8

Transport Across
Membranes: Overcoming
Formation of a
Blood Clot. A clot of
fibrin strands (gray) is
entangling a number
of erythrocytes (red),
a model cell type for
studying membrane
transport processes

the Permeability Barrier

(colorized SEM).

W e have previously (Chapter 7) focused on the structure and chemistry of

membranes. We have noted that its hydrophobic interior makes a membrane
an effective barrier to the passage of most molecules and ions, thereby keeping some
substances inside the cell and others out. Within eukaryotic cells, membranes also delineate
organelles by retaining the appropriate molecules and ions needed for specific functions. www.masteringbiology.com
However, it is not enough to think of membranes simply as permeability barriers.
Crucial to the proper functioning of a cell or organelle is the ability to overcome the
permeability barriers for specific molecules and ions so that they can be moved into and
out of the cell or organelle selectively. In other words, membranes are not simply barriers
to the indiscriminate movement of substances into and out of cells and organelles. They are
also selectively permeable, or semipermeable, because they allow the controlled passage
of specific molecules and ions from one side of the membrane to the other. In this chapter
we will look at the ways substances are moved selectively across membranes, and we will
consider the significance of such transport processes to the life of the cell.
209
 8.1 Cells and Transport Processes facilitated diffusion of solutes (sometimes called passive trans-
port) and requires no input of energy.
An essential feature of every cell is its ability to accumulate a In other cases, transport proteins mediate the active trans-
variety of substances at concentrations that are often strik- port of solutes, moving them against their respective free en-
ingly different from those in the surrounding environment. ergy gradients in an energy-requiring process. Active trans-
For cells to survive and function properly, they typically must port must be driven by an energy-yielding process such as the
maintain a constant internal environment very different from hydrolysis of ATP or the simultaneous transport of another
the extracellular environment, a process called homeostasis. solute, usually an ion such as H+ or Na+, down its free energy
Proper regulation of cellular transport is essential to ensure gradient. As we discuss each of these three transport processes
homeostasis and proper cell function. in turn, it will be useful to refer back to Table 8-1.
Most of the substances that move across membranes are
dissolved gases, ions, and small organic molecules—solutes, The Movement of a Solute Across a
in other words. Gases include oxygen, carbon dioxide, and ni-
Membrane Is Determined by Its Concentration
trogen. Common ions transported across membranes are so-
Gradient or Its Electrochemical Potential
dium (Na+), potassium (K+), calcium (Ca2+), chloride (Cl-),
and hydrogen (H+) ions. Most of the small organic molecules The movement of a molecule that has no net charge is de-
are metabolites—substrates, intermediates, and products in termined by the concentration gradient of that molecule
the various metabolic pathways that operate within cells or across the membrane. The concentration gradient is the mag-
specific organelles. Sugars, amino acids, and nucleotides are nitude of the difference in concentration of a substance on op-
some common examples. posite sides of a membrane. A larger concentration difference
These solutes are almost always present at higher con- represents a greater concentration gradient, which creates a
centrations on the inside of the cell or organelle than on the larger driving force for that substance. Simple or facilitated
outside. Very few cellular reactions or processes could occur if diffusion of a molecule involves exergonic and spontaneous
they had to depend on the low concentrations at which essen- movement “down” the concentration gradient from higher to
tial substrates are present in the cell’s surroundings. In some lower concentration (negative ∆G), whereas active transport
cases, such as electrical signaling in nerve and muscle tissue, involves endergonic movement “up” the concentration gradi-
the controlled movement of ions across the membrane is cen- ent (positive ∆G). Because it is not spontaneous, active trans-
tral to the function of the cell. In addition, many drugs, in- port requires some driving force.
cluding prescribed medications, have intracellular targets and The movement of an ion, on the other hand, is deter-
therefore must be able to cross membranes to enter the cell. mined by its electrochemical potential, which is the sum
A central aspect of cell function, then, is transport— of the chemical and the electrical driving forces. It is the com-
the ability to move ions and organic molecules across mem- bined effect of the concentration gradient of an ion and the
branes selectively. The importance of membrane transport is charge gradient of all ions across the membrane. Facilitated
evidenced by the fact that about 20% of the genes that have diffusion of an ion involves exergonic movement in the direc-
been identified in the bacterium Escherichia coli are involved in tion dictated by its electrochemical potential, so we must con-
some aspect of transport. Figure 8-1 summarizes a few of the sider both the concentration gradient of that ion and the net
many transport processes that occur within eukaryotic cells. difference in charge across the membrane.
For example, many cells have an excess of negative
charges inside the cell, so that if a positive ion such as Na+
Solutes Cross Membranes by were in equal concentration both inside and outside the cell,
Simple Diffusion, Facilitated Diffusion, the charge gradient would drive the inward movement of Na+,
and Active Transport even though its concentration inside the cell becomes higher
Three fundamentally different mechanisms are involved in than outside. At thermodynamic equilibrium, there would be
the movement of solutes across membranes, as shown in more Na+ inside the cell than outside, and the concentration
Table 8-1. A few types of molecules move across membranes gradient favoring outward movement of Na+ would exactly
by simple diffusion—direct, unaided movement of solute mol- balance the charge gradient favoring inward movement.
ecules into and through the lipid bilayer in the direction dic- In contrast, active transport of an ion involves endergonic
tated by the difference in the concentrations of the solute on movement against the electrochemical potential for that ion.
the two sides of the membrane. The active transport of ions across a membrane creates the
For most solutes, however, movement across biological charge gradient, or membrane potential (Vm), across the
membranes at a significant rate is possible only because of the membrane that is present in most cells. Most cells have a
presence of transport proteins—integral membrane proteins negative Vm, which by convention means they have an excess
that recognize substances with great specificity and speed of negatively charged solutes inside the cell. For example, a
their movement across the membrane. To see how these trans- resting nerve cell has a Vm of approximately - 60 mV. This
port proteins can be studied in live cells, see Key Technique, charge difference favors the inward movement of cations
page 220. In some cases, transport proteins move solutes such as Na+and the outward movement of anions such as
down their free energy gradient in the direction of thermo- Cl- . It also opposes the outward movement of cations and the
dynamic equilibrium. This gradient represents the difference, inward movement of anions. (In Chapter 22, you will learn
on opposite sides of the membrane, in concentration, charge, about the role of cellular membrane potential in neuron
or both concentration and charge. This process is known as function in detail.)
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M08_HARD7694_09_GE_C08.indd 210 13/02/17 4:28 pm

 Glucose

Carrier protein
Plasma
membrane
H+
Glucose
H+
Pyruvate
mRNA Pi
Proteins
Nucleotides OH- ADP
Mitochondrion

Proteins ATP

ADP

ATP
Nucleus
Organic O2 O2
acids

CO2
CO2
Succinate
Deoxynucleotides
Fatty acids

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

Aquaporin
Glyoxysome
Water

Triose phosphates
Glycine
O2
Glycolate
CO2

Peroxisome

Serine
3-phosphoglycerate
Chloroplast

H+
Lysosome
Carrier
Na+
Amino protein
Protein acids
Pump
protein Amino
K+ acids Amino
acids

H+
H+

ATP
ADP

Figure 8-1 Transport Processes Within a Composite Eukaryotic Cell. The molecules and ions shown
in this composite plant/animal cell are some of the many kinds of solutes that are transported across the
membranes of eukaryotic cells. The enlargements depict a small portion of a mitochondrion (upper right) and a
chloroplast (lower left), illustrating the pumping of protons across membranes during electron transport and the
use of the resulting electrochemical potential to drive ATP synthesis in these organelles.
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M08_HARD7694_09_GE_C08.indd 211 13/02/17 4:28 pm

 Table 8-1 Comparison of Simple Diffusion, Facilitated Diffusion, and Active Transport

Properties Simple Diffusion Facilitated Diffusion Active Transport

Solutes transported
Small polar (H2O, glycerol) Small polar (H2O, glycerol)
Small nonpolar (O2, CO2) Large polar (glucose) Large polar (glucose)
Large nonpolar (oils, steroids) Ions (Na+, K+, Ca2+) Ions (Na+, K+, Ca2+)
Thermodynamic properties
Direction relative to electrochemical Down Down Up
gradient
Metabolic energy required No No Yes
Intrinsic directionality No No Yes
Kinetic properties
Membrane protein required No Yes Yes
Saturation kinetics No Yes Yes
Competitive inhibition No Yes Yes

In all organisms, active transport of ions across the plas- concentrations of Na+ and Cl- are more than 10-fold higher
ma membrane results in asymmetric distributions of ions outside the cell than inside, whereas the concentration of K+
inside and outside of cells, creating an electrochemical gradi- is approximately 40-fold higher inside the cell.
ent for many ions. For example, in human skeletal muscle, the
The Erythrocyte Plasma Membrane Provides
Examples of Transport Mechanisms
(a) Simple diffusion. (b) Facilitated diffusion using In our discussion of membrane transport processes, we will
Oxygen, carbon dioxide, and carrier proteins.
water diffuse directly across GLUT1 transports glucose into the
use the transport proteins of the erythrocyte as examples.
the plasma membrane in erythrocyte, where the glucose These are some of the most extensively studied and best un-
response to their relative concentration is lower. An anion derstood of all cellular transport proteins. Vital to the erythro-
concentrations inside and exchange protein transports cyte’s role in providing oxygen to body tissues is the movement
outside the cell. No transport chloride (Cl-) and bicarbonate
protein is required. (HCO3-) in opposite directions.
across its plasma membrane of O2, CO2, and bicarbonate ion
(HCO3-), as well as glucose, which serves as the cell’s main en-
(a) Glucose ergy source. Also important is the membrane potential main-
transporter tained across the plasma membrane by the active transport of
CO2 (GLUT1) (b) potassium ions inward and sodium ions outward. In addition,
Glucose special pores, or channels, allow water and ions to enter and
HCO3- Anion exchange leave the cell rapidly in response to cellular needs. These trans-
H2O CO2
Cl- protein
port activities are summarized in Figure 8-2 and will be used
H 2O Glucose Sodium/potassium as examples in the following discussion.
HCO3- pump
O2 O2 Cl-
H2O ADP CONCEPT CHECK 8.1
RED BLOOD CELL
What is the difference between the concentration gradient
3 Na+
2 K+ and the electrochemical potential? For which of the three
3 Na+
types of transport mechanisms is the magnitude of the con-
(c) ATP
H2O 2 K+ centration gradient relevant? For which is the electrochemi-
Aquaporin cal gradient important?
(d)

(c) Facilitated diffusion using (d) Active transport using

channel proteins. ATP-requiring pumps. 8.2 Simple Diffusion: Unassisted
Aquaporin channel proteins can Driven by the hydrolysis of ATP,
facilitate the rapid inward or the Na+/K+ pump moves sodium Movement Down the Gradient
outward movement of water ions outward and potassium ions
depending on the relative solute inward, establishing an electro- The most straightforward way for a solute to get from one side
concentration on opposite chemical potential across the of a membrane to the other is simple diffusion, which is the
sides of the membrane. plasma membrane for both ions. unassisted net movement of a solute from a region where its
Figure 8-2 Important Transport Processes of the concentration is higher to a region where its concentration is
Erythrocyte. Depicted here are several types of transport pro- lower (Figure 8-2a). Because membranes have a hydrophobic
cesses that are vital to erythrocyte function. interior, simple diffusion is typically a means of transport only
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 for gases, nonpolar molecules, and very small polar molecules (a) In the capillaries of body tissues (low [O2] and high [CO2]
such as water, glycerol, and ethanol. Large molecules, most relative to the erythrocytes), O2 is released by hemoglobin
polar molecules, and all ions require an intrinsic membrane within the erythrocytes and diffuses outward to meet tissue
needs. CO2 diffuses inward and is converted to bicarbonate
protein, known as a transporter, to move across the mem- by carbonic anhydrase in the cytosol. Bicarbonate ions are
brane, as we will see later in the chapter. transported outward by the anion exchange protein,
Oxygen is a gas that traverses the hydrophobic lipid bilayer accompanied by the inward movement of chloride ions to
readily and therefore moves across membranes by simple dif- maintain charge balance. Carbon dioxide therefore returns
to the lungs as bicarbonate ions.
fusion. This behavior enables erythrocytes in the circulatory
system to take up oxygen in the lungs and release it in body
tissues. In the capillaries of body tissues, where the oxygen (a) Low [O2]
concentration is low, oxygen is released from hemoglobin and High [CO2]
diffuses passively from the cytoplasm of the erythrocyte into
the blood plasma and from there into the cells lining the capil-
CO2
laries (Figure 8-3a).
In the capillaries of the lungs, the opposite occurs: oxy- CO2 + H2O H+ + HCO3- HCO3-
gen diffuses from the inhaled air in the lungs, where its con- Carbonic
centration is higher, into the cytoplasm of the erythrocytes,
anhydrase Cl- Cl-
O2
where its concentration is lower (Figure 8-3b). Carbon dioxide RED BLOOD CELL
is also able to cross membranes by simple diffusion. However,
O2
most CO2 is actually transported in the form of bicarbonate In body tissues
ion, as we will see later in the chapter. Not surprisingly, carbon Blood plasma
dioxide and oxygen move across the erythrocyte membrane in
opposite directions. Carbon dioxide diffuses inward in body tis-

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

sues and outward in the lungs.
(b)
High [O2]
Diffusion Always Moves Solutes
Low [CO2]
Toward Equilibrium In the lungs

No matter how a population of molecules is distributed ini-

tially, diffusion always tends to create a uniform solution CO2
in which the concentration of all solutes is the same every-
CO2 + H2O H+ + HCO3- HCO3-
where. To illustrate this point, consider the apparatus shown Carbonic
in Figure 8-4a, consisting of two chambers separated by a anhydrase Cl- Cl-
membrane that is freely permeable to molecules of an un- O2 RED BLOOD CELL
charged solute represented by the black dots. Initially, the
concentration of the solute is higher in chamber A than in O2
chamber B. Other conditions being equal, random move- Blood plasma
ments of solute molecules back and forth through the mem-
brane will lead to a net movement of solute from chamber A to (b) In the capillaries of the lungs (high [O2] and low [CO2]
relative to the erythrocytes), O2 diffuses inward and binds
chamber B. When the concentration of solute is equal on both to hemoglobin. Bicarbonate moves inward from the blood
sides of the membrane, the system is at equilibrium. Random plasma, accompanied by an outward movement of
back-and-forth movement of individual molecules continues, chloride ions. Incoming bicarbonate is converted into CO2,
but no further net change in concentration occurs. Thus, which diffuses out of the erythrocytes and into the cells
lining the capillaries of the lungs. The CO2 is now ready to
diffusion is always movement toward equilibrium and is therefore be expelled from the body.
a spontaneous process. No energy is required and no trans-
port protein is needed. Figure 8-3 Directions of Oxygen, Carbon Dioxide, and
Another way to express this is to say that diffusion always ­Bicarbonate Transport in Erythrocytes. The directions in which
tends toward minimum free energy. Chemical reactions and O2, CO2, and HCO3- move across the plasma membrane of the
erythrocyte depend on the location of the erythrocyte in the body.
physical processes always proceed in the direction of decreas-
ing free energy, in accordance with the second law of thermo-
dynamics (see Chapter 5). Diffusion through membranes is no Osmosis Is the Diffusion of Water Across
exception: free energy is minimized as molecules move down a Selectively Permeable Membrane
their concentration gradient. The driving force for diffusion Most of the discussion in this chapter focuses on the transport
is entropy, the randomization of molecules as concentrations of solutes—ions and small molecules that are dissolved in the
equalize on both sides of the membrane. We will apply this aqueous environment of cells, their organelles, and their sur-
principle later in the chapter when we calculate ∆G, the free roundings. That emphasis is quite appropriate because most
energy change that accompanies the transport of molecules of the traffic across membranes involves ions such as K+, Na+,
and ions across membranes. At thermodynamic equilibrium, and H+ and hydrophilic molecules such as sugars, amino ac-
no further net movement occurs because the free energy of ids, and a variety of metabolic intermediates. But to under-
the system is at a minimum. stand solute transport fully, we also need to understand the
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 (a) Simple diffusion takes place when the membrane separating in Figure 8-4a, but with the two chambers now separated by a
chambers A and B is permeable to molecules of dissolved selectively permeable membrane that is permeable to water but not
solute, represented by the black dots. Net movement of solute to the dissolved solute. Under these conditions, water diffuses
molecules across the membrane is from chamber A to B (high
to low solute concentration). Equilibrium is reached when the across the membrane from chamber B to chamber A. Although
solute concentration is the same in both chambers. looking very different from the tube shown in Figure 8-4a, cells
behave in much the same way. Because the concentration of
solutes is almost always higher inside a cell than outside, for
A B A B most cells this means that water will tend to move inward. If
not controlled, that inward movement of water will cause cells
that lack a cell wall to swell and perhaps burst.
Osmotic movement of water into and out of a cell is re-
Solute lated to the relative osmolarity, or total solute concentration,
movement of the cytoplasm versus the extracellular solution. A solution
from A to B
with a higher solute concentration than that inside a cell is
called a hypertonic solution, whereas a solution with a sol-
Membrane
(permeable to solute) ute concentration lower than that inside a cell is referred to
as a hypotonic solution. Hypertonic solutions cause water
molecules to diffuse out of the cell, dehydrating it. In contrast,
(b) Osmosis occurs when the membrane between the two hypotonic solutions cause water to diffuse into the cell, in-
chambers is not permeable to the dissoved solute, creasing the internal pressure. A solution with the same sol-
represented by the black triangles. Because solute cannot ute concentration as the cell is called an isotonic solution;
cross the membrane, water diffuses from chamber B, where
the solute concentration is lower (more water), to chamber A,
in this case, there will be no net movement of water in either
where the solute concentration is higher (less water). At direction.
equilibrium, the solute concentration will be equal on both Osmosis accounts for a well-known observation: cells
sides of the membrane. tend to shrink or swell as the solute concentration of the
extracellular medium changes. Consider, for example, the
scenario of Figure 8-5. An animal cell that starts out in an
A B A B isotonic solution will shrink and shrivel if it is transferred to a
hypertonic solution (Figure 8-5a). On the other hand, the cell
will swell if placed in a hypotonic solution. And it will actually
lyse—or burst—if placed in a very hypotonic solution, such as
Water pure water containing no solutes (Figure 8-5b).
movement
from B to A
HYPERTONIC ISOTONIC HYPOTONIC
SOLUTION SOLUTION SOLUTION
Membrane
(not permeable to solute) H2O
H2O H2O H2O
Figure 8-4 Comparison of Simple Diffusion and Osmosis.
In both examples shown, there is initially more solute in chamber
A than in chamber B. The membrane in example (a) is permeable (a) (b)
to the solute, and the membrane in example (b), like a typical cell
membrane, is not.
Shriveled Normal Lysed
ANIMAL CELL
forces that act on the water in which the solute is dissolved,
H2O
thereby determining its movement into and out of cells. H2O H2O H2O
Because most solutes cannot cross cell membranes by
diffusion, water tends to move across membranes in response
to differences in solute concentrations on the two sides of the
(c) (d)
membrane. Specifically, water will diffuse from the side of
the membrane with the lower solute concentration (slightly
Plasmolyzed Flaccid Turgid
higher water concentration) to the side with the higher sol-
PLANT CELL
ute concentration (slightly lower water concentration). This
diffusion of water in response to differences in solute concen- Figure 8-5 Responses of Animal and Plant Cells to Changes
in Osmolarity. (a) If an animal cell (or other cell not surrounded by
tration is called osmosis and is readily observed when a selec-
a cell wall) is transferred from an isotonic solution to a hypertonic
tively permeable membrane separates two compartments, one
solution, water leaves the cell, and the cell shrivels. (b) If the cell is
of them containing a solute that cannot cross the membrane.
transferred to a hypotonic solution, water enters the cell, and the
Water will move across the membrane to equalize the concen- cell swells, sometimes to the point that it bursts. (c) Plant cells (or
tration of solutes on both sides of the membrane. other cells with rigid cell walls) also shrivel (plasmolyze) in a hyper-
This principle is illustrated by Figure 8-4b. Solutions of dif- tonic solution, but (d) they will only become turgid—and will not
fering solute concentrations are placed in chambers A and B, as burst—in a hypotonic solution.
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 The osmotic movements of water shown in Figure 8-5 uncharged molecules such as oxygen exchanged so rapidly
occur because of differences in the osmolarity of the cyto- that the rates could hardly be measured. The inescapable con-
plasm and the extracellular solution. Usually, the solute con- clusion was that the lipid bilayer represents the primary per-
centration is greater inside a cell than outside. This is due meability barrier of a membrane. Small, uncharged molecules
both to the high concentrations of ions and small organic can pass through the barrier by simple diffusion, whereas
molecules required for normal cellular functions and to the sodium and potassium ions can barely pass at all. Based on
large numbers of macromolecules dissolved in the cytosol. In subsequent experiments by many investigators using a variety
addition, most of these molecules are charged, and the coun- of lipid bilayer systems and thousands of different solutes, we
terions that balance these charges contribute significantly to can predict with considerable confidence how readily a solute
the intracellular osmolarity. Thus, most cells are hypertonic will diffuse across a lipid bilayer. The three main factors affect-
compared with their surroundings, so water will move inward ing diffusion of solutes are size, polarity, and charge. We will
across the plasma membrane, causing the cells to swell. consider each of these factors in turn.
How do cells cope with the problem of high osmolar-
ity and the resulting influx of water due to osmosis? Cells of Solute Size. Generally speaking, lipid bilayers are more perme-
plants, algae, fungi, and many bacteria have rigid cell walls able to smaller molecules than to larger molecules. The smallest
that keep the cells from swelling and bursting in a hypotonic molecules relevant to cell function are water, oxygen, and car-
solution (Figure 8-5d). Instead, the cells become very firm bon dioxide. Membranes are quite permeable to these molecules,
from the turgor pressure that builds up due to the inward which require no specialized transport processes for moving
movement of water. The resulting turgidity accounts for the into and out of cells, although we will see later that there is a
firmness, or turgor, of fully hydrated plant tissue. Without this specialized transporter in some cells allowing rapid transport of
turgidity, the tissue will wilt. water molecules across certain membranes. But, in the absence
In a hypertonic solution, on the other hand, the out- of a transporter, even such small molecules do not move across
ward movement of water causes the plasma membrane to membranes freely. Water molecules, for example, diffuse across a

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

pull away from the cell wall by a process called plasmolysis bilayer 10,000 times more slowly than they move when allowed
(Figure 8-5c). The wilting of a plant or a plant part during wa- to diffuse freely in the absence of a membrane.
ter deprivation is due to the plasmolysis of its cells. You can Still, water diffuses across membranes at rates much
demonstrate plasmolysis readily by dropping a piece of cel- higher than would be expected for such a polar molecule. The
ery into a solution with a high concentration of salt or sugar. reason for this behavior is not well understood. One proposal
Plasmolysis can be a practical problem when plants are grown is that membranes contain tiny pores that allow the passage
under conditions of high salinity, as is sometimes the case in of water molecules but are too small for any other polar sub-
locations near an ocean. stance. An alternative suggestion is that in the continual
Cells without cell walls solve the osmolarity problem by movements of membrane lipids, transient “holes” are created
continuously and actively pumping out inorganic ions, thereby in the lipid monolayers that allow water molecules to move
reducing the intracellular osmolarity and thus minimizing the first through one monolayer and then through the other.
difference in solute concentration between the cell and its sur- There is little experimental evidence to support either of these
roundings. Animal cells continuously remove sodium ions. This hypotheses, however, and simple diffusion of water across
is, in fact, one important purpose of the Na+/K+ pump. Animal membranes remains an enigma.
cells swell and sometimes even lyse when treated with ouabain, In addition to water, small polar molecules with a mo-
an inhibitor of the Na+/K+ pump. When medications are lecular weight (MW) of up to about 100—such as ethanol
given intravenously in a hospital, they are typically dissolved in (CH3CH2OH; MW = 46) and glycerol (C3H8O3; MW = 92)—
phosphate-buffered saline, which has the same osmolarity as the are able to diffuse across membranes. However, larger polar mol-
blood, avoiding potential problems of cell lysis or dehydration. ecules such as glucose (C6H12O6; MW = 180) cannot. Cells
therefore need specialized proteins in their plasma membranes to
facilitate the entry of glucose and most other polar solutes.
Simple Diffusion Is Typically Limited
to Small, Nonpolar Molecules Solute Polarity. There is a correlation between the lipid solu-
To investigate the factors influencing the diffusion of solutes bility of a molecule and its membrane permeability. In general,
through membranes, scientists frequently use membrane lipid bilayers are more permeable to nonpolar molecules and
models. An important advance in developing such models was less permeable to polar molecules. This is because nonpolar
provided in 1961 by Alec Bangham and his colleagues, who molecules dissolve more readily in the hydrophobic phase of the
found that when lipids are extracted from cell membranes and lipid bilayer and can therefore cross the membrane much more
dispersed in water, they form liposomes. Liposomes are small rapidly than can polar molecules of similar size. For example,
vesicles about 0.1 mm in diameter, each consisting of a closed, the steroid hormones estrogen and testosterone are largely non-
spherical, lipid bilayer that is devoid of membrane proteins polar and therefore can diffuse across membranes despite hav-
(see Figure 4-2). Bangham showed that it is possible to trap ing molecular weights of 370 and 288, ­respectively.
solutes such as potassium ions in the liposomes as they form A simple measure of the polarity (or nonpolarity) of a
and then measure the rate at which the solute escapes by dif- solute is its partition coefficient, which is the ratio of its solu-
fusion across the liposome bilayer. bility in an organic solvent (such as vegetable oil or octanol)
The results were remarkable. Ions such as potassium and to its solubility in water. In general, the more nonpolar—
sodium were trapped in the vesicles for days, whereas small or hydrophobic—a substance is, the higher its partition
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 coefficient and the more readily and rapidly it can move across
(or dissolve in) a membrane. For example, the partition coef-
ficients of the various amino acids can be used to calculate the
Facilitated diffusion
hydropathy index of a protein (see Figure 7-21).

v = rate of diffusion
(hyperbolic)
When incorporated into proteins as amino acid residues,
the amino acids no longer have the two charged ends as do
free amino acids, and the degree of polarity is determined
solely by the side chain. Amino acids with nonpolar side
chains (see Figure 3-2), such as tryptophan, leucine, and va-
Simple diffusion
line, have high partition coefficients and are likely to be found (linear)
in transmembrane regions of a membrane protein, in contrast
to those with polar side chains and low partition coefficients
¢3S4 = solute concentration gradient
(serine and threonine).
Figure 8-6 Comparison of the Kinetics of Simple Diffusion
Solute Charge. The relative impermeability of polar sub- and Facilitated Diffusion. For simple diffusion across a mem-
stances in general and of ions in particular is due to their brane, the relationship between v, the rate of diffusion, and ∆[S],
strong association with water molecules, forming a shell of the solute concentration gradient, is linear over a broad concentra-
hydration. For such solutes to move into the membrane, the tion range (red line). For facilitated diffusion, the relationship is linear
water molecules must be stripped off, and an input of energy when the concentration gradient is small but exhibits saturation
kinetics and is therefore hyperbolic (green line), eventually reaching
is required to eliminate the bonding between the ions and the
a maximum value at very high ∆[S].
water molecules. Therefore, the association of ions with wa-
ter molecules to form shells of hydration dramatically restricts
ion transport across membranes. shape, and polarity of S; and the equilibrium distribution of
The impermeability of membranes to ions is important to S in the membrane and aqueous phases. As either the perme-
cell activity because for it to function, every cell must maintain ability or the concentration gradient increases, the rate of in-
an electrochemical potential across its plasma membrane. In ward transport increases.
most cases this potential is a gradient of either sodium ions As Equation 8-1 indicates, simple diffusion is characterized
(animal cells) or protons (most other cells). On the other hand, by a linear relationship between the inward flux of the solute
membranes must also allow ions to cross the barrier in a con- across the membrane and the concentration gradient of the
trolled manner. As we will see later in the chapter, the proteins solute, with no evidence of saturation at high concentrations.
that facilitate ion transport serve as hydrophilic channels This relationship is seen as the straight red line in Figure 8-6.
that provide a low-energy pathway for movement of the ions Simple diffusion differs in this respect from facilitated diffusion,
across the membrane. which is subject to saturation and generally follows hyperbolic
Michaelis–Menten kinetics, as we will see shortly.
The Rate of Simple Diffusion Is Directly We can summarize simple diffusion by noting that it is rel-
Proportional to the Concentration Gradient evant only to molecules such as ethanol and O2 that are small
So far, we have focused on qualitative aspects of simple diffu- enough and/or nonpolar enough to cross membranes without
sion. We can be more quantitative by considering the thermo- the aid of transport proteins. Simple diffusion proceeds exer-
dynamic and kinetic properties of the process (see Table 8-1, gonically in the direction dictated by the concentration gra-
page 212). Thermodynamically, simple diffusion is always an dient, with a linear, nonsaturating relationship between the
exergonic process, requiring no input of energy. Individual diffusion rate and the concentration gradient.
molecules simply diffuse randomly in both directions, but net
CONCEPT CHECK 8.2
flux will always be in the direction of minimum free energy—
How is osmosis different from the simple diffusion of molecular
which in the case of uncharged molecules means down the
oxygen (O2) across a membrane? How are they similar?
concentration gradient.
Kinetically, a key feature of simple diffusion is that the net
rate of transport for a specific substance is directly proportional
to the concentration difference for that substance on opposite 8.3 Facilitated Diffusion:
sides of the membrane.. For the diffusion of solute S from the Protein-Mediated Movement
outside to the inside of a cell, the expression for the rate, or
velocity, of inward diffusion through the membrane, vinward, is Down the Gradient
Most substances in cells are too large or too polar to cross mem-
vinward = P∆ 3S 4 (8-1)
branes at reasonable rates by simple diffusion, even if the pro-
where vinward is the rate of inward diffusion (in moles/ cess is exergonic. Such solutes can move into and out of cells
second-cm2 of membrane surface), and ∆[S] is the con- and organelles at appreciable rates only with the assistance of
centration gradient of the solute across the membrane transport proteins that mediate the movement of solute mol-
(∆ 3S 4 = 3S 4 outside − 3S 4 inside). P is the permeability coef- ecules across the membrane. If such a process is exergonic, it
ficient, an experimentally determined parameter that depends is called facilitated diffusion because the solute still diffuses
on the thickness and viscosity of the membrane; the size, in the direction dictated by the concentration gradient (for
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 uncharged molecules) or by the electrochemical gradient (for a carrier protein is an allosteric membrane-spanning protein
ions), with no input of energy needed. The role of the transport that alternates between two conformational states. In one
protein is simply to provide a path through the hydrophobic state, the solute-binding site of the protein is open or acces-
lipid bilayer, facilitating the “downhill” diffusion of a polar or sible on one side of the membrane. Following solute binding,
charged solute across an otherwise impermeable barrier. the protein changes to an alternate conformation in which
Unlike simple diffusion, whose rate increases proportionally the solute-binding site is on the other side of the membrane,
with the concentration difference across the membrane, facili- triggering its release. We will encounter an example of this
tated diffusion can become saturated at high solute concentra- mechanism shortly when we discuss the facilitated diffusion
tions because there are a limited number of transport proteins. of glucose into erythrocytes.
Thus, whereas a graph of rate versus concentration differ-
ence will be linear for simple diffusion, it will be hyperbolic for Carrier Proteins Are Analogous to Enzymes
facilitated diffusion (Figure 8-6, green curve). Note that this re- in Their Specificity and Kinetics
sembles the hyperbolic shape of the curve for the initial velocity
As we noted earlier, carrier proteins are sometimes called
of an enzyme versus substrate concentration (see Figure 6-8).
permeases. This term is apt because the suffix –ase suggests
As an example of facilitated diffusion, consider the move-
a similarity between carrier proteins and enzymes. Like an
ment of glucose across the plasma membrane of a cell in your
enzyme-catalyzed reaction, facilitated diffusion involves an
body. The concentration of glucose is typically higher in the
initial binding of the “substrate” (the solute to be transported)
blood than in the cell, so the inward transport of glucose is
to a specific site on a protein surface (the solute’s binding site
exergonic—that is, it does not require the input of energy.
on the carrier protein) and the eventual release of the “prod-
However, glucose is too large and too polar to diffuse across
uct” (the transported solute), with an “enzyme-substrate”
the membrane unaided. A transport protein is required to fa-
complex (solute bound to carrier protein) as an intermediate.
cilitate its inward movement.
Like enzymes, carrier proteins can be regulated by external

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

factors that bind and modulate their activity.
Carrier Proteins and Channel Proteins
Facilitate Diffusion by Different Mechanisms Specificity of Carrier Proteins. Another property that
Transport proteins involved in facilitated diffusion of small carrier proteins share with enzymes is specificity. Like enzymes,
molecules and ions are integral membrane proteins that transport proteins are highly specific, often for one compound
contain several, or even many, transmembrane segments and or a small group of closely related compounds and sometimes
therefore traverse the membrane multiple times. Functionally, even for a specific stereoisomer. A good example is the carrier
these proteins fall into two main classes that transport solutes protein that facilitates the diffusion of glucose into erythrocytes
in quite different ways. Carrier proteins (also called trans- (see Figure 8-2b). This protein recognizes only glucose and a
porters or permeases) bind one or more solute molecules on one few closely related monosaccharides, such as galactose and
side of the membrane and then undergo a conformational mannose. Moreover, the protein is stereospecific: it accepts the
change that transfers the solute to the other side of the mem- d- but not the l-isomer of these sugars. This specificity is pre-
brane. In so doing, a carrier protein binds the solute molecules sumably a result of the precise stereochemical fit between the
in such a way as to shield the polar or charged groups of the solute and its binding site on the carrier protein.
solute from the nonpolar interior of the membrane. Thus, the properties of carrier proteins explain the char-
Channel proteins, on the other hand, form hydrophilic acteristic features of facilitated diffusion: movement of polar
channels through the membrane that allow the passage of molecules and ions down a concentration gradient, specific-
solutes without a major change in the conformation of the ity for the particular substrate transported, the ability to be
protein. Some of these channels are relatively large and non- saturated at high levels of substrate, and sensitivity to specific
specific, such as the pores found in the outer membranes of inhibitors of transport.
bacteria, mitochondria, and chloroplasts. Pores are formed
by transmembrane proteins called porins and allow selected Kinetics of Carrier Protein Function. As you might ex-
hydrophilic solutes with molecular weights up to about 600 pect from the analogy with enzymes, carrier proteins become
to diffuse across the membrane. However, most channels are saturated as the concentration of the transportable solute
small and highly selective. Most of these smaller channels are is raised. This is because the number of transport proteins is
involved in the transport of ions rather than molecules and limited, and each transport protein functions at some finite
are therefore referred to as ion channels. The movement of sol- maximum velocity. As a result, carrier-facilitated transport,
utes through ion channels is much more rapid than transport like enzyme catalysis, exhibits saturation kinetics. This type of
by carrier proteins, presumably because complex conforma- transport has an upper limiting velocity Vmax and a constant
tional changes are not necessary. Km corresponding to the concentration of transportable sol-
ute needed to achieve one-half of the maximum rate of trans-
Carrier Proteins Alternate Between Two port. This means that the initial rate of solute transport, v, can
Conformational States be described mathematically by the same equation we used to
describe enzyme kinetics (see Equation 6-7 on page 163):
An important topic of contemporary membrane research con-
cerns the mechanisms whereby transport proteins facilitate
the movement of solutes across membranes. For carrier pro- Vmax 3S 4
v= (8-2)
teins, the alternating conformation model describes how Km + 3S 4
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 where [S] is the initial concentration of solute on one side of this simplified illustration, but alternate between two confor-
the membrane (for example, on the outside of the membrane if mations as solutes are transported.
the initial rate of inward transport is to be determined). A plot
of transport rate versus initial solute concentration is there-
The Erythrocyte Glucose Transporter
fore hyperbolic for facilitated diffusion instead of linear as for
and Anion Exchange Protein Are Examples
simple diffusion (see Figure 8-6, green curve). This difference in
saturation kinetics is an important means of distinguishing be-
of Carrier Proteins
tween simple and facilitated diffusion (see Table 8-1, page 212). Now that we have described the general properties of carrier
A further similarity between enzymes and carrier pro- proteins, we will consider two specific examples: the uniport
teins is that proteins are often subject to competitive inhibition carrier for glucose and the antiport anion carrier for Cl- and
by molecules or ions that are structurally related to the in- HCO3- . Both of these transporters are present in the cell mem-
tended “substrate.” For example, the transport of glucose by a brane of erythrocytes.
glucose carrier protein is competitively inhibited by the other
monosaccharides that the protein also accepts—that is, the The Glucose Transporter: A Uniport Carrier. As we noted
rate of glucose transport is reduced in the presence of other earlier, the movement of glucose into an erythrocyte is an
transportable sugars. example of facilitated diffusion mediated by a uniport carrier
protein (see Figure 8-2b). The concentration of glucose in blood
plasma is usually in the range of 65–90 mg/100 mL, or about
Carrier Proteins Transport Either 3.6–5.0 mM. The erythrocyte (or almost any other cell in con-
One or Two Solutes tact with the blood, for that matter) is capable of glucose uptake
Although carrier proteins are similar in their kinetics and by facilitated diffusion because of its low intracellular glucose
their presumed mechanism of action involving alternate concentration and the presence in its plasma membrane of a
conformations, they may differ in significant ways. The most glucose carrier protein, or glucose transporter (GLUT). The
important differences concern the number of solutes trans- GLUT of erythrocytes is called GLUT1 to distinguish it from
ported and the direction they move. When a carrier protein related GLUTs in other mammalian tissues. GLUT1 allows glu-
transports a single solute across the membrane, the process is cose to enter the cell about 50,000 times faster than it would
called uniport (Figure 8-7a). The glucose carrier protein we enter by free diffusion through a lipid bilayer.
will discuss shortly is a uniporter. When two solutes are trans- GLUT1-mediated uptake of glucose displays all the classic
ported simultaneously and their transport is coupled such features of facilitated diffusion: it is specific for glucose (and
that transport of either stops if the other is absent, the process a few related sugars, such as galactose and mannose), it pro-
is called coupled transport (Figure 8-7b). Coupled transport ceeds down a concentration gradient without energy input, it
is referred to as symport (or cotransport) if the two solutes are exhibits saturation kinetics, and it is susceptible to competi-
moved in the same direction or as antiport (or countertrans- tive inhibition by related monosaccharides. GLUT1 is an inte-
port) if the two solutes are moved in opposite directions across gral membrane protein with 12 hydrophobic transmembrane
the membrane. The transport proteins that mediate these pro- segments. These are presumably folded and assembled in the
cesses are called symporters and antiporters, respectively. Note membrane to form a cavity lined with hydrophilic side chains
that transporters are not simply open channels, as depicted in that form hydrogen bonds with glucose molecules as they
move through the membrane.
GLUT1 is thought to transport glucose by an alternating
conformation mechanism, as illustrated in Figure 8-8. The
two conformational states are called T1, which has the bind-
OUTER Sb ing site for glucose open to the outside of the cell, and T2, with
SURFACE S Sa Sb Sa the binding site open to the interior of the cell. The process
begins when a molecule of D-glucose collides with and binds
to a GLUT1 molecule that is in its T1 conformation ● 1 . With
glucose bound, GLUT1 now shifts to its T2 conformation ● 2.
The conformational change allows the release of the glucose
molecule to the interior of the cell ● 3 , after which the GLUT1
INNER molecule returns to its original conformation, with the bind-
SURFACE Sb ing site again facing outward ● 4.
Sa Sb Sa
S The example shown in Figure 8-8 is for inward transport,
Symport Antiport but the process is readily reversible because carrier proteins
function equally well in either direction. A carrier protein is
(a) Uniport (b) Coupled transport
really just a gate in an otherwise impenetrable wall, and, like
most gates, it facilitates traffic in either direction. Individual
Figure 8-7 Comparison of Uniport, Symport, and Antiport
Transport by Carrier Proteins. (a) In uniport transport, a mem-
solute molecules may be transported either inward or out-
brane transport protein moves a single solute (S) across a mem- ward, depending on the relative concentrations of the solute
brane. (b) In coupled transport, a membrane transport protein on the two sides of the membrane. If the concentration is
simultaneously moves two solutes (Sa and Sb) either in the same higher outside, net flow will be inward. If the higher concen-
direction (symport) or in opposite directions (antiport). tration occurs inside, net flow will be outward.
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 1 Glucose binds to a 2 Glucose binding causes 3 Glucose is released to 4 Loss of bound glucose
GLUT1 transporter protein the GLUT1 transporter to the interior of the cell, causes GLUT1 to return to
that has its binding site shift to its T2 conformation initiating a second its original (T1) confor-
open to the outside of the with the binding site open conformational change in mation, ready for a
cell (T1 conformation). to the inside of the cell. GLUT1. further transport cycle.

O
T2

O
O O
OUTSIDE
Glucose Glucose OF CELL

T1 T1
Glucose transporter
(GLUT1) INSIDE OF CELL

Figure 8-8 The Alternating Conformation Model for Facilitated Diffusion of Glucose by the
Glucose Transporter GLUT1 in the Erythrocyte Membrane. The inward transport of glucose by GLUT1
is shown here in four steps, arranged at the periphery of a cell.

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

The low intracellular glucose concentration that makes facilitate glucose transport out of liver cells to maintain blood
facilitated diffusion possible for most animal cells exists be- glucose at a constant level.
cause incoming glucose is quickly phosphorylated to glucose- Later in the chapter we will discuss another type of glu-
6-phosphate by the enzyme hexokinase, with ATP as the phos- cose transporter, the Na+/glucose symporter. In contrast to
phate donor and energy source: GLUT1, which carries only glucose, the Na+/glucose sym-
porter simultaneously transports Na+ and glucose molecules
ATP ADP
across a membrane in the same direction. This provides a
glucose glucose-6-phosphate means to transport glucose into a cell that already has a higher
hexokinase (8-3) concentration of glucose than its environment. Typically, the
facilitated diffusion of Na+ down its electrochemical gradient
This hexokinase reaction is the first step in glucose metabo- provides the energy to transport glucose up its concentration
lism (which we will discuss in Chapter 9). The low Km of hexo- gradient.
kinase for glucose (1.5 mM) and the highly exergonic nature
of the reaction (∆G°′ = - 4.0 kcal/mol) ensure that the con- The Erythrocyte Anion Exchange Protein: An Antiport
centration of free glucose within the cell is kept low, maintain- Carrier. Another well-studied example of facilitated diffusion
ing the concentration gradient across the cell membrane. For is the anion exchange protein of the erythrocyte plasma
many mammalian cells, the intracellular glucose concentra- membrane (see Figure 8-2b). This antiport protein, also called
tion ranges from 0.5 to 1.0 mM, which is about 15–20% of the chloride-bicarbonate exchanger, facilitates the reciprocal ex-
the glucose level in the blood plasma outside the cell. change of chloride and bicarbonate ions in opposite directions
The phosphorylation of glucose also has the effect of across the plasma membrane. The coupling of chloride and bi-
locking glucose in the cell, because the plasma membrane of carbonate transport is obligatory, and transport will stop if ei-
the erythrocyte does not have a transport protein for glucose- ther anion is absent. Moreover, the anion exchange protein is
6-phosphate. GLUT1, like most sugar transporters, does not very selective. It exchanges bicarbonate for chloride in a strict
recognize the phosphorylated form of the sugar. In addition, 1:1 ratio, and it accepts no other anions.
due to phosphorylation, the level of free glucose is kept low in The anion exchange protein is thought to function by al-
the cell, ensuring that equilibrium is not reached and allowing ternating between two conformational states in what is termed
the cell to continue to import glucose. a “ping-pong” mechanism. In one conformation, the anion
The GLUT1 of erythrocytes is just one of several glu- exchange protein binds chloride on one side of the membrane.
cose transporters in mammals. In humans there are 14 dif- Chloride binding causes a change in conformation such that
ferent GLUT proteins, each encoded by a separate gene. Each chloride is released on the other side of the membrane, where
transporter has distinct physical and kinetic characteristics the protein binds bicarbonate. This causes a second conforma-
appropriate for its function in the particular cell type where tional change, releasing bicarbonate on the other side of the
it is found. For example, GLUT3 and GLUT4 are found in ce- membrane, where the protein again binds chloride. Repeated
rebral neurons and skeletal muscle, respectively, where they cycles of binding and release transport the two ions in opposite
import glucose for energy. On the other hand, GLUT2 is the directions.
glucose transporter in liver cells, which break down glycogen Either anion can bind to the protein on either side of
to produce glucose for the blood. GLUT2 has properties that the membrane, so the direction of transport depends upon
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 KEY TECHNIQUE
Expression of Heterologous
Membrane Proteins in Frog Oocytes

Details: In the 1970s, John Gurdon and colleagues at Oxford

University showed that oocytes of Xenopus laevis, the African
clawed toad, provided a very useful in vivo experimental sys-
tem for the translation of heterologous mRNA (mRNA from
other organisms). In their pioneering work, they showed that,
following microinjection of rabbit hemoglobin into the cells,
these oocytes could efficiently synthesize rabbit hemoglobin.
This powerful system has been used for more than 40 years to
express a wide variety of heterologous proteins, including the
often difficult-to-study membrane proteins.
Xenopus oocytes are large cells, having a diameter of about
1 mm, making them visible to the naked eye. Using a dissecting
microscope, the eggs can be injected with mRNA of a protein
under study. For multisubunit proteins, several mRNAs—each
representing one of the subunits—can be co-injected, often
­producing an intact, multisubunit protein in a functional form.
Peter Agre and colleagues, in collaboration with Bill Guggino,
1 cm used Xenopus oocytes to show that the aquaporin membrane
protein was a water channel, as had been predicted by numer-
Screening Xenopus oocytes in a 96-well microplate. ous physiological studies.
Xenopus oocytes have a number of practical advantages that
PROBLEM: Studying the function of transport proteins is make them extremely useful as an experimental system. They
difficult because, by nature, they are integral membrane proteins are large and easily microinjected with a very fine needle. The
embedded in the membrane. As a consequence, they are diffi- frogs themselves are relatively easy to raise in captivity, and
cult to purify and study in an intact, functional form. So how can harvesting the oocytes is a relatively routine procedure. Once
the function of a specific membrane protein be studied in vivo in isolated from female frogs, oocytes can be maintained without
a living cell? additional nutrients in little more than fresh water.
About a decade after Gurdon and colleagues showed that
SOLUTION: Frog oocytes (egg cell precursors) are a Xenopus oocytes could be used to express rabbit hemoglobin,
common cell type that is used to produce functional integral they became a popular system for the expression of functional
membrane proteins. The mRNA encoding the membrane protein ion channels for studying electrophysiology in neurons and other
can be microinjected into an individual oocyte, where the cell’s cells. Later, they became a popular host cell for nuclear trans-
ribosomes translate the mRNA into the desired protein. For plantation.
multisubunit membrane proteins, the different mRNAs of the To study the expression of heterologous proteins, an oocyte
individual subunits can be co-injected. is typically injected under a dissecting microscope using a fine,
hollow needle (Figure 8A-1, ● 1 ). Once the tip of the needle is
inside the cell, the mRNA is injected in a very small volume of
Key Tools: Xenopus laevis oocytes; a micropipette; mRNA buffer, typically about 50 nL.
for the desired membrane protein; a fine, hollow needle to inject The mRNA (or mRNAs) is then translated by the endogenous
the mRNA into the oocyte, a voltage recorder and computer for Xenopus ribosomes to synthesize an intact, often functional pro-
ion channel proteins. tein (●2 ). In the case of membrane proteins, the protein is often

the relative concentrations of the ions on opposite sides of outward movement of each negatively charged bicarbonate
the membrane. In cells with a high bicarbonate concentra- ion is accompanied by the uptake of one negatively charged
tion, bicarbonate leaves the cell and chloride enters. In cells chloride ion.
with a low bicarbonate concentration, the reciprocal process In the lungs, this entire process is reversed: chloride ions
occurs—bicarbonate enters the cell and chloride leaves. are transported out of erythrocytes accompanied by the up-
The anion exchange protein plays a central role in the take of bicarbonate ions, which are then converted back to
process by which waste CO2 produced in metabolically active CO2 by carbonic anhydrase. The net result is the movement
tissues is delivered to the lungs to be expelled. In these tissues, of CO2 (in the form of bicarbonate ions) from tissues to the
CO2 diffuses into erythrocytes, where the cytosolic enzyme car- lungs, where the CO2 is exhaled from the body. In addition, the
bonic anhydrase converts it to bicarbonate ions (see Figure 8-3). import of bicarbonate into erythrocytes in the lungs increases
As the concentration of bicarbonate in the erythrocyte rises, it the cellular pH, which enhances oxygen binding to hemoglo-
moves out of the cell. To prevent a net charge imbalance, the bin in the lungs. When the erythrocytes reach the tissues and
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 Needle Oocyte Microelectrodes Voltage recorder

3 If the protein is an ion channel,

the oocyte’s membrane voltage
properties can be recorded to
1 An mRNA encoding a 2 Wait for the protein to be study the activity of the channel.
membrane protein or a inserted into the membrane,
synthesized protein mixed typically within 4 to 24 hours.
with liposomes is injected INSIDE OF CELL Plasma membrane
into an oocyte.

Ion channels

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

OUTSIDE OF CELL

Figure 8A-1 Microinjection of an Oocyte to Produce a Membrane Protein. In this example, an ion
channel is produced and inserted into the oocyte membrane, whose electrical properties can then be measured.

inserted into the membrane in the proper orientation as dictated some interference with the production or function of the desired
by the protein’s structure. If the protein inserted is an ion chan- protein. In addition, Xenopus oocytes may lack specific signaling
nel, the voltage difference across the oocyte membrane can be molecules that interact with the membrane protein in its native
measured to study the properties of the ion channel (● 3 ). cell type that are necessary for its proper function. Despite a
Although injecting mRNA into oocytes is common, in other few inevitable shortcomings, Xenopus oocytes have proven to
cases, rather than allowing the oocyte to synthesize the cor- be an extremely valuable “living test tube” for the expression
responding protein, it is easier to synthesize a membrane and functional study of integral membrane proteins.
protein directly in the laboratory. The protein is then mixed with
liposomes, and the protein-carrying liposomes are then injected QUESTION: A researcher is studying a fern that is
into the oocyte. shown to have a membrane protein that transports the
Although they are convenient, there are several limitations arsenate ion (HAsO42− ), a common pollutant in ground
of Xenopus oocytes for expression of heterologous proteins.
water, across membranes and may be useful to clean polluted
They are best kept and used at 20°C, which may not be the
optimum temperature for mammalian proteins, which typically sites. However, after having injected the correct mRNA for the
function optimally at 37°C. In addition, endogenous proteins and protein, the researcher failed to detect arsenate transport in
channels normally expressed by Xenopus oocytes may cause the oocytes. What are the possible reasons for this?

bicarbonate is exported, the cellular pH drops and oxygen and aquaporins. Because these transport proteins are integral
binding decreases, allowing more rapid release to the tissues. membrane proteins, they are often difficult to purify and ana-
lyze. Key Technique, page 220, shows how the function of
an integral membrane protein can be studied in vivo by ex-
Channel Proteins Facilitate Diffusion by
pressing the protein in frog egg cells, using aquaporin as an
Forming Hydrophilic Transmembrane Channels example.
Whereas some transport proteins facilitate diffusion by func-
tioning as carrier proteins that alternate between different Ion Channels: Transmembrane Proteins That Allow
conformational states, others do so by forming hydrophilic Rapid Passage of Specific Ions. Despite their apparently
transmembrane channels that allow specific solutes to move simple design—a tiny pore lined with hydrophilic atoms—
across the membrane directly. We will consider three kinds ion channels are remarkably selective. Most allow passage
of transmembrane protein channels: ion channels, porins, of only one kind of ion, so separate channels are needed for
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 Human Connections
MEMBRANE TRANSPORT, CYSTIC FIBROSIS, AND THE PROSPECTS FOR GENE THERAPY

A young mother sits in the waiting room of her doctor’s office with ions across the membrane. Ultimately, this leads to less water
her young daughter. Her daughter often has difficulty breathing and flow as well, so CF patients have unusually thick mucus in their
hasn’t been gaining weight like most other children her age. Moreo- lungs. Bacteria are able to colonize and grow within the mucus
ver, when the mother kisses the girl’s forehead, she is left with a layer, leading to chronic bacterial infections (Figure 8B-1b).
salty taste on her lips. As she will soon discover, the young girl has In addition, gas exchange in the lungs is impaired by the thick
cystic fibrosis (CF). One hundred years ago the girl’s outlook would mucus, so patients are unable to achieve normal levels of physi-
have been bleak; she would have suffered from chronic weakness, cal activity. In the pancreas, a different secretory problem results:
difficulty breathing, and malnourishment and likely would have died digestive enzymes that should reach the small intestine fail to do
by the age of 10. Today, with modern therapies and treatments, so. This accounts for the inability of CF patients to gain weight.
patients with CF have an average life span of about 40 years. Treatments for CF are varied. Patients take oral pancreatic
CF is an incurable disease that affects roughly one out of every enzymes to help digest and absorb more of the necessary build-
9000 individuals in the United States. It primarily affects the secre- ing blocks for biological macromolecules that allow normal weight
tory cells in epithelial tissues of the lungs, pancreas, and skin gain. Visits to a respiratory therapist involve lowering the head
(although the disease manifests itself in many different tissues of and physical pounding on the back to loosen mucus so it may be
the body). The disease is named for its typical pancreatic cysts and expelled (Figure 8B-2a). More recently, a mechanical vest has
fibrosis, or hardening, of lung tissue in affected individuals. been developed that is worn for a half hour twice a day and pro-
Common to all of the affected tissues is a transmembrane pro- vides high-frequency chest wall oscillation to loosen the mucus
tein integral to the transport of chloride ions across the membrane: (Figure 8B-2b). Unfortunately, over many years, lung tissue
the cystic fibrosis transmembrane conductance regulator (CFTR), typically becomes damaged and scarred, further reducing efficient
a cAMP-regulated ion channel. In normal individuals, the channel gas exchange. If this occurs, lung transplant is often a last resort.
pumps chloride ions into the lumen of lung airways and other tissues These treatments address the most severe symptoms of CF.
(Figure 8B-1a). Sodium ions then flow passively across the mem- But is a cure for the disease possible? Current efforts toward
brane into the lumen, and water follows via osmosis. This results in curing CF involve directed gene therapies. From a layperson’s per-
relatively thin mucus that keeps the tissue moist and can be expelled spective, getting a functional copy of the CFTR gene into affected
from the lungs and bronchial passages through the beating of cilia, tissues might seem the “easiest” route to a cure. For example,
microtubule-based structures that generate gentle fluid currents. there are many influenza viruses that preferentially infect lung
Patients with CF, however, have mutations in the CFTR gene cells; in fact, many people receive yearly vaccinations against
that render the ion channel encoded by this gene nonfunctional. these viruses via inhalers. What if one of these viruses could be
To date, more than 1000 different mutations have been identified, engineered to carry a functional CFTR gene? In theory, after infec-
all with a similar effect: CF patients are unable to move chloride tion, the viral genes would integrate into the host cell. Through

Transmembrane domains OUTSIDE

CFTR
protein Cl- OF CELL

Cl-

H2O Airway
lumen
Plasma
Na+ membrane
Cells lining
airways Mucus
N
(a) Normal cells lining airways; hydrated mucus

Cl-
Regulatory
Airway domain
Cl-
lumen
ATP
H2O molecules
Nucleotide-binding
domain
Na+ Nucleotide-
Cells lining binding domain INSIDE
airways Mucus C OF CELL

(b) Cells with cystic fibrosis infected with bacteria (c) Proposed CFTR structure

Figure 8B-1 Cystic Fibrosis and Chloride Ion Secretion. (a) Normal lungs have hydrated mucus lining
the airways. (b) Cystic fibrosis is caused by a defect in the secretion of chloride ions in cells lining the lungs,
leading to insufficient hydration, thick mucus, and the promotion of bacterial growth. (c) Proposed structure and
orientation of CFTR in the lung cell membrane.
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 transporting such ions as Na+, K+, Ca2+, and Cl- . This se-
lectivity is remarkable given the small differences in size and
charge among some of these ions. Selectivity results both from
ion-specific binding sites involving specific amino acid side
chains and polypeptide backbone atoms inside the channel
and from a constricted center of the channel that serves as a
size filter. The rate of transport is equally remarkable: in some
cases, a single channel can conduct almost a million ions of a
specific type per second!
Most ion channels are gated, which means that the pore
opens and closes in response to some stimulus. In animal cells,
three kinds of stimuli control the opening and closing of gated
channels: voltage-gated channels open and close in response to
changes in the membrane potential; ligand-gated channels are
triggered by the binding of specific substances to the channel
protein; and mechanosensitive channels respond to mechanical
forces that act on the membrane. (You will learn about gated
channels in detail in Chapter 22.)
How do researchers study the function of ion channels
(a) Percussion therapy for CF
such as these in living cells? A technique known as patch clamp-
ing allows researchers to monitor the ion flow through individ-
ual ion channels in a small patch of membrane on the surface
of a cell such as a neuron (see Figure 22A-1, page 690). This

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

technique is sensitive enough that it can record the opening
and closing of a single ion channel and thus has given us many
insights into what external influences and chemical regulators
affect channel activity. It has also allowed structural studies of
the channels themselves to determine which parts of the channel
protein are essential for proper functioning in living cells.
Regulation of ion movement across membranes plays
an important role in many types of cellular processes. Muscle
contraction and many cellular responses require regulation of
Ca2+ levels via calcium-specific ion channels. Also, the trans-
mission of electrical signals by nerve cells depends critically
on rapid, controlled changes in the movement of Na+ and
K+ through their respective channels. These changes are so
rapid that they are measured in milliseconds. In addition to
such short-term regulation, most ion channels are subject to
longer-term regulation, usually in response to external stimuli
(b) Vest for chest wall oscillation
such as hormones.
Figure 8B-2 Respiratory Physiotherapy. (a) Standard percus- Ion channels are also necessary for maintaining the
sion therapy for treatment of cystic fibrosis. (b) Therapy using a proper salt balance in the cells and airways lining our lungs.
vibrating vest that provides mechanical oscillation of the chest wall. In lung epithelial cells, a specific chloride ion channel known
as the cystic fibrosis transmembrane conductance regulator
normal cellular processes, the functional protein would then be
expressed and integrated into the lung epithelium. In fact, cur-
(CFTR) protein helps maintain the proper Cl- concentration
rent avenues for gene therapy do include replacing some of the in these airways. We have long known that people with the
genome of infectious adenoviruses with the CFTR gene and then life-threatening disease known as cystic fibrosis accumulate
administering the virus through an inhaler. Advantages of this type unusually thick mucus in their lungs—a condition that often
of gene therapy are that adenoviruses are tissue-specific, are able leads to pneumonia and other lung disorders. Now we under-
to incorporate large amounts of foreign DNA and their epidemiol- stand that the underlying problem is an inability to secrete
ogy does not cause serious symptoms in the host. chloride ions and that the genetic defect is in a protein that
Unfortunately, the very hallmarks of the disease are where functions as a chloride ion channel (a topic we discuss in detail
such theory and practice collide. The mucous layer in many CF in Human Connections, page 222).
patients is so thick that the engineered virus may never reach
An especially important protein involved in ion trans-
the lung cells, so the functional CFTR would never be expressed
port down a concentration gradient is the ATP synthase en-
by the cells that need it. Another potential risk of adenoviral
gene therapy is that the genome may be integrated randomly
zyme (which we will encounter in more detail in Chapters 10
into the genome of the host cell and may disrupt the function of and 11; see Figure 10-23). ATP synthase acts as a proton
other genes needed in these cells for their normal activities. In channel that allows protons to travel down their concentra-
spite of these serious challenges, research and clinical trials with tion gradient by facilitated diffusion. This protein is found
the goal of better gene delivery systems continue. in mitochondria and chloroplasts, which use energy either
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 Figure 8-9 The Porin Channel Protein. (a)
N Side view and (b) end view of an E. coli porin
C protein showing the 14-stranded transmem-
(a) Porin side view (b) Porin end view brane b barrel and the N- and C-termini.

from the oxidation of sugars (mitochondria) or from the sun It was not until 1992 that Peter Agre and colleagues at
(chloroplasts) to create a transmembrane proton gradient. Johns Hopkins University finally isolated the long-sought-after
The exergonic flow of protons down their concentration water channel protein, which they named aquaporin (AQP).
gradient through the ATP synthase allows the endergonic They found that this particular erythrocyte protein, when in-
conversion of ADP and inorganic phosphate into the high- troduced into membranes of relatively water-impermeable
energy ATP molecule. frog egg cells, would cause the cells to explode when placed in
pure water due to the rapid influx of water. Control eggs with-
Porins: Transmembrane Proteins That Allow Rapid out the protein placed in the pure water were unchanged. In
Passage of Various Solutes. Compared with ion chan- 2003, Agre shared a Nobel Prize in chemistry with Roderick
nels, the pores found in the outer membranes of mitochon- MacKinnon, who determined the first three-dimensional
dria, chloroplasts, and many bacteria are somewhat larger structure of an ion channel.
and much less specific. These pores are formed by ­multipass Aquaporins facilitate the rapid movement of water mol-
transmembrane proteins called porins. However, the ecules into or out of cells in specific tissues that require this
transmembrane segments of porin molecules cross the mem- capability. For example, your kidneys reabsorb water as part of
brane not as a helices but as a closed cylindrical b sheet called urine formation, and specialized cells in this organ have a high
a b barrel (Figure 8-9). The b barrel has a water-filled pore density of AQPs in their plasma membrane, allowing the kid-
at its center. Polar side chains (not shown) line the inside of ney to filter more than 100 L of blood plasma every day. Aqua-
the pore, whereas the outside of the barrel consists mainly of porins are also abundant in erythrocytes, which must be able
nonpolar side chains that interact with the hydrophobic inte- to expand or shrink rapidly in response to sudden changes in
rior of the membrane. The pore allows passage of various hy- osmotic pressure as they move through the kidney or other
drophilic solutes. The upper size limit for the solute molecules arterial passages. Erythrocytes have approximately 200,000
is determined by the pore size of the particular porin—only aquaporin molecules per cell.
solutes smaller than about 600 Da can pass through the Esch- In plants, AQPs are a prominent feature of root cell
erichia coli porin shown in the figure. Mutations in certain bac- plasma membranes and the vacuolar membrane, reflecting
terial porins can lead to antibiotic resistance in these bacteria the rapid movement of water that is required to develop tur-
by effectively blocking entry of the antibiotics that would nor- gor pressure, as discussed previously. Bacteria have recently
mally be used to fight an infection. been shown to contain AQPs. One type of bacterial AQP has
a slightly larger channel and can transport glycerol as well as
Aquaporins: Transmembrane Channels That Allow water and is thus termed an aquaglyceroporin.
Rapid Passage of Water. Whereas water can diffuse slowly Aquaporins are tetrameric integral membrane proteins
across cell membranes in the absence of a protein transporter, that have four identical monomers, with each monomer
movement of water across membranes in some tissues is much containing six helical transmembrane segments. The four
more rapid than can be accounted for by diffusion alone. In monomers associate side by side in the membrane to form
fact, the existence of water channels in cell membranes was four identical water channels lined with amino acid residues
postulated as early as the mid- to late 1800s. Despite a century having hydrophilic side chains (Figure 8-10a). The space in
of experiments suggesting their existence, water channels the center of the tetramer is blocked by a lipid molecule. The
remained elusive, and their very existence was sometimes in diameter of each of the four water channels narrows to about
doubt. 0.3 nm (3 Å) (Figure 8-10b), which is just large enough for
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 Lipid molecule Water channels Extracellular

Water
Water
channel

0.3 nm
Figure 8-10 The Aquaporin Channel Protein.
(a) End view of the tetrameric human aquaporin
Molecular
Model: channel, showing the six a helices in each of the
Aquaporin-1 four identical monomers. Two of the four water
channels in the tetramer are labeled. (b) Space-
filling model of the side view of a single aquaporin
channel, showing the entry and exit sites for water Cytoplasmic
molecules (red). (a) Aquaporin tetramer (end view) (b) Aquaporin monomer (side view)

water molecules to pass through one at a time in single file. the concentration inside. Third, it enables the cell to maintain
Even with this constraint, water molecules flow through an constant, nonequilibrium intracellular concentrations of spe-
aquaporin channel at the rate of several billion per second. cific inorganic ions, notably K+, Na+, Ca2+, and H+.
The specific amino acid residues lining the water channel allow This ability to create an internal cellular environment
only H2O molecules through and discriminate against the sim- whose solute concentrations are far removed from equilib-
ilar-sized OH- and H3O + ions. In addition, the narrowest re- rium is a crucial feature of active transport. In contrast to

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

gion of the water channel contains positively charged arginine simple and facilitated diffusion, which create conditions that
residues, which repel and block the passage of any H + ions. are the same on opposite sides of a membrane, active trans-
port is a means of establishing differences in solute concen-
CONCEPT CHECK 8.3 tration and/or electrical potential across membranes. The end
How are carrier proteins and channel proteins similar? How result is a nonequilibrium steady state, without which life as
do they differ? Consider both structure and mechanism. we know it would be impossible. Many membrane proteins in-
volved in active transport are called pumps to emphasize that
an input of energy is required to move materials against their
8.4 Active Transport: Protein- concentration or electrochemical gradients.
Mediated Movement Up the Gradient An important distinction between active transport and
simple or facilitated diffusion concerns the direction of trans-
Facilitated diffusion is an important mechanism for speeding port. Simple and facilitated diffusion are both nondirectional
up the movement of substances across cellular membranes. with respect to the membrane. For both types of diffusion,
But it accounts for the transport of molecules only toward solutes can move in either direction, depending entirely on
equilibrium, which means down a concentration or electro- the prevailing concentration or electrochemical gradient.
chemical gradient. What happens when a substance needs to Active transport, on the other hand, usually has intrinsic
be transported against a gradient, as when a nutrient or other directionality. An active transport system that moves a sol-
substance is accumulated in a cell or organelle? Such situa- ute across a membrane in one direction will not usually move
tions require active transport, a process that differs from fa- that solute in the other direction and is typically a unidirec-
cilitated diffusion in one crucial aspect: active transport makes tional process. However, in some cases, we will see that a trans-
it possible to move solutes away from thermodynamic equilib- porter can work in either direction depending on the cellular
rium (that is, up a concentration gradient or against an electro- environment.
chemical potential). Therefore, it always requires an input of
energy. In other words, active transport couples a thermody-
namically unfavorable process (movement up a concentration The Coupling of Active Transport to an Energy
gradient) to an exergonic process (usually ATP hydrolysis). As Source May Be Direct or Indirect
a result, membrane proteins involved in active transport must Active transport mechanisms can be divided into two related
provide mechanisms not only for moving desired solute mol- categories that differ primarily in the source of energy and
ecules across the membrane but also for coupling such move- whether or not two solutes are transported simultaneously.
ments to energy-yielding reactions. Depending on the energy source, active transport is regarded
Active transport performs three major functions in cells as being either direct or indirect (Figure 8-11). In direct
and organelles. First, it makes possible the uptake of essen- ­active transport (also called primary active transport), the
tial nutrients from the environment or surrounding fluid, accumulation of solute molecules or ions on one side of the
even when their concentrations in the cell are already higher. membrane is coupled directly to an exergonic chemical reac-
Second, it allows various substances, such as secretory prod- tion, most commonly the hydrolysis of ATP (Figure 8-11a).
ucts and waste materials, to be removed from the cell or or- Transport proteins driven directly by ATP hydrolysis are called
ganelle, even when the concentration outside is greater than transport ATPases or ATPase pumps.
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 (a) Direct active transport (b) Indirect active transport to transport solutes against a concentration gradient or an
involves a transport system involves the coupled electrochemical potential.
coupled to an exergonic transport of a solute S and
chemical reaction, most ions—protons, in this case.
P-Type ATPases. P-type ATPases (P for “phosphorylation”)
commonly the hydrolysis of The exergonic inward
ATP. As shown here, ATP movement of protons are members of a large family of proteins that are reversibly
hydrolysis drives the outward provides the energy to move phosphorylated by ATP as part of the transport mechanism,
transport of protons, thereby the transported solute, S, with a specific aspartic acid residue becoming phosphorylated
establishing an electro- against its concentration
in each case. P-type ATPases have eight to ten transmembrane
chemical potential for protons gradient or electrochemical
across the membrane. potential. segments in a single polypeptide that crosses the membrane
multiple times (see Figure 7-5b). Also, they are sensitive to in-
H+ H+ + H+
hibition by the vanadate ion, VO3- 4 , which closely resembles
H+ H+ H+
H+ H
H+
the phosphate ion (PO3- 4 ) and thus interferes with phosphory-
OUTSIDE H+
H+ H+ H+ H+ lation. Vanadate sensitivity can therefore be used by research-
OF CELL H+ S
ers as a means of identifying P-type ATPases.
Most P-type ATPases are located in the plasma mem-
brane and, based on sequence and structural similarities, fall
into one of five subfamilies. P1-ATPases have been found in all
organisms and are mainly responsible for transporting ions
of heavy metals. Several kinds of P2-ATPases are responsible
INSIDE
OF CELL H+
for maintaining gradients of ions such as Na+, K+, H+, and
(a) H+ (b)
S S Ca2+ across the plasma membrane of many eukaryotic cells.
ATP S S The best-known example is the Na+/K+ pump found in almost
S
ADP + Pi S all animal cells. We will consider this ATPase in more detail
shortly. Another example is the Ca2+/H+ ATPase involved in
muscle contraction (see Problem 8-10 and Chapter 14). A
Figure 8-11 Comparison of Direct and Indirect Active third P2-ATPase is the H+/K+ ATPase responsible for acidifi-
Transport. Note the circulation of protons across the membrane cation of the gastric juice in your stomach. This protein is the
that results from the coupling of direct and indirect mechanisms of
target of medications known as proton pump inhibitors, which
active transport. Note also that transporters are not simply the open
are used to combat excess stomach acid. Similar to these are
channels that are shown in the simplified illustration.
the P3-H+ ATPases of plants and fungi, which pump protons
outward across the plasma membrane of these cells, acidify-
ing the external medium.
Indirect active transport also requires energy but de- The P4-ATPases differ from the preceding types in that
pends on the simultaneous transport of two solutes, with the they do not pump ions but instead pump relatively hydrophobic
favorable movement of one solute down its gradient driving molecules such as cholesterol and fatty acids. Also, they do not
the unfavorable movement of the other solute up its gradient. transport materials completely across membranes but trans-
The coupling of a favorable process or reaction with an unfa- port lipids from one leaflet of the membrane bilayer to the other,
vorable one allows both to proceed with an overall decrease in acting as flippases that help to maintain membrane asymmetry.
free energy. This dual transport process can be described as ei- P5-ATPases are less well characterized, but several are known to
ther a symport or an antiport, depending on whether the two transport cations. They share sequence homology but not sol-
solutes move in the same or opposite directions. In most cases, ute specificity. Some are found in the ER, where they appear to
one of the two solutes is an ion that moves exergonically down function in protein processing, and others are associated with
its electrochemical gradient—Na+ in animals and H+ in most the vacuole (yeast) or lysosome (animals) and have been impli-
other organisms. As it moves, it drives the simultaneous end- cated in hereditary neuronal diseases in humans.
ergonic transport of the second solute (often a monosaccha-
ride or an amino acid) against its concentration gradient or, in V-Type ATPases. V-type ATPases (V for “vacuole”) pump
the case of ions, its electrochemical potential (Figure 8-11b). protons into such organelles as vacuoles, vesicles, lysosomes,
Thus indirect active transport is often called secondary active endosomes, and the Golgi apparatus. Typically, the proton
transport. gradient across the membranes of these organelles ranges
from tenfold to more than 10,000-fold. V-type pumps are
not inhibited by vanadate because they do not undergo phos-
Direct Active Transport Depends on Four
phorylation as part of the transport process. They have two
Types of Transport ATPases multisubunit components: an integral component embedded
The most common mechanism employed for direct active within the membrane and a peripheral component that juts
transport involves transport ATPases that link active trans- out from the membrane surface. The peripheral component
port to the hydrolysis of ATP. Four main types of transport contains the ATP-binding site and hence the ATPase activity.
ATPases have been identified: P-type, V-type, F-type, and ABC-
type ATPases (Table 8-2). These four types of transport pro- F-Type ATPases. F-type ATPases (F for “factor”) are found
teins differ in structure, mechanism, localization, and physi- in bacteria, mitochondria, and chloroplasts. F-type ATPases
ological roles, but all of them use the energy of ATP hydrolysis are involved in proton transport and have two components,
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 Table 8-2 Main Types of Transport ATPases (Pumps)

Solutes Transported Kind of Membrane Kind of Organisms Example of ATPase function

P-type ATPases (P for “phosphorylation”)
P1
K+, Cu+, Zn2+, Cd2+, Pb2+ Plasma membrane Bacteria, archaea, plants, fungi, Transport of potassium or heavy metal
animals ions
P2
Ca2+ / H+ SR* or plasma membrane Eukaryotes Keeps 3Ca2+4 low in cytosol
Na+/ K+ Plasma membrane Animals Maintains membrane potential
( - 60 mV)
H+/ K+ Plasma membrane Animals Pumps H+ to acidify stomach
P3
H+ Plasma membrane Plants, fungi Pumps protons out of cell to generate
membrane potential ( - 180 mV)
P4
Phospholipids Plasma membrane Eukaryotes Flippases that maintain asymmetry in
the lipid bilayer
P5
Various cations ER, vacuole, lysosome Eukaryotes Not well characterized

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

V-type ATPases (V for “vacuole”)
H+ Lysosomes, secretory Animals Keeps pH of compartment low, which
vesicles activates hydrolytic enzymes
Vacuolar membrane Plants, fungi
F-type ATPases (F for “factor”); also called ATP synthases
H+ Inner mitochondrial Eukaryotes Uses H+ gradient to drive ATP
membrane synthesis
Plasma membrane Bacteria
Thylakoid membrane Plants
ABC-type ATPases (ABC for “ATP-binding cassette”)
Importers Plasma membrane, Bacteria Nutrients such as vitamin B12
organellar membranes
A variety of solutes**
Exporters Plasma membrane Bacteria, archaea, eukaryotes Multidrug resistance transporter
removes drugs and antibiotics from cell
Antitumor drugs, toxins,
antibiotics, lipids

*Sarcoplasmic reticulum, a specialized type of ER found in animal muscle cells

**Solutes include ions, sugars, amino acids, carbohydrates, vitamins, peptides, and proteins.

both of them multisubunit complexes. The integral membrane F-type ATPases illustrate an important principle: Not
component, called Fo, is a transmembrane pore for protons. only can ATP be used as an energy source to generate and main-
The peripheral membrane component, called F1, includes tain ion gradients, but such gradients can be used as an energy
the ATP-binding site. F-type ATPases can use the energy of source to synthesize ATP. This principle, which was discovered
ATP hydrolysis to pump protons against their electrochemical in studies of F-type pumps, is the basis of ATP-synthesizing
­potential. mechanisms in all eukaryotic organisms and in most bacte-
These transport proteins can also facilitate the reverse ria as well.
process to synthesize ATP (as we will see when we study aer-
obic respiration and photosynthesis in Chapters 10 and 11, ABC-Type ATPases. The fourth major class of ATP-
­respectively). In the reverse direction, the exergonic flow of driven pumps is the ABC-type ATPases, also called ABC
protons down their gradient is used to drive ATP synthesis. ­transporters. The ABC designation is for “ATP-binding cas-
When they function in this latter mode, these F-type ATPases sette,” where the term cassette describes the catalytic domain of
are more appropriately called ATP synthases. As ATP the protein that binds ATP as an integral part of the transport
­synthases, these proteins function to use either the energy process. The more than 150 known ABC-type ATPases com-
from sugar oxidation (respiration) or the energy of solar ra- prise a very large family of transport proteins that are found in
diation (photosynthesis) to produce a transmembrane proton all organisms. They are related to each other in sequence and
gradient that drives ATP synthesis. probably also in molecular mechanism. Most of the ABC-type
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 ATPases discovered initially were from bacterial species and Indirect Active Transport Is Driven
were importers that are involved in uptake of nutrients. But by Ion Gradients
increasing numbers of ABC-type ATPases known as exporters,
In contrast to direct active transport, which is powered by
some of great clinical importance, are being reported in eukary-
energy released from a chemical reaction such as ATP hy-
otes as well. As we will soon see, some have been found in hu-
drolysis, indirect active transport (also called secondary ac-
man tumor cells, where, unfortunately, they can export antitu-
tive transport) is driven by the movement of an ion down its
mor medications. At last count, 48 different genes for ABC-type
electrochemical gradient. This principle has emerged from
ATPases have been identified in the human genome.
studies of the active uptake of sugars, amino acids, and other
The typical ABC-type ATPase has four protein domains.
organic molecules into cells: the inward transport of mole-
Two of these domains are highly hydrophobic and are embed-
cules up their electrochemical gradients is often coupled to,
ded in the membrane; the other two are peripheral and are
and driven by, the simultaneous inward movement of either
associated with the cytoplasmic side of the membrane. Each
sodium ions (for animal cells) or protons (for most plant, fun-
of the two embedded domains consists of six membrane-span-
gal, and bacterial cells) down their respective electrochemical
ning segments that form the channel through which solute
gradients.
molecules pass. The two peripheral domains are the cassettes
The widespread existence of such symport mechanisms
that bind ATP and couple its hydrolysis to the transport pro-
explains why most cells continuously pump either sodium
cess. These four domains are separate polypeptides in most
ions or protons out of the cell. In animals, for example, the
cases, especially in bacterial cells. However, examples are also
relatively high extracellular concentration of sodium ions
known in which the four domains are part of a large, multi-
maintained by the Na+/K+ pump serves as the driving force
functional polypeptide.
for the uptake of a variety of sugars and amino acids. For
Although the other three classes of ATPases transport
example, to obtain the data shown in Figure 8-12, investi-
only cations, the different ABC-type ATPases handle a remark-
gators varied the extracellular concentration of sodium ions
able variety of solutes. Most ABC transporters are specific for
and measured the transport rate of the amino acid glycine
a particular solute or class of closely related solutes. But the
into erythrocytes or the sugar 7-deoxy-d-glucoheptose into
variety of solutes transported by the many members of this
cells lining the intestine. There is a clear correlation between
superfamily is great, including ions, sugars, amino acids, and
increasing Na+ concentration and the rate of transport of
even peptides and polysaccharides.
the relevant molecule in each tissue. Such experiments
ABC transporters are of considerable medical interest
showed that transport of amino acids and sugars into cells
because some of them pump antibiotics or other drugs out
can be stimulated by a high concentration of sodium ions in
of the cell, thereby making the cell resistant to the drug. For
the extracellular medium.
example, some human tumors are remarkably resistant to a
The uptake of sugars and amino acids in this manner is
variety of drugs that are normally quite effective at arrest-
regarded as indirect active transport because it is not directly
ing tumor growth. Cells of such tumors have unusually high
driven by the hydrolysis of ATP or a related “high-energy”
concentrations of a large protein called the multidrug re-
compound. Ultimately, however, uptake still depends on ATP
sistance (MDR) transport protein, which was in fact the
because the Na+/K+ pump that maintains the sodium ion
first ABC-type ATPase to be identified in humans. The MDR
gradient is itself driven by ATP hydrolysis. The continuous
transport protein uses the energy of ATP hydrolysis to pump
hydrophobic drugs out of cells, thereby reducing the cyto-
plasmic concentration of the drugs and hence their effective-
ness as therapeutic agents. Unlike most ABC transporters, the
MDR protein has a remarkably broad specificity: It can export
a wide range of chemically dissimilar drugs commonly used
in cancer chemotherapy, so that a cell with the MDR protein
in its plasma membrane becomes resistant to a wide variety
Relative transport rate

of therapeutic agents. Similarly, MDR proteins of some bacte- Amino acid

ria can transport a variety of antibiotics out of the cell, giving
these cells resistance to multiple antibiotics.
Medical interest in this class of transport proteins was Sugar
heightened when cystic fibrosis was shown to be caused by a ge-
netic defect in a plasma membrane protein that is structurally
related to the ABC transporters, the cystic fibrosis transmem-
brane conductance regulator (CFTR) that we encountered
earlier in this chapter. CFTR is similar in sequence and likely in
topology to the core domains of ABC transporters. However,
CFTR is an ion channel, and, unlike most of the ABC-type 0 50 100 150
ATPases, it does not use ATP to drive transport. Instead, ATP Na+ concentration (mM)
hydrolysis appears to be involved in opening the channel. (Re- Figure 8-12 Effect of External Sodium Ion Concentration on
cent developments in our understanding of cystic fibrosis at Amino Acid and Sugar Transport. As the extracellular Na+ con-
the molecular level and attempts at gene therapy to treat this centration is increased, the rate of transport of certain nutrients into
disease are reported in Human Connections, page 222.) the cell increases proportionately due to indirect active transport.
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 outward pumping of Na+ by the ATP-driven Na+/K+ pump The protein responsible for this process was discovered in
and inward movement of Na+ by symport (coupled to the 1957 by Jens Skou, a Danish physiologist, who was awarded
uptake of another solute) establishes a circulation of sodium a Nobel Prize in chemistry in 1997. The discovery of this ion-
ions across the plasma membrane of every animal cell, similar transporting enzyme was in fact the first documented case of
to the cycling of H+ ions shown in Figure 8-11. active transport. This Na+ /K + ATPase, or Na+ /K + pump,
Whereas animal cells use sodium ions to drive indirect ac- as this P-type ATPase is usually called, uses the exergonic hy-
tive transport, most other organisms rely on a proton gradient drolysis of ATP to drive the endergonic inward transport of
instead. For example, fungi and plants utilize proton symport potassium ions and the outward transport of sodium ions
for the uptake of organic solutes, with an ATP-driven proton against their concentration gradients. The Na+/K+ pump is
pump responsible for the generation and maintenance of the primarily responsible for the asymmetric distribution of these
proton electrochemical potential. Many kinds of bacterial cells ions across the plasma membrane in animal cells. Like most
also make extensive use of proton cotransport to drive the up- other active transport systems, this pump has inherent direc-
take of solute molecules, as do mitochondria. In all such cases, tionality: potassium ions are always pumped inward and so-
however, the proton gradient is maintained by an electron dium ions are always pumped outward. In fact, sodium and
transfer process that accompanies cellular respiration (as we potassium ions activate the ATPase only on the side of the
will see in Chapter 10). membrane they are transported from—sodium ions from the
In addition to the symport uptake of organic molecules inside, potassium ions from the outside. Three sodium ions are
such as sugars and amino acids, sodium ions or proton gra- moved out and two potassium ions are moved in per molecule
dients can be used to drive the export of other ions, including of ATP hydrolyzed.
Ca2+ and K+. This type of indirect active transport is usually Figure 8-13 is a schematic illustration of the Na+/K+
antiport and may involve the exchange of potassium ions for pump. The pump is a trimeric transmembrane protein with
protons or the exchange of calcium ions for sodium ions, for one a, one b, and one g subunit. The a subunit, which forms
example. the ion channel itself, contains binding sites for ATP and so-

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

dium ions on the inner side of the membrane and binding sites
CONCEPT CHECK 8.4 for potassium ions on the external side. The functions of the b
How would you determine whether a specific integral subunit, which is glycosylated, and the g subunit are not yet
membrane transporter is operating by facilitated diffusion or clear.
active transport? The Na+/K+ pump is an allosteric protein exhibiting
two alternative conformational states, referred to as E1 and
E2. The E1 conformation is open to the inside of the cell and
8.5 Examples of Active Transport
Having considered some general features of active transport,
we are now ready to look at three specific examples: one ex-
ample each of direct and indirect active transport from animal
cells, and an unusual type of light-driven transport in a bacte-
rium. In each case we will note what kinds of solutes are trans-
ported, what the driving force is, and how the energy source is K+ K+
coupled to the transport mechanism. We will focus first on the Oligosaccharides
OUTSIDE
Na+/K+ ATPase (or pump) present in all animal cells because OF CELL
it is a well-understood example of direct active transport by a
P-type ATPase. Then we will consider a second example from
animal cells: the indirect active transport of glucose by a Na+/ g a a b
glucose symporter, using the energy of the sodium ion gradient
established by the Na+/K+ ATPase. Finally, we will look briefly
at light-driven proton transport in certain bacteria.

Direct Active Transport: The Na+/K+ Pump

INSIDE
Maintains Electrochemical Ion Gradients OF CELL
Binding site
Na+ Na+ for ATP
A characteristic feature of most animal cells is a high intracellu- Na+
lar level of potassium ions and a low intracellular level of sodium
ions. In a mammalian neuron, the 3K+ 4 inside/ 3K+ 4 outside
ratio is about 30:1, and the 3Na+ 4 inside/ 3Na+ 4 outside ratio
is about 0.08:1. The resulting electrochemical potentials for
Figure 8-13 The Na + /K + Pump. The trimeric Na+/K+ pump
potassium and sodium ions are essential as the driving force found in most animal cells contains a, b, and g transmembrane
for coupled transport as well as for the transmission of nerve subunits. The pump is shown in the E1 conformation, which is open
impulses. Both the inward pumping of potassium ions and to the inside of the cell. Binding of sodium ions causes a conforma-
the outward pumping of sodium ions are therefore energy- tional change to the E2 form, which opens to the outside to accept
requiring processes, as both ions are being moved up their potassium ions, which are transported inward. The transport mecha-
electrochemical gradients. nism in this pump is shown in detail in Figure 8-14.
229

M08_HARD7694_09_GE_C08.indd 229 13/02/17 4:29 pm

 has a high affinity for sodium ions, whereas E2 is open to the ions are moved to the inner surface, where they dissociate,
outside, with a high affinity for potassium ions. Phosphoryla- leaving the carrier ready to accept more sodium ions ● 6.
tion of the pump by ATP, a sodium-triggered event, stabilizes The Na+/K+ pump is not only one of the best-understood
it in the E2 conformation. Dephosphorylation, on the other transport systems but also one of the most important for ani-
hand, is triggered by K+ and stabilizes the enzyme in the E1 mal cells. Besides maintaining the appropriate intracellular
conformation. concentrations of both potassium and sodium ions, it is respon-
As illustrated schematically in Figure 8-14, the actual sible for maintaining the membrane potential that exists across
trans­port mechanism involves an initial binding of three sodi- the plasma membrane. The Na+/K+ pump assumes still more
um ions to E1 on the inner side of the membrane ● 1 , upper right. significance when we take into account the vital role that so-
The binding of sodium ions triggers autophosphorylation of dium ions play in the inward transport of organic substrates, a
the a subunit of the pump using ATP as the phosphate donor topic we now come to as we consider sodium ­symport.
●2 , resulting in a conformational change from E1 to E2. As a
result, the bound sodium ions are moved through the mem-
brane to the external surface, where they are released to the Indirect Active Transport: Sodium Symport
outside ● 3 . Then, potassium ions from the outside bind to the a Drives the Uptake of Glucose
subunit ● 4 , triggering dephosphorylation and a return to the As an example of indirect active transport, let us consider
original conformation ● 5 . During this process, the potassium the uptake of glucose by the Na+/glucose symporter.

Initial state:
pump open to inside
(E1 conformation) OUTSIDE
6 Two K+ expelled to OF CELL
the inside as ATP binds b 1 Three Na+ from
and the pump returns inside the cell bind to E1
to initial state g a a

ATP

3 Na+
2 K+
K+ Na+
K+ Na+
Na+
ATP
ATP

2 Na+ binding triggers

5 K+ binding triggers P
INSIDE
autophosphorylation
dephosphorylation, of the a subunit using ATP
OF CELL and ADP is released
causing a
conformational ADP
change back to E1

P Na+
P
Na+
Na+
K+
K+

4 Two K+ from 3 A conformational

outside the cell change to E2 expels
bind to E2 three Na+ to the outside
of the cell
2 K+ 3 Na+

Pump open to outside,

ready to start second
half of cycle
(E2 conformation)

Figure 8-14 Model Mechanism for the Na + /K + Pump. The transport process is shown here in six steps
arranged at the periphery of a cell. The outward transport of sodium ions is coupled to the inward transport of
potassium ions, both against their respective electrochemical potentials. The driving force is provided by ATP
hydrolysis, which is required for phosphorylation of the a subunit of the pump at step ● 2.
230

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 Although most glucose transport into or out of cells in your c­ onformational change in the protein that exposes the sodium
body occurs by facilitated diffusion, as shown in Figure 8-8, ions and the glucose molecule to the inner surface of the mem-
the epithelial cells lining your intestines have transport pro- brane ● 3 . There the two sodium ions dissociate in response to
teins that are able to take up glucose and certain amino acids the low intracellular sodium ion concentration ● 4 . This locks
from the intestines even when their concentrations there are the transporter in its inward-facing conformation until the
much lower than in the epithelial cells. This energy-requir- glucose molecule dissociates ● 5 . Then the empty transporter
ing process is driven by the simultaneous uptake of sodium is free to return to its outward-facing conformation ● 6.
ions, which is exergonic because of the steep electrochemi- The sodium ion gradient is in turn maintained by the con-
cal gradient for sodium that is maintained across the plasma tinuous outward extrusion of sodium ions (dashed arrow) by
membrane by the Na+/K+ pump (higher [Na+] outside the the Na+/K+ pump shown in Figure 8-14. As a result, sodium
cell). These sodium-dependent glucose transporters are often re- ions circulate across the plasma membrane, pumped outward
ferred to as SGLT proteins. by the Na+/K+ pump and flowing back into the cell as the driv-
Figure 8-15 depicts the transport mechanism for the ing force for sodium symport of molecules such as glucose. Sim-
Na+/glucose symporter, which requires the inward move- ilar mechanisms are involved in the uptake of amino acids and
ment of two sodium ions to drive the simultaneous uptake of other organic substrates by sodium symport in animal cells and
one glucose molecule. Transport is initiated by the binding of by proton symport in plant, fungal, and bacterial cells.
two external sodium ions to their binding sites on the sym- This detailed understanding of the Na+/glucose sym-
porter, which is open to the outer surface of the membrane port mechanism has helped in the treatment of the disease
●1 . This allows a molecule of glucose to bind ●
2 , followed by a cholera. Cholera is caused by the bacterium Vibrio cholera,

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

Initial state: OUTSIDE
symporter open to outside OF CELL
6 Release of glucose
Na+ Na+
allows the empty
1 Two Na+ from outside
symporter to return
the cell are bound
to initial state

Na+
Na+

Glucose
O
Glucose
5 Loss of Na+ O
is followed by INSIDE
glucose release to 2 Binding of Na+
OF CELL allows glucose
inside
binding and a subsequent
conformational change

Na+
Na+
O

Na+ Na+
O

4 Na+ are released

inside, but are continually
Na+
extruded to outside by a 3 Symporter opens to inside
separate Na+-K+ Na+
O

pump (dashed line) Na+ Na+

Figure 8-15 Model Mechanism for the Na + /Glucose Symporter. The transport process is shown here
in six steps arranged at the periphery of a cell. The inward transport of glucose against its concentration gradi-
ent is driven by the simultaneous inward transport of sodium ions down their electrochemical gradient. The
sodium ion gradient is in turn maintained by the continuous outward transport of sodium ions (dashed arrow) by
the Na+/K+ pump of Figure 8-14.
231

M08_HARD7694_09_GE_C08.indd 231 13/02/17 4:29 pm

 which produces a toxin that paralyzes intestinal cells and can the plasma membrane of halophilic (“salt-loving”) archaea
lead to death via dehydration. Oral rehydration is the standard belonging to the genus Halobacterium. In contrast to transport
treatment for cholera, and it has been shown that rehydration proteins that utilize energy derived from ATP or ion gradients,
with a salt and sugar solution is the most effective remedy. Ad- bacteriorhodopsin uses energy derived from photons of light
ministration of NaCl helps the body retain water as it strives to drive active transport. Bacteriorhodopsin contains a pig-
to maintain salt balance in the tissues, and providing glucose ment molecule that traps light energy and uses it to drive the
along with the salt therefore allows for more efficient uptake active transport of protons outward across the plasma mem-
of salt via the Na+/glucose symporter. brane. It thereby creates an electrochemical proton gradient
Note that both types of glucose transporters that we have that powers the synthesis of ATP by an ATP synthase analo-
discussed, the GLUT uniporter and the Na+/glucose sym- gous to the F-type ATP synthases found in chloroplasts and
porter, can be found in the same cell. For example, cells lining mitochondria.
the small intestine may have a Na+/glucose symporter on the The light-absorbing pigment of bacteriorhodopsin is
intestinal side to transport glucose by active transport from retinal, a carotenoid derivative related to vitamin A that also
the intestinal lumen, where its concentration is high, into the serves as the visual pigment in the retina of your eyes. Reti-
cells, which have a lower glucose concentration. Then on the nal makes bacteriorhodopsin bright purple in color, which is
opposite side of the cell, a GLUT transporter can transport why halobacteria have been called purple photosynthetic bacte-
glucose into the bloodstream by facilitated diffusion, where its ria (Figure 8-16a). Bacteriorhodopsin appears in the plasma
concentration is lower than in the cell. membrane of the Halobacterium cell as colored patches called
purple membrane (Figure 8-16b).
The Bacteriorhodopsin Proton Pump Uses Bacteriorhodopsin is an integral membrane protein with
Light Energy to Transport Protons seven a-helical membrane-spanning segments oriented in
The final active transport system we will consider is the sim- the membrane to form an overall cylindrical shape (Figure
plest. Nothing more is involved than a small integral mem- 8-16c). A molecule of retinal is attached to a lysine residue
brane protein called bacteriorhodopsin. This protein of the protein. When the retinal, which is a chromophore (a
(briefly introduced in Chapter 7) is a proton pump found in colored, light-absorbing pigment), absorbs a photon of light,

Purple membrane

Photon of light
(b) Halobacterium cell
with patches of purple
membrane

H+

OUTSIDE OF CELL

H3N+

(a) Halobacterium (purple color) grows in

the high-salt concentration of solar Retinal
evaporation ponds used for
manufacturing salt around San
Francisco Bay

Figure 8-16 The Bacteriorhodopsin Proton

Pump of Halobacteria. (a) Archaea belonging to the
INSIDE OF CELL
genus Halobacterium are purple due to the protein bac-
teriorhodopsin. (b) Bacteriorhodopsin is a light-activated
proton pump in the plasma membrane of Halobacte- O
rium cells that is found in bright purple patches known C O−
as purple membrane. (c) The seven a-helical transmem-
H+
brane segments of bacteriorhodopsin form a cylinder
that contains a molecule of the light-absorbing pigment (c) Bacteriorhodopsin molecule embedded in
retinal. the plasma membrane
232

M08_HARD7694_09_GE_C08.indd 232 13/02/17 4:29 pm

 it becomes photoactivated. The activated bacteriorhodopsin from the outside of a membrane-bounded compartment to the
protein then becomes capable of transferring protons from the inside can be represented as
inside to the outside of the cell. The electrochemical potential
produced by pumping protons out of the cell in this way is Soutside ¡ Sinside (8-4)
used by membrane-located halobacterial ATPases to synthe-
size ATP as the protons flow down their concentration gradi- The free energy change for this inward transport reaction (see
ent back into the cell. Chapter 5) can be written as
Because it is a light-driven proton pump, the bacteri-
orhodopsin molecule is being exploited for use in biomolecu- 3S 4 inside
lar electronics. This is a relatively new field in which biological ∆Ginward = ∆G° + RT ln (8-5)
3S 4 outside
molecules with electron- and ion-conducting properties are
being used as structural elements of biooptical, biocomputing, where ∆G is the free energy change, ∆G° is the standard free
and biosensor devices. One potential application of biomo- energy change, R is the gas constant (1.987 cal/mol@K), T
lecular electronics is the design of more efficient solar cells to is the absolute temperature, and 3S 4 inside and 3S 4 outside are
collect solar energy and convert it to electricity. the prevailing concentrations of S on the inside and outside
Energy-dependent proton pumping is one of the most of the membrane, respectively. However, the equilibrium con-
basic concepts in cellular energetics. Proton pumping, which stant Keq for the transport of an uncharged solute is always 1
occurs in all bacteria, mitochondria, and chloroplasts, repre- because at equilibrium the solute concentrations on the two
sents the driving force for all of life on Earth because it is an sides of the membrane will be the same:
absolute requirement for the efficient synthesis of ATP. (We
will discuss the mechanisms underlying the generation of pro- 3S 4 inside
ton gradients and the use of the energy of such gradients in Keq = = 1.0 (8-6)
3S 4 outside
more detail when we discuss respiration and photosynthesis

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

in Chapters 10 and 11.) This means that ∆G° is always zero:

CONCEPT CHECK 8.5

∆G° = - RT ln Keq = - RT ln 1 = 0 (8-7)
Both the Na /glucose symporter and the Na+/K+ pump move
+

sodium ions across a membrane. How is the movement dif-

ferent for the two types of transporters? So the expression for ∆G of inward transport of an uncharged
solute shown in Equation 8-5 simplifies to

3S 4 inside
8.6 The Energetics of Transport ∆Ginward = RT ln
3S 4 outside
(8-8)

Every transport event in the cell is an energy transaction.

Notice that if 3S 4 inside is less than 3S 4 outside, ∆G is negative,
Energy either is released as transport occurs or is required to
indicating that the inward transport of substance S is exer-
drive transport. To understand the energetics of transport,
gonic. It can therefore occur spontaneously, as would be ex-
we must recognize that two different factors may be involved.
pected for facilitated diffusion down a concentration gradient.
For uncharged solutes, the only variable is the concentration
But if 3S 4 inside is greater than 3S 4 outside, inward transport of
gradient across the membrane, which determines whether
S is against the concentration gradient, and ∆G will be posi-
transport is “down” a gradient (exergonic) or “up” a gradi-
tive. In this case, inward transport of S is endergonic, and the
ent (endergonic). For charged solutes, however, there is both
amount of energy required to drive the active transport of S
a concentration gradient and an electrical potential across
into the cell is indicated by the magnitude of the positive value
the membrane. The two may either reinforce each other or
of ∆G.
oppose each other, depending on the charge on the ion and
the direction of transport. We will first look at the transport
of uncharged substances and then consider the additional An Example: The Uptake of Lactose. Suppose that
complication that arises when charged substances are moved the concentration of lactose within a bacterial cell is to be
across membranes. maintained at 10 mM, whereas the external lactose con-
centration is only 0.20 mM. The energy requirement for the
inward transport of lactose at 25°C can be calculated from
For Uncharged Solutes, the 𝚫G of Transport Equation 8-8 as
Depends Only on the Concentration Gradient
For solutes with no net charge, we need only be concerned 3lactose 4 inside
∆Ginward = RT ln
with the concentration gradient across the membrane. We can 3lactose 4 outside
therefore treat the transport process as we would treat a sim- 0.010
= (1.987)(273 + 25) ln
ple chemical reaction, and we can calculate ∆G accordingly. 0.0002
= 592 ln 50
Calculating 𝚫G for the Transport of Molecules. The = 2316 cal/mol
general “reaction” for the transport of molecules of solute S = 2.32 kcal/mol (8-9)
233

M08_HARD7694_09_GE_C08.indd 233 13/02/17 4:30 pm

 In many bacterial cells, the energy to drive lactose uptake is ion transport. Because it is almost always negative, the mem-
provided by an electrochemical proton gradient, so lactose up- brane potential typically favors the inward movement of cat-
take in such cells is an example of indirect active transport. ions and opposes their outward movement. As we mentioned
As written, Equation 8-8 on page 233 applies to inward earlier, the net effect of both the concentration gradient and
transport. For outward transport, the positions of Sinside and the potential gradient for an ion is called the electrochemical
Soutside are simply interchanged within the logarithm. As a re- potential for that ion.
sult, the absolute value of ∆G remains the same, but the sign
is changed. As for any other process, a transport reaction that Calculating 𝚫G for the Transport of Ions. Both compo-
is exergonic in one direction will be endergonic to the same de- nents of the electrochemical potential must be considered
gree in the opposite direction. The equations for calculating when determining the energetics of ion transport. To calculate
∆G of inward and outward transport of uncharged solutes ∆G for the transport of ions therefore requires an equation
are summarized in Table 8-3. with two terms, one to express the effect of the concentration
gradient across the membrane and the other to take into ac-
For Charged Solutes, the 𝚫G of Transport count the membrane potential.
Depends on the Electrochemical Potential If we let Sz represent a solute with a charge z, then we can
calculate ∆G for the inward transport of Sz as
For charged solutes—ions, in other words—we need to
consider the electrochemical potential for that ion, which is
3S 4 inside
influenced by both the concentration gradient for that ion ∆Ginward = +RT ln + zFVm (8-10)
and the membrane potential of the cell itself, Vm. For ani- 3S 4 outside
mal cells, Vm usually falls in the range of - 60 to - 90 mV. where R, T, 3S 4 inside, and 3S 4 outside are defined as before; z is
In bacterial and plant cells, it is significantly more negative, the charge on S (such as +1, +2, - 1, - 2); F is the Faraday
often about - 150 mV in bacteria and between - 200 and constant (23,062 cal/mol@V); and Vm is the membrane poten-
- 300 mV in plants. By convention, the minus sign indicates tial (in volts). Note that Equation 8-8 on page 233 is just the
that the excess of negative charge is on the inside of the cell. simplified version of Equation 8-10 for solutes with a z value
Thus, the Vm value indicates how negative (or positive, in the of zero—that is, for molecules with no net charge.
case of a plus sign) the inside of the cell is compared with the For a typical cell, the membrane potential Vm is nega-
outside. tive (an excess of negative charge inside the cell). A positive
The membrane potential obviously has no effect on un- ion (z 7 0) will cause the term zFVm to be negative, lowering
charged solutes, but it significantly affects the energetics of the ∆G for inward transport. This indicates that uptake of
the positive ion is energetically favorable, as you would ex-
pect if the interior of the cell has a net negative charge. The
uptake of positive ions becomes more exergonic and more
favorable as the membrane potential of the cell decreases.
The opposite is true for a negative ion (z 6 0), which gives
Table 8-3 Calculation of 𝚫G for the Transport of a positive value for zFVm and a positive change in free en-
Charged and Uncharged Solutes ergy, indicating an unfavorable process that will not occur
spontaneously.
Transport Process For the outward transport of S, ∆G has the same value
Membrane as for inward transport but is opposite in sign, so we can write

Inward ∆Goutward = - ∆Ginward (8-11)

S S
An Example: The Uptake of Chloride Ions. To illustrate
Outward the use of Equation 8-10 and to point out that intuition does
not always serve us well in predicting the direction of ion
Outside Inside transport, consider what happens when nerve cells with an
intracellular chloride ion concentration of 50 mM are placed
∆G for Transport of Uncharged Solutes: in a solution containing 100 mM Cl- . Because the Cl- con-
3 S4 inside R = 1.987 cal/mol@K centration is twice as high outside the cell as inside, you might
∆Ginward = RT ln T = K = °C + 273 expect chloride ions to diffuse passively into the cell without
3 S4 outside
the need for active transport. However, this expectation ig-
∆Goutward = - ∆Ginward
nores the membrane potential of about - 60 mV ( - 0.06 V)
𝚫G for Transport of Charged Solutes: that exists across the plasma membrane of nerve cells. The
3 S4 inside z = charge on ion
minus sign reminds us that the inside of the cell is negative
∆Ginward = RT ln + zFVm with respect to the outside, which means that the inward
3 S4 outside F = 23,062 cal/mol@V
movement of negatively charged anions such as Cl- will be
Vm = membrane against the membrane potential. Thus, the inward movement
potential (in volts)
∆Goutward = - ∆Ginward
of chloride ions is down the concentration gradient but up the
charge gradient.
234

M08_HARD7694_09_GE_C08.indd 234 13/02/17 4:30 pm

 To quantify the relative magnitudes of these two oppos- determine the magnitude of the membrane potential, Vm, if
ing forces at 25°C, we can use Equation 8-10 on page 234 the ion concentrations on opposite sides of the membrane are
with the relevant values inserted: known. This equation is very similar in basic form to the equa-
tion that allows us to calculate the free energy change of a
3S 4 inside chemical reaction by knowing the concentrations of reactants
∆Ginward = +RT ln + zFVm and products (see Equation 5-17 on page 141). Extensions of
3S 4 outside
this equation are important for determining the contributions
0.05 of various ions to the overall resting membrane potential of a
= +(1.987)(273 + 25) ln a b
0.10 cell (discussed in detail in Chapter 22).
+ ( - 1)(23,062)( - 0.06) In this chapter, we have focused on the movement of ions
= + 592 ln(0.5) + (23,062)(0.06) and small molecules into and out of cells and organelles and
= - 410 + 1384 have seen that these solutes pass directly through the mem-
brane, though often with the aid of specific transport proteins.
= 974 cal/mol
In addition to such traffic, however, many cells are able to take
= + 0.97 kcal/mol (8-12) up and release substances that are too large to pass through a
membrane regardless of its permeability properties. Exocyto-
The sign of ∆G is positive, meaning that even though the sis is the process whereby cells release proteins that are syn-
chloride ion concentration is twice as high outside the cell as thesized within the cell and sequestered within membrane-
inside, energy will still be required to drive the movement of bounded vesicles, whereas endocytosis involves the uptake
chloride ions into the cell. This is due to the excess of negative of macromolecules and other substances by trapping and
charge inside the cell, represented by the negative value of Vm. engulfing them within an invagination of the plasma mem-
The movement of a mole of chloride ions up the charge gra- brane. Both of these processes are unique to eukaryotic cells.
(We will consider exocytosis and endocytosis in detail when we

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

dient represented by the membrane potential requires more
energy (+1384 calories) than is released by the movement of get to Chapter 12.)
the mole of chloride ions down their concentration gradient
( - 410 calories). To guide you in such calculations, the equa- CONCEPT CHECK 8.6
tions for inward and outward transport of charged solutes You are studying the energetics of transport of the amino
are included along with those for the transport of uncharged acid aspartic acid, whose side chain can exist in the non-
solutes in Table 8-3, which summarizes the thermodynamic ionized neutral state ( ¬ COOH) or the ionized, negatively
properties of each of these processes. charged state ( ¬ COO- ). To calculate the ∆G of transport
At equilibrium, ∆G = 0. By performing a few algebraic into a cell, why would you need to know the ionization state
manipulations, Equation 8-10 on page 234 then allows us to of the side chain?

Summary of Key Points For activities, animations, and review quizzes, go to

the study area at www.masteringbiology.com.

8.1 Cells and Transport Processes ■■ Membranes are permeable to lipids, which can pass through the
nonpolar interior of the lipid bilayer. Membrane permeability
■■ The selective transport of molecules and ions across membrane of most compounds is directly proportional to their partition
barriers ensures that necessary substances are moved into and coefficient—their relative solubility in oil versus water.
out of cells and cell compartments at the appropriate time and at
■■ The direction of diffusion of a solute across a membrane is
useful rates.
­determined by its concentration gradient and always moves
■■ Nonpolar molecules and small, polar molecules cross the mem- toward equilibrium. The solute will diffuse down the gradi-
brane by simple diffusion. Transport of all other solutes, includ- ent from a region of higher concentration to a region of lower
ing ions and many molecules of biological relevance, is mediated concentration.
by specific transport proteins that provide passage through an
■■ If the membrane is impermeable to the solute, water will move
otherwise impermeable membrane.
by osmosis from the area of lower solute concentration (higher
■■ Each such transport protein has at least one, and frequently sev- 3H2O4 ) to the area of higher solute concentration (lower 3H2O4 ).
eral, hydrophobic membrane-spanning sequences that embed the
protein within the membrane and often act as the channel itself.
Typically, a separate regulatory domain controls channel open- 8.3 Facilitated Diffusion: Protein-Mediated
ing and closing. Movement Down the Gradient
8.2 Simple Diffusion: Unassisted Movement ■■ Transport can either be downhill or uphill in relation to an
uncharged solute’s concentration gradient. For an ion, we must
Down the Gradient consider its electrochemical potential—the combined effect of the
■■ Simple diffusion through biological membranes is limited to small ion’s concentration gradient and the charge gradient across the
or nonpolar molecules such as O2, CO2, and lipids. Water mol- membrane.
ecules, although polar, are small enough to diffuse across mem- ■■ Downhill transport of large, polar molecules and ions, called
branes in a manner that is not entirely understood. facilitated diffusion, must be mediated by carrier proteins and
235
 channel proteins because these molecules and ions cannot diffuse protons (plant, fungal, and many bacterial cells). For example,
through the membrane directly. the inward transport of nutrients across the plasma membrane
■■ Carrier proteins function by alternating between two conforma- is often driven by the symport of sodium ions that were pumped
tional states. Examples include the glucose transporter and the outward by the Na+/K+ pump. As they flow back into the cell,
anion exchange protein found in the plasma membrane of the they drive inward transport of sugars, amino acids, and other
erythrocyte. organic molecules.

■■ Transport of a single kind of molecule or ion is called uniport.

The coupled transport of two or more molecules or ions at a time 8.5 Examples of Active Transport
may involve movement of both solutes in the same direction
(symport) or in opposite directions (antiport). ■■ The Na+/K+ pump uses direct active transport to move sodium
■■ Channel proteins facilitate diffusion by forming transmembrane and potassium ions in opposite directions against their concen-
channels lined with hydrophilic amino acids. Three important tration gradients, with ATP hydrolysis providing the necessary
categories of channel proteins are ion channels (used mainly for input of energy.
transport of H+, Na+, K+, Ca2+ , Cl- , and HCO3- ), porins (for vari- ■■ The Na+/glucose symporter uses indirect active transport to
ous high-molecular-weight solutes), and aquaporins (for water). move glucose into the cell, along with Na+. The driving force is
the movement of Na+down a steep concentration gradient main-
tained by the Na+/K+ pump.
8.4 Active Transport: Protein-Mediated ■■ In Halobacterium, active transport is powered by light energy. As
Movement Up the Gradient photons of light are absorbed by bacteriorhodopsin, protons are
pumped across the cell membrane and out of the cell. As the pro-
■■ Active transport—the uphill transport of large, polar molecules
tons flow back into the cell, ATP is synthesized.
and ions—requires a protein transporter and an input of energy.
It may be powered by ATP hydrolysis, the electrochemical poten-
tial of an ion gradient, or light energy.
8.6 The Energetics of Transport
■■ Active transport powered by ATP hydrolysis utilizes four major
classes of transport proteins: P-type, V-type, F-type, and ABC- ■■ The ∆G for transport can be readily calculated. If ∆G 6 0, trans-
type ATPases. One widely encountered example is the ATP- port will be spontaneous. If ∆G 7 0, an input of energy will be
powered Na+/ K+ pump (a P-type ATPase), which maintains required to drive transport. If ∆G = 0, there will be no net move-
electrochemical potentials for sodium and potassium ions across ment of the solute.
the plasma membrane of animal cells. ■■ For uncharged solutes, ∆G depends only on the concentration
■■ Active transport driven by an electrochemical potential usually gradient. For charged solutes, both the concentration gradient
depends on a gradient of either sodium ions (animal cells) or and the membrane potential must be taken into account.

Problem Set For activities, animations, and review quizzes, go

to the study area at www.masteringbiology.com.

8-1 True or False? Indicate whether each of the following state- (h) Transport channel proteins have a high level of specificity for a
ments about membrane transport is true (T) or false (F). If false, solute.
reword the statement to make it true.
(a) Facilitated diffusion of glucose occurs rapidly because the con- 8-2 Telling Them Apart. From the following list of properties,
centration gradient is maintained by packaging intracellular indicate which one(s) can be used to distinguish between each of the
glucose into vesicles. following pairs of transport mechanisms.

(b) The exergonic movement of an ion coupled with the movement Transport Mechanisms
of a solute down a concentration gradient is an example of pri- (a) Simple diffusion; facilitated diffusion
mary active transport.
(b) Facilitated diffusion; active transport
(c) The Keq value for the diffusion of polar molecules out of the cell (c) Simple diffusion; active transport
is less than one because membranes are essentially impermeable
(d) Direct active transport; indirect active transport
to such molecules.
(e) Symport; antiport
(d) Aquaporins facilitate the rapid movement of water molecules
(f) Uniport; coupled transport
into or out of cells.
(g) P-type ATPase; V-type ATPase
(e) Oxygen can move freely across the plasma membrane by simple
diffusion. Properties
(f) In simple diffusion, the net rate of transport for a specific sub- 1. Directions in which two transported solutes move
stance is indirectly proportional to the concentration difference 2. Direction the solute moves relative to its concentration gradient
for that substance across the membrane. or its electrochemical potential
(g) ABC transporters are of medical interest because they are known 3. Kinetics of solute transport
to be involved in drug resistance.
236
 4. Requirement for metabolic energy (c) Assume that the sodium ion concentration is 12 mM inside a cell
5. Requirement for simultaneous transport of two solutes and 145 mM outside the cell and that the membrane potential
is -90 mV. Can a cell use ATP hydrolysis to drive the outward
6. Intrinsic directionality
transport of sodium ions on a 2:1 basis (two sodium ions trans-
7. Competitive inhibition ported per ATP hydrolyzed) if the ATP/ADP ratio is 5:1, the inor-
8. Sensitivity to the inhibitor vanadate ganic phosphate concentration is 50 mM, and the temperature is
37°C? What about on a 3:1 basis? Explain your answers.
8-3 Mechanisms of Transport. For each of the following state- (d) Assume that a bacterial cell maintains a proton gradient across
ments, answer with a D if the statement is true of simple diffusion, its plasma membrane such that the pH inside the cell is 8.0
with an F if it is true of facilitated diffusion, and with an A if it is when the outside pH is 7.0. Can the cell use the proton gradient
true of active transport. Any, all, or none (N) of the choices may be to drive ATP synthesis on a 1:1 basis (one ATP synthesized per
appropriate for a given statement. proton transported) if the membrane potential is - 180 mV, the
(a) Requires the presence of an integral membrane protein temperature is 25°C, and the ATP, ADP, and inorganic phosphate
concentrations are as in part c? What about on a 1:2 basis?
(b) Solutes move down their free energy gradient in the direction of
Explain your answers.
thermodynamic equilibrium
(c) Is not subject to saturation 8-7 QUANTITATIVE Sodium Ion Transport. A marine protozoan
(d) Requires the hydrolysis of ATP is known to pump sodium ions outward by a simple ATP-driven Na+
(e) Is a way of establishing a difference in the concentration gradi- pump that operates independently of potassium ions. The intra-
ent of solutes across a membrane cellular concentrations of ATP, ADP, and Pi are 20, 2, and 1 mM,
respectively, and the membrane potential is -75 mV.
(f) Applies only to small, nonpolar solutes
(a) Assuming that the pump transports three sodium ions outward
(g) Applies only to ions per molecule of ATP hydrolyzed, what is the lowest internal
(h) Transport can occur in either direction across the membrane, sodium ion concentration that can be maintained at 25°C when

Chapter 8 | Transport Across Membranes: Overcoming the Permeability Barrier

depending on the prevailing concentration gradient the external sodium ion concentration is 150 mM?
(i) Has a positive ∆G (b) If you were dealing with an uncharged molecule rather than an
(j) Usually has intrinsic directionality ion, would your answer for part a be higher or lower, assuming
all other conditions remained the same? Explain.
8-4 Discounting the Transverse Carrier Model. At one time,
membrane biologists thought that transport proteins might act by 8-8 QUANTITATIVE The Case of the Acid Stomach. The gastric
binding a solute molecule or ion on one side of the membrane and juice in your stomach has a pH of 2.0. This acidity is due to the
then diffusing across the membrane to release the solute molecule secretion of protons into the stomach by the epithelial cells of the
on the other side. We now know that this transverse carrier model is gastric mucosa. Epithelial cells have an internal pH of 7.0 and a
almost certainly wrong. Suggest two reasons that argue against such membrane potential of -70 mV (inside is negative) and function at
a model. One of your reasons should be based on our current under- body temperature (37°C).
standing of membrane structure and the other on thermodynamic (a) What is the concentration gradient of protons across the epithe-
considerations. lial membrane?
(b) Do you think that proton transport can be driven by ATP hydrol-
8-5 QUANTITATIVE Potassium Ion Transport. Most of the cells of ysis at the ratio of one molecule of ATP per proton transported?
your body pump potassium ions inward to maintain an internal K+ (c) If protons were free to move back into the cell, calculate the
concentration that is 30 to 40 times the external concentration. membrane potential that would be required to prevent them
(a) What is ∆G (at 37°C) for the transport of potassium ions into from doing so.
a cell that maintains no membrane potential across its plasma (d) Calculate the free energy change associated with the secretion of
membrane? 1 mole of protons into gastric juice at 37°C.
(b) For a nerve cell with a membrane potential of -60 mV, what is
∆G for the inward transport of potassium ions at 37°C? 8-9 Charged or Not: Does It Make a Difference? Many solutes
(c) For the nerve cell in part b, what is the maximum number of that must move into and out of cells exist in either a protonated
potassium ions that can be pumped inward by the hydroly- or ionized form, or they have functional groups that can be either
sis of one ATP molecule if the ATP/ADP ratio in the cell is protonated or ionized. Simple molecules such as CO2, H3PO4 (phos-
5:1 and the inorganic phosphate concentration is 10 mM? phoric acid), and NH3 (ammonia) are in this category, as are organic
(Assume ∆G°′ = -7.3 kcal/mol for ATP hydrolysis.) molecules with carboxylic acid groups, phosphate groups, and/or
amino groups.
8-6 QUANTITATIVE Ion Gradients and ATP Synthesis. The ion (a) Consider ammonia as a simple example of such compounds.
gradients maintained across the plasma membranes of most cells What is the charged form of ammonia called? What is its chemi-
play a significant role in cellular energetics. Ion gradients are often cal formula?
either generated by the hydrolysis of ATP or used to make ATP by the (b) Which of these two forms will predominate in a solution with a
phosphorylation of ADP. highly acidic pH? Explain your answer.
(a) Cite an example in which ATP is used to generate and maintain (c) For which of these two forms will the uptake across the plasma
an ion gradient. What is another way that an ion gradient can be membrane of a cell be affected by the concentration gradient of
generated and maintained? that form on the inside versus the outside of the cell? For which,
(b) Cite an example in which an ion gradient is used to make ATP. if either, of these two forms will uptake be affected by the mem-
What is another use for ion gradients? brane potential of the plasma membrane? Explain your answers.
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M08_HARD7694_09_GE_C08.indd 237 27/02/17 3:22 pm

 (d) For a cell that must take up ammonia from its environment, will (a) What is the ATPase activity, calculated as micromoles of ATP
uptake of the charged form require more or less energy than hydrolyzed per milligram of protein per minute?
uptake of the uncharged form, assuming that the cell has a nega- (b) The ATPase is calcium-activated, as shown by the increase in
tive membrane potential? Explain your answer. ATP hydrolysis when the calcium was added and the decrease
(e) Instead of ammonia, consider the uptake of acetic acid, in hydrolysis when all the added calcium was taken up into the
CH3COOH, an important intermediate in several biological vesicles 1 minute after it was added. How many calcium ions are
pathways. What is the charged form in this case? For which of taken up for each ATP hydrolyzed?
the two forms will the uptake across the plasma membrane of a (c) The final addition is an ionophore that carries calcium ions
cell be affected by the concentration gradient of that form on the across membranes. Why does ATP hydrolysis begin again?
inside versus the outside of the cell? For which, if either, of these
two forms will uptake be affected by the membrane potential of
the plasma membrane? Explain your answers.
• 8-11 Inverted Vesicles. An important advance in transport
research was the development of methods for making closed
(f) For a cell that must take up acetic acid from its environment, membrane vesicles that retain the activity of certain transport
will uptake of the charged form require more or less energy than systems. One such system uses resealed vesicles from red blood cell
uptake of the uncharged form, assuming that the cell has a nega- membranes, in which the orientation of membrane proteins may be
tive membrane potential? Explain your answer. either the same as in the intact cell (right side out) or inverted (inside
out). Such vesicles have demonstrable ATP-driven Na+/K+ pump
8-10 QUANTITATIVE The Calcium Pump of the Sarcoplasmic activity. By resealing the vesicles in one medium and then placing
Reticulum. Muscle cells use calcium ions to regulate the contrac- them in another, it is possible to have ATP, sodium ions, and potas-
tile process. Calcium is both released and taken up by the sarco- sium ions present inside the vesicle, present outside the vesicle, or not
plasmic reticulum (SR). Release of calcium from the SR activates present at all.
muscle contraction, and ATP-driven calcium uptake causes the
(a) Suggest one or two advantages that such vesicles might have
muscle cell to relax afterward. When muscle tissue is disrupted by
compared to intact red blood cells for studying the Na+/K+
homogenization, the SR forms small vesicles called microsomes that
pump. Can you think of any possible disadvantages?
maintain their ability to take up calcium. To obtain the data shown
in Figure 8-17, a reaction medium was prepared to contain 5 mM (b) For the inverted vesicles, indicate whether each of the following
ATP and 0.1 M KCl at pH 7.5. An aliquot of SR microsomes con- should be present inside the vesicle, outside the vesicle, or not
taining 1.0 mg protein was added to 1 mL of the reaction mixture, present at all in order to demonstrate ATP hydrolysis: Na+, K+,
followed by 0.4 mmol of calcium. Two minutes later, a calcium and ATP.
ionophore was added. (An ionophore is a substance that facilitates (c) If you were to plot the rate of ATP hydrolysis as a function of
the movement of an ion across a membrane.) ATPase activity was time after initiating transport in such inverted vesicles, what sort
monitored during the additions, with the results shown in the figure. of a curve would you expect to obtain?

• 8-12 Ouabain Inhibition. Ouabain is a specific inhibitor of the

(mmol ATP hydrolyzed/mg protein-min)

active transport of sodium ions out of the cell and is therefore a

0.3 valuable tool in studies of membrane transport mechanisms. Which
of the following processes in your own body would you expect to be
sensitive to inhibition by ouabain? Explain your answer in each case.
ATPase activity

0.2 (a) Active uptake of lactose by the bacteria in your intestine

SR 2+
Ca (b) Facilitated diffusion of glucose into a muscle cell
0.1 (c) Active transport of dietary phenylalanine across the intestinal
Ionophore
mucosa

0 (d) Uptake of potassium ions by red blood cells

0 1 2 3 4 5
Time (min)

Figure 8-17 Calcium Uptake by the Sarcoplasmic

Reticulum. See Problem 8-10.

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M08_HARD7694_09_GE_C08.indd 238 13/02/17 4:30 pm