of DNA and RNA.

Electron micrographs of animal tissue sections haveshown that a space of about 20 nm ordinarily is present be-tween the nonjunctional regions of plasma membranes ofadjacent cells. This space contains integral membrane andextracellular surface glycoproteins that assist junctions in in-tercellular adhesion.


▲ FIGURE 15-29 Experimental demonstration that tightjunctions prevent passage of water-soluble substances.Pancreatic acinar tissue is fixed and prepared for microscopy afew minutes after electron-opaque lanthanum hydroxide isinjected into the blood of an experimental animal. As shown inthis electron micrograph of adjacent acinar cells, the lanthanumhydroxide can penetrate between the cells but is arrested at thelevel of the tight junction. [Courtesy of D. Friend.]

An understanding of the structure and function ofdesmosomes requires knowledge about actin microfilamentsand intermediate filaments. Likewise, an understanding ofgap junctions and their equivalent in plant cells (plasmo-desmata) depends on knowledge of cellular metabolism andsignaling. Therefore, we defer detailed discussion of thesejunctions until later chapters when these related topics areexamined.

S U M M A R Y Transport across Epithelia

• The apical and basolateral plasma membrane domainsof epithelial cells contain different transport proteinsand carry out quite different transport processes.

• In the intestinal epithelial cell, Na1/glucose andNa1/amino acid symporters are in the apical membraneregion facing the intestinal lumen, while Na1/K1

ATPases and glucose and amino acid uniporters are inthe basolateral membrane region facing the blood capil-laries. The coordinated operation of these membranetransport proteins allows the uphill transepithelialmovement of amino acids and glucose from the lumento the blood, powered by ATP hydrolysis by theNa1/K1 ATPase (see Figure 15-25).

• Parietal cells in the stomach lining, which secreteHCl into the lumen, have ATP-powered H1/K1 pumps,K1 channels, and Cl2 channels on the apical membraneand pH-sensitive Cl2/HCO3

2 antiporters on the baso-lateral membrane. The combined action of these pro-teins allows the cytosolic pH to be maintained near neutrality, despite the active export of protons from thecells into the stomach lumen, causing its acidification(see Figure 15-26).

• The plasma membrane contains specialized regionsthat form various types of cell junctions between adja-cent cells (see Figure 15-23).

• Tight junctions interconnecting epithelial and otherpolarized cells seal off body cavities and restrict diffusionof plasma-membrane proteins from the apical to the ba-solateral surfaces. Tight junctions also prevent diffusionof lipids in the exoplasmic (but not the cytosolic leaflet)from the apical to the basolateral domains of theplasma membrane.

• Adherens junctions and spot desmosomes bind theplasma membranes of adjacent cells in a way that givesstrength and rigidity to the entire tissue. Hemidesmo-somes help connect cells to the extracellular matrix.

• Gap junctions in animal cells and plasmodesmata in plant cells interconnect the cytosol of two adjacentcells, allowing small molecules and ions to pass between them.

15.8 Osmosis, Water Channels,and the Regulation ofCell Volume

In this section, we examine two types of transport pheno-mena that, at first glance, may seem unrelated: the regula-tion of cell volume in both plant and animal cells, and thebulk flow of water (the movement of water containing dis-solved solutes) across one or more layers of cells. In humans,for example, water moves from the blood filtrate that willform urine across a layer of epithelial cells lining the kidneytubules and into the blood, thus concentrating the urine. (Ifthis did not happen, one would excrete several liters of urinea day!) In higher plants, water and minerals are absorbedby the roots and move up the plant through conducting tubes(the xylem); water is lost from the plant mainly by evapo-ration from the leaves. What these processes have in com-mon is osmosis—the movement of water from a region oflower solute concentration to a region of higher solute con-centration. We begin with a consideration of some basic factsabout osmosis, and then show how they explain severalphysiological properties of animals and plants.

Osmotic Pressure Causes Water to Move across MembranesAs noted early in this chapter, most biological membranesare relatively impermeable to ions and other solutes, but likeall phospholipid bilayers, they are somewhat permeable towater (see Figure 15-1). Permeability to water is increasedby water-channel proteins discussed below. Water tends tomove across a membrane from a solution of low solute con-centration to one of high. Or, in other words, since solu-tions with a high amount of dissolved solute have a lowerconcentration of water, water will move from a solution ofhigh water concentration to one of lower. This process isknown as osmotic flow.

Osmotic pressure is defined as the hydrostatic pressurerequired to stop the net flow of water across a membraneseparating solutions of different compositions (Figure 15-30).In this context, the “membrane” may be a layer of cells ora plasma membrane. If the membrane is permeable to wa-ter but not to solutes, the osmotic pressure across the mem-brane is given by

(15-11)

where p is the osmotic pressure in atmospheres (atm) or mil-limeters of mercury (mmHg); R is the gas constant; T is theabsolute temperature; and DC is the difference in total soluteconcentrations, 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 Na1

ions and 0.5 M Cl2 ions and has approximately the sameosmotic pressure as a 1 M solution of glucose or lactose.


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

Different Cells Have Various Mechanisms for Controlling Cell VolumeAnimal cells will swell when they are placed in a hypotonicsolution (i.e., one in which the concentration of solutes islower than it is in the cytosol). Some cells, such as erythro-cytes, will actually burst as water enters them by osmoticflow. Rupture of the plasma membrane by a flow of waterinto the cytosol is termed osmotic lysis. Immersion of all an-imal cells in a hypertonic solution (i.e., one in which theconcentration of solutes is higher than it is in the cytosol)causes them to shrink as water leaves them by osmotic flow.

Consequently, it is essential that animal cells be maintainedin an isotonic medium, which has a solute concentrationclose to that of the cell cytosol (see Figure 5-22).

Even in an isotonic environment, all animal cells face aproblem in maintaining their cell volume. Cells contain a largenumber of charged macromolecules and small metabolitesthat attract ions of opposite charge (e.g., K1, Ca21, PO4

32).Also recall that there is a slow leakage of extracellular ions,particularly Na1 and Cl2, into cells down their concentra-tion gradient. As a result of these factors, in the absence ofsome countervailing mechanism, the cytosolic solute concen-tration would increase, causing an osmotic influx of waterand eventually cell lysis. To prevent this, animal cells activelyexport inorganic ions as rapidly as they leak in. The exportof Na1 by the ATP-powered Na1/K1 pump plays the majorrole in this mechanism for preventing cell swelling. If culturedcells are treated with an inhibitor that prevents production ofATP, they swell and eventually burst, demonstrating the im-portance of active transport in maintaining cell volume.

Unlike animal cells, plant, algal, fungal, and bac-terial cells are surrounded by a rigid cell wall. Be-cause of the cell wall, the osmotic influx of water

that occurs when such cells are placed in a hypotonic solu-tion (even pure water) leads to an increase in intracellularpressure but not in cell volume. In plant cells, the concen-tration of solutes (e.g., sugars and salts) usually is higher inthe vacuole than in the cytosol, which in turn has a highersolute concentration than the extracellular space. The os-motic pressure, called turgor pressure, generated from theentry of water into the cytosol and then into the vacuolepushes the cytosol and the plasma membrane against the resistant cell wall. Cell elongation during growth occurs bya hormone-induced localized loosening of a region of thecell wall, followed by influx of water into the vacuole, in-creasing its size (see Figure 22-33).

Although most protozoans (like animal cells) do not have a rigid cell wall, many contain a contractile vacuolethat permits them to avoid osmotic lysis. A contractile vacuole takes up water from the cytosol and, unlike a plant vacuole, periodically discharges its contents throughfusion with the plasma membrane (Figure 15-31). Thus,

Osmosis, Water Channels, and the Regulation of Cell Volume 609

▲ FIGURE 15-30 Experimental system for demonstratingosmotic pressure. Solutions A and B are separated by amembrane that is permeable to water but impermeable to allsolutes. If CB (the total concentration of solutes in solution B) isgreater than CA, water will tend to flow across the membranefrom solution A to solution B. The osmotic pressure p betweenthe solutions is the hydrostatic pressure that would have to beapplied to solution B to prevent this water flow. From the van’tHoff equation, p 5 RT (CB 2 CA).

FIGURE 15-31 The contractile vacuolein Paramecium caudatum, a typical ciliatedprotozoan, as revealed by Nomarskimicroscopy of a live organism. The vacuoleis filled by radiating canals that collect fluidfrom the cytosol. When the vacuole is full, it fuses for a brief period with the plasmamembrane and expels its contents. (a) A full vacuole and system of radiating canals. (b) A nearly empty vacuole; the radiatingcanals are collecting more fluid from thecytosol to refill it.

▲

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

Water Channels Are Necessary for Bulk Flow of Water across Cell MembranesEven though a pure phospholipid bilayer is only slightlypermeable to water, small changes in extracellular osmoticstrength cause most animal cells to swell or shrink rapidly.In contrast, frog oocytes and eggs, which have an internalsalt concentration comparable to other cells (<150 mM),do not swell when placed in pond water of very low osmoticstrength. These observations led investigators to suspect thatthe plasma membranes of erythrocytes and other cell typescontain water-channel proteins that accelerate the osmoticflow of water. The absence of these water channels in frogoocytes and eggs protects them from osmotic lysis.

Microinjection experiments with mRNA encoding aqua-porin, an erythrocyte membrane protein, provided convinc-ing evidence that this protein increases the permeability ofcells to water (Figure 15-32). In its functional form, aqua-porin is a tetramer of identical 28-kDa subunits, each ofwhich contains six transmembrane a helices that form threepairs of homologs in an unusual orientation (Figure 15-33a).The channel through which water moves is thought to belined by eight transmembrane a helices, two from each sub-unit (Figure 15-33b). Aquaporin or homologous proteins areexpressed in abundance in erythrocytes and in other cells

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

Simple Rehydration Therapy Depends on Osmotic Gradient Created by Absorption of Glucose and Na1

An understanding of osmosis and the intestinal absorption of glucose forms the basis for a simpletherapy that has saved millions of lives, particularly

in less-developed countries. In these countries, diarrhea causedby cholera and other intestinal pathogens is a major cause of death of young children. A cure demands not only killingthe bacteria with antibiotics, but also rehydration—replace-ment of the water that is lost from the blood and other tissues.

Simply drinking water does not help, because it is ex-creted from the gastrointestinal tract almost as soon as itenters. To understand the simple therapy that is used, recallthat absorption of glucose by the small intestine involves thecoordinated movement of Na1; one cannot be transportedwithout the other (see Figure 15-25). The movement of NaCland glucose from the intestinal lumen, across the epithelialcells, and into the blood creates a transepithelial osmoticgradient, forcing movement of water from the intestinal lu-men into the blood. Thus, giving affected children a solu-tion of sugar and salt to drink (but not sugar or salt alone)causes the bulk flow of water into the blood from the in-testinal lumen and leads to rehydration.


▲ FIGURE 15-32 Experimental demonstration that aquaporinis a water-channel protein. Frog oocytes, which normally donot express aquaporin, were microinjected with erythrocytemRNA encoding aquaporin. These photographs show controloocytes (bottom image in each panel) and microinjected oocytes(top image in each panel) at the indicated times after transferfrom an isotonic salt solution (0.1 mM) to a hypotonic salt

solution (0.035 M). The volume of the control oocytes remainedunchanged, because they are poorly permeable to water. Incontrast, the microinjected oocytes expressing aquaporin swelled because of an osmotic influx of water, indicating thataquaporin increases their permeability to water. [Courtesy of

Gregory M. Preston and Peter Agre, Johns Hopkins University School

of Medicine.]

Changes in Intracellular Osmotic Pressure Cause Leaf Stomata to Open

Although most plants cells do not change their vol-ume or shape because of the osmotic movement ofwater, the opening and closing of stomata—the

pores through which CO2 enters a leaf—provides an impor-tant exception. The external epidermal cells of a leaf arecovered by a waxy cuticle that is largely impenetrable to water and to CO2, a gas required for photosynthesis by the

chlorophyll-laden mesophyll cells in the leaf interior. As CO2

enters a leaf, water vapor is simultaneously lost—a processthat can be injurious to the plant. Thus it is essential thatthe stomata open only during periods of light, when pho-tosynthesis occurs; even then, they must close if too muchwater vapor is lost.

Two guard cells surround each stomate (Figure 15-34a).Changes in turgor pressure lead to changes in the shape ofthese guard cells, thereby opening or closing the pores.Stomatal opening is caused by an increase in the concen-tration of ions or other solutes within the guard cells be-cause of (1) opening of K1 and Cl2 channels and the sub-sequent influx of K1 and Cl2 ions from the environment,(2) the metabolism of stored sucrose to smaller compounds,or (3) a combination of these two processes. The resultingincrease in the intracellular solute concentration causes water to enter the guard cells osmotically, increasing their

Osmosis, Water Channels, and the Regulation of Cell Volume 611

FIGURE 15-33 The structure of aquaporin, a water-channel protein in the erythrocyte plasma membrane. Thistetrameric protein has four identical subunits. (a) Schematicmodel of an aquaporin subunit showing the three pairs ofhomologous transmembrane a helices, A and A9, B and B9, andC and C9. As indicated by the arrows showing the N-terminal nC-terminal directionality of the helices, the homologous segmentsare oriented in the opposite direction. (b) Head-on view oftetrameric aquaporin showing the packing of the transmembranea helices in the plane of the membrane, as determined by x-raycrystallography. The helices (represented as circles) in each ofthe four subunits are shown in different colors. Although theidentity of the two helices from each subunit that line the centralchannel is not known, they probably are a pair of homologoussegments. The opposite orientation of the two helices in a pairwithin the membrane would account for the ability of thechannel to transport water equally in both directions across themembrane. [Adapted from A. Chang et al., 1997, Nature 387:627.]

▲

FIGURE 15-34 The opening and closing of stomata. (a) Light micrographof a leaf of a wandering Jew (Tradescantia sp) plant shows two stomata, eachsurrounded by a pair of guard cells. (b) Opening of K1 and Cl2 channels in theplasma membrane of the guard cells is followed by an influx of K1 and Cl2 intothe 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.]

▲

turgor pressure (Figure 15-34b). Since the guard cells areconnected to each other only at their ends, the turgor pres-sure causes the cells to bulge outward, opening the stomatalpore between them. Stomatal closing is caused by the reverseprocess—a decrease in solute concentration and turgor pres-sure within the guard cells.

Stomatal opening is under tight physiological control byat least two mechanisms. A drop in CO2 within the leaf, resulting from active photosynthesis, causes the stomata toopen, permitting additional CO2 to enter the leaf interior sothat photosynthesis can continue. When more water exitsthe leaf than enters it from the roots, the mesophyll cellsproduce the hormone abscissic acid, which causes K1 effluxfrom 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 towater than to ions or other solutes, and water movesacross them by osmosis from a solution of lower soluteconcentration to one of higher solute concentration.

• Animal cells swell or shrink when placed in hypo-tonic or hypertonic solutions, respectively. To maintaintheir normal cytosolic osmolarity and hence cell volume,animal cells must export Na1 and other ions that leakor are transported from the extracellular space into thecytosol.

• The rigid cell wall surrounding plant cells preventstheir swelling and leads to generation of turgor pressurein response to the osmotic influx of water.

• In response to the entry of water, protozoans main-tain their normal cell volume by extruding water fromcontractile vacuoles.

• Aquaporin in the erythrocyte plasma membrane andother water-channel proteins increase the water perme-ability of biomembranes (see Figure 15-33).

• Opening and closing of K1 and Cl2 channels andthe resulting changes in cytosolic solute concentrationsof guard cells cause stomata in leaves to open and close(see Figure 15-34).

PERSPECTIVES for the Future

In this chapter, we have explained certain aspectsof human physiology in terms of the action of spe-cific membrane transport proteins. Such a molec-

ular physiology approach has many medical applications.Even today, specific inhibitors or activators of channels,pumps, and transporters constitute the largest single classof drugs. For instance, an inhibitor of the gastric H1/K1

ATPase that acidifies the stomach is the most widely useddrug for treating stomach ulcers. As we discuss in Chapter 21,the plasma membrane of nerve cells contains Na1-symportproteins that are specifically inhibited by many drugs ofabuse (e.g., cocaine) and antidepression medications (e.g.,Prozac). Inhibitors of kidney channel proteins are widelyused to control hypertension (high blood pressure); by block-ing resorption of water from the forming urine into theblood, these drugs reduce blood volume and thus blood pres-sure. Calcium-channel blockers are widely employed to con-trol the intensity of contraction of the heart.

Within the next years, the human genome project willgenerate the sequences of all human membrane transportproteins. Soon after, researchers will discover in which typesof cells and tissues these proteins are expressed. Using recom-binant DNA techniques, scientists will be able to generatelines of cultured cells that express these in abundance, sothat their molecular properties can be studied. Gene-knock-out studies in mice will provide clues to their role in humanphysiology and disease.

This basic knowledge will enable drug company re-searchers to identify new types of compounds that inhibitor activate just one of these transport proteins and not itshomologs expressed in other types of cells. In this way, newand highly specific drugs will be developed to treat a vari-ety of diseases. Physicians will also be able to identify indi-viduals who may be at risk for certain types of diseases (e.g.,hypertension or diabetes) because they have mutations incertain membrane transport proteins. And at the level of ba-sic biology, we will all learn precisely how the human bodydigests and metabolizes all kinds of food and controls thelevels of sugars, salts, fats, and other essential molecules inthe blood and tissues.

PERSPECTIVES in the Literature

The Escherichia coli lactose permease is one of the most ex-tensively studied membrane transport proteins. Encoded bythe y gene of the lac operon (see Figure 10-1), this proton–lactose symporter is essential for transport of lactose intothe bacterial cell against its concentration gradient. Despiteextensive efforts, the lactose permease has not been crystal-lized, and thus no three-dimensional structure is available.Nonetheless, Cysteine-scanning mutagenesis, in which everyamino acid, in turn, is changed to cysteine, has been par-ticularly revealing. The “new” cysteine residue can be sub-jected to a number of chemical modifications to determineits location relative to other amino acids and its involvementin lactose binding and transport. As you read the recent pa-pers listed below, and earlier ones referenced in Frillingos etal., focus on the following questions:

1. What types of chemical, biochemical, and biophysicaltechniques were applied to the mutant proteins? What typesof data do these different techniques produce?


2. How did such studies lead to a detailed three-dimensionalmodel of the arrangements of the twelve membrane-span-ning a-helices?

3. How did these studies identify amino acid residues cru-cial for binding of lactose at the exoplasmic surface andresidues thought to conduct protons through the protein?

4. How did the authors determine that binding of the sub-strate induced tilting of several membrane-spanning a-helicesthat might accompany transport of the proton and sugar?

5. What other experimental techniques might be applied toshed additional light on the structure and function of thisprotein?

Frillingos, S., et al. 1998. Cys-scanning mutagenesis: a novelapproach to structure function relationships in polytopicmembrane proteins. FASEB J. 12:1281–1299. (A re-view.)

Venkatesan, P., and H. R. Kaback. 1998. The substrate-bind-ing site in the lactose permease of Escherichia coli. Proc.Nat’l Acad. Sci. USA 95:9802–9807.

Wang, Q., et al. 1998. Proximity of helicesVIII (Ala 273)and IX (Met 299) in the lactose permease of Escherichiacoli. Biochemistry 37:4910–4915.

Wu, J., D. Hardy, and H. R. Kaback. 1998. Transmembranehelix tilting and ligand-induced conformational changesin the lactose permease determined by site-directed chem-ical crosslinking in situ. J. Mol. Biol. 282:959–967.

1. The concentration of glucose in the mammalian blood-stream is about 5 mM and varies within the range of 3 mMafter a few days of fasting to 7 mM after a feast. Consid-ering these to be typical glucose levels, do you expect theKm for a glucose transporter to be 10–6 M or 10–3 M. Why?

2. Chemically, steroid hormones are lipids. On the basis oftheir expected permeability properties in biological mem-branes, predict whether receptor proteins for steroid hor-mones would be expected to be cell-surface or intracellularproteins.

3. Membrane transport proteins, be they uniports, symports,antiports, or ATPases, all have several transmembrane he-lices. How does this contribute to the function of the trans-port protein?

4. A Na1/glucose symport can transport glucose against aconcentration gradient. How is this energetically unfavor-able process linked to ATP consumption, be it direct or in-direct?

5. How might water channels be important in the openingof leaf stomata?

MCAT/GRE-Style Questions

Key Concept Please read the section titled “Na1 Entryinto Mammalian Cells Has a Negative DG” (p. 587) andanswer the following questions (assume a membrane po-tential of 270 mV and the Na1 and K1 concentrationsshown in Figure 15-8):

1. The Na1/K1 ATPase pumps 2 moles of Na1 out of thecell for every 3 moles of K1 pumped into the cell. What isthe DG for pumping 1 mole of Na1 out of the cell?

a. 23.03 kcal/mol.b. 1.41 kcal/mol.c. 1.61 kcal/mol.d. 3.03 kcal/mol.

2. What is the DG for pumping 1 mole of K1 into the cell?a. 20.19 kcal/mol.b. 1.41 kcal/mol.c. 21.61 kcal/mol.d. 3.03 kcal/mol.

3. What is the overall energetics of one complete round oftransport of 2 moles of Na1 and 3 moles of K1?

a. 26.63 kcal.b. 5.49 kcal.c. 6.03 kcal.d. 6.63 kcal.

4. How many ATPs are minimally consumed during onecomplete round of transport?

a. 1.b. 2.c. 3.d. 5.

5. You treat the cell with a drug, a K1 ionophore, that se-lectively equilibrates K1 concentrations across the mem-brane. What now is the DG for K1 transport by the ATPase?Assume that the membrane potential stays the same.

a. 21.61 kcal/mol.b. 20.19 kcal/mol.c. 1.42 kcal/mol.d. 3.03 kcal/mol.

Key Experiment Please read the section titled “TightJunctions Seal Off Body Cavities and Restrict Diffusion ofMembrane Components” (p. 604) and answer the follow-ing questions:

6. Functions of tight junctions include all the following except:

a. Separation of extracellular fluids.b. Sealing of body cavities.

MCAT/GRE-Style Questions 613

Testing Yourself on the Concepts

c. Prevention of diffusion of membrane proteins andlipids between apical and basolateral regions.

d. Tight communication and exchange of small mole-cules between neighboring cells.

7. Molecules present in or associated with tight junctionsinclude 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 bya. Mg21 removal and addition.b. Ca21 removal and addition.c. Glycosidase treatment.d. Trypsin treatment.

9. What is the expected effect on the distribution of plasma-membrane proteins between the apical and basolateral re-gions 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 thecytosolic leaflet of the apical membrane of epithelial cells ina two-step procedure. After completing the procedure, whatis 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-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 theprotein was thought not to be a Cl2 channel itself but rathera regulator of the Cl2 channel. What experimental obser-vation shows that the CFTR protein itself is a Cl2 channel?

a. The ability of recombinant CFTR expressed in the ep-ithelial cells of cystic fibrosis patients to restore theCl2 transport properties of these cells.

b. The ability of recombinant CFTR expressed in testCOS cells to alter the Cl2 transport properties of thesecells.

c. The ability of recombinant CFTR expressed to confercAMP sensitivity to C2 transport.

d. The ability of recombinant CFTR inserted into lipo-somes to form Cl2 transport channels.

13. Introduction of CFTR into the lung is an attractive routefor genetic correction of at least some of the symptoms ofcystic fibrosis. This is true for all the following reasons except:

a. DNA introduced into the lung rather than the blood-stream easily crosses the blood-brain barrier.

b. The lung is easily accessible to DNA-containingaerosols.

c. The lung is one of the major organs affected in CF.d. The introduction of DNA into lung cells does not al-

ter the DNA of germ cells.

Key Terms

active transport 580antiport 598basal lamina 602cell junctions 602cotransport 598epithelium 602hypertonic 609hypotonic 609

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Transport Across Cell Membrane