1. health and fitness
2. disease
3. cholesterol

chapter
12
Biological Membranes
and Transport
The first cell probably came into being when a membrane formed, enclosing a small volume of aqueous solution and separating it from the rest of the
universe. Membranes define the external boundaries of cells and regulate
the molecular traffic across that boundary; in eukaryotic cells, they divide
the internal space into discrete compartments to segregate processes and
components (Fig. 12–1). They organize complex reaction sequences and
are central to both biological energy conservation and cell-to-cell communication. The biological activities of membranes flow from their remarkable
physical properties. Membranes are flexible, self-sealing, and selectively
permeable to polar solutes. Their flexibility permits the shape changes that
accompany cell growth and movement (such as amoeboid movement). With
their ability to break and reseal, two membranes can fuse, as in exocytosis,
or a single membrane-enclosed compartment can undergo fission to yield
two sealed compartments, as in endocytosis or cell division, without creating gross leaks through cellular surfaces. Because membranes are selectively permeable, they retain certain compounds and ions within cells and
within specific cellular compartments, while excluding others.
Membranes are not merely passive barriers. They include an array of
proteins specialized for promoting or catalyzing various cellular processes.
At the cell surface, transporters move specific organic solutes and inorganic
ions across the membrane; receptors sense extracellular signals and trigger
molecular changes in the cell; adhesion molecules hold neighboring cells together. Within the cell, membranes organize cellular processes such as the
synthesis of lipids and certain proteins, and the energy transductions in
mitochondria and chloroplasts. Membranes consist of just two layers of
molecules and are therefore very thin; they are essentially two-dimensional.
Because intermolecular collisions are far more probable in this twodimensional space than in three-dimensional space, the efficiency of enzymecatalyzed processes organized within membranes is vastly increased.
In this chapter we first describe the composition of cellular membranes
and their chemical architecture—the dynamic molecular structures that
underlie their biological functions. We describe several classes of membrane
proteins defined by the ways in which they associate with the lipid bilayer.
Cell adhesion, endocytosis, and the membrane fusion that occurs with viral
infection will illustrate the dynamic role of membrane proteins. We then
turn to the protein-mediated passage of solutes across membranes via
transporters and ion channels. In later chapters we will discuss the role of
membranes in signal transduction (Chapters 13 and 23), energy transduction (Chapter 19), lipid synthesis (Chapter 21), and protein synthesis
(Chapter 27).
(a)
(b)
(c)
(d)
(e)
(f)
figure 12–1
Biological membranes. Viewed in cross section, all intracellular membranes share a characteristic trilaminar
appearance. The protozoan Paramecium contains a
variety of specialized membrane-bounded organelles.
When a thin section of a Paramecium is stained with
osmium tetroxide to highlight membranes, each of the
membranes appears as a three-layer structure, 5 to 8 nm
(50 to 80 Å) thick. The trilaminar images consist of two
electron-dense layers on the inner and outer surfaces
separated by a less dense central region. Above are highmagnification views of the membranes of (a) a cell body
(plasma and alveolar membranes tightly apposed),
(b) a cilium, (c) a mitochondrion, (d) a digestive vacuole,
(e) the endoplasmic reticulum, and (f) a secretory vesicle.
389
390
Part II
Structure and Catalysis
table 12–1
Major Components of Plasma Membranes in Various Organisms
Components (% by weight)
Protein
Phospholipid
Sterol
30
45
47
52
56
75
30
27
26
7
40
25
19
25
7
4
4
0
Human myelin sheath
Mouse liver
Maize leaf
Yeast
Paramecium (ciliated protist)
E. coli
Sterol type
Other lipids
Cholesterol
Cholesterol
Sitosterol
Ergosterol
Stigmasterol
—
Galactolipids, plasmalogens
—
Galactolipids
Triacylglycerols, steryl esters
—
—
The Molecular Constituents of Membranes
One approach to understanding membrane function is to study membrane
composition—to determine, for example, which components are common
to all membranes and which are unique to membranes with specific functions. Before describing membrane structure and function, we therefore
consider the molecular components of membranes: proteins and polar
lipids, which account for almost all the mass of biological membranes, and
carbohydrate present as part of glycoproteins and glycolipids.
Rat hepatocyte membrane type
Plasma
Inner
mitochondrial
Outer
mitochondrial
Lysosomal
Each Type of Membrane Has Characteristic Lipids and Proteins
Nuclear
Rough ER
Smooth ER
Golgi
0
20
40
60
80
100
Percent membrane lipid
Cholesterol
Sphingolipids
Cardiolipin
Phosphatidylcholine
Minor lipids
Phosphatidylethanolamine
figure 12–2
Lipid composition of the plasma membrane and
organelle membranes of a rat hepatocyte. The functional specialization of each membrane type is reflected
in its unique lipid composition. Cholesterol (orange) is
prominent in plasma membranes but barely detectable in
mitochondrial membranes. Cardiolipin (blue) is a major
component of the inner mitochondrial membrane but not
of the plasma membrane. Phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol (yellow) are relatively minor components of most membranes but may
serve critical functions. Phosphatidylinositol and its derivatives, for example, are important in signal transductions
triggered by hormones. Sphingolipids (red), phosphatidylcholine (purple), and phosphatidylethanolamine (green)
are present in most membranes, but in varying proportions. Glycolipids, which are major components of the
chloroplast membranes of plants, are virtually absent
from animal cells.
The relative proportions of protein and lipid vary with the type of membrane (Table 12–1), reflecting the diversity of biological roles. For example,
certain neurons have a myelin sheath, an extended plasma membrane that
wraps around the cell many times and acts as a passive electrical insulator.
The myelin sheath consists primarily of lipids, whereas the plasma membranes of bacteria and the membranes of mitochondria and chloroplasts, in
which many enzyme-catalyzed processes take place, contain more protein
than lipid.
For studies of membrane composition, the first task is to isolate a selected membrane. When eukaryotic cells are subjected to mechanical shear,
their plasma membranes are torn and fragmented, releasing cytosolic components and membrane-bounded organelles such as mitochondria, chloroplasts, lysosomes, and nuclei. Plasma membrane fragments and intact organelles can be isolated by centrifugal techniques described in Chapter 2
(see Fig. 2–20).
Chemical analysis of membranes isolated from various sources reveals
certain common properties. Each kingdom, each species, each tissue or cell
type, and the organelles of each cell type have a characteristic set of membrane lipids. The plasma membranes of hepatocytes, for example, are enriched in cholesterol and contain no detectable cardiolipin (Fig. 12–2); in
the inner mitochondrial membrane of the hepatocytes, this distribution is
reversed. Cells clearly have mechanisms to control the kinds and amounts
of membrane lipids they synthesize and to target specific lipids to particular organelles. In many cases, we can surmise the adaptive advantages of
distinct combinations of membrane lipids; in other cases, the functional significance of these combinations remains to be discovered.
The protein composition of membranes from different sources varies
even more widely than their lipid composition, reflecting functional specialization. In a rod cell of the vertebrate retina, one portion of the cell is highly
specialized for the reception of light; more than 90% of the plasma membrane protein in this region is the light-absorbing glycoprotein rhodopsin.
The less specialized plasma membrane of the erythrocyte has about 20
Chapter 12 Biological Membranes and Transport
391
prominent types of proteins as well as dozens of minor ones; many of these
are transporters, each moving a specific solute across the membrane. The
plasma membrane of Escherichia coli contains hundreds of different
proteins, including transporters and many enzymes involved in energyconserving metabolism, lipid synthesis, protein export, and cell division.
The outer membrane of E. coli has a different function (protection) and a
different set of proteins.
Some membrane proteins are covalently linked to complex arrays of
carbohydrate. For example, in glycophorin, a glycoprotein of the erythrocyte plasma membrane, 60% of the mass consists of complex oligosaccharide units covalently attached to specific amino acid residues. Ser, Thr, and
Asn residues are the most common points of attachment (see Fig. 9–25). At
the other end of the scale is rhodopsin of the rod cell plasma membrane,
which contains a single hexasaccharide. The sugar moieties of surface glycoproteins influence the folding of the protein, as well as its stability and intracellular destination, and they play a significant role in the specific binding of ligands to glycoprotein surface receptors (see Fig. 9–29). Unlike
plasma membranes, intracellular membranes such as those of mitochondria and chloroplasts rarely contain covalently bound carbohydrates.
Some membrane proteins are covalently attached to one or more lipids,
which serve as hydrophobic anchors that hold the proteins to the membrane, as we shall see.
figure 12–3
Fluid mosaic model for membrane structure. The fatty
acyl chains in the interior of the membrane form a fluid,
hydrophobic region. Integral proteins float in this sea of
lipid, held by hydrophobic interactions with their nonpolar
amino acid side chains. Both proteins and lipids are free
to move laterally in the plane of the bilayer, but movement
of either from one face of the bilayer to the other is
restricted. The carbohydrate moieties attached to some
proteins and lipids of the plasma membrane are exposed
on the extracellular face of the membrane.
The Supramolecular Architecture of Membranes
All biological membranes share certain fundamental properties. They are
impermeable to most polar or charged solutes, but permeable to nonpolar
compounds; they are 5 to 8 nm (50 to 80 Å) thick and appear trilaminar
when viewed in cross section with the electron microscope (Fig. 12–1). The
combined evidence from electron microscopy and studies of chemical composition, as well as physical studies of permeability and the motion of individual protein and lipid molecules within membranes, led to the development of the fluid mosaic model for the structure of biological membranes
(Fig. 12–3). Phospholipids and sterols form a lipid bilayer in which the nonpolar regions of the lipid molecules face each other at the core of the bilayer
and their polar head groups face outward. In this bilayer sheet, proteins are
Oligosaccharide
chains of
glycoprotein
Outside
Glycolipid
Lipid
bilayer
Nonpolar
fatty acyl
chains
Phospholipid
polar heads
Sterol
Inside
Peripheral
protein
Integral protein
(single transmembrane helix)
Peripheral protein
covalently linked
to lipid
Integral protein
(multiple transmembrane helices)
392
Part II
Structure and Catalysis
embedded at irregular intervals, held by hydrophobic interactions between
the membrane lipids and hydrophobic domains in the proteins. Some proteins protrude from only one side of the membrane; others have domains
exposed on both sides. The orientation of proteins in the bilayer is asymmetric, giving the membrane “sidedness”; the protein domains exposed on
one side of the bilayer are different from those exposed on the other side,
reflecting functional asymmetry. The individual lipid and protein units in a
membrane form a fluid mosaic whose pattern, unlike a mosaic of ceramic
tile and mortar, is free to change constantly. The membrane mosaic is fluid
because most of the interactions among its components are noncovalent,
leaving individual lipid and protein molecules free to move laterally in the
plane of the membrane.
We now look at some of these features of the fluid mosaic model in more
detail and consider the experimental evidence that confirms the model.
A Lipid Bilayer Is the Basic Structural Element of Membranes
figure 12–4
Amphipathic lipid aggregates that form in water. (a) In
micelles, the hydrophobic chains of the fatty acids are
sequestered at the core of the sphere. There is virtually
no water in the hydrophobic interior. (b) In an open
bilayer, all acyl side chains except those at the edges
of the sheet are protected from interaction with water.
(c) When a two-dimensional bilayer folds on itself, it forms
a closed bilayer, a three-dimensional hollow vesicle (liposome) enclosing an aqueous cavity.
Individual units are
wedge-shaped
(cross-section of head
greater than that
of side chain)
Micelle
(a)
Glycerophospholipids, sphingolipids, and sterols are virtually insoluble in
water. When mixed with water, they spontaneously form microscopic lipid
aggregates in a phase separate from their aqueous surroundings, clustering
together with their hydrophobic moieties in contact with each other and
their hydrophilic groups interacting with the surrounding water. Recall that
lipid clustering reduces the amount of hydrophobic surface exposed to water and thus minimizes the number of molecules in the shell of ordered water at the lipid-water interface (see Fig. 4–7), resulting in an increase in entropy. Hydrophobic interactions among lipid molecules provide the
thermodynamic driving force for the formation and maintenance of these
clusters.
Depending on the precise conditions and the nature of the lipids, three
types of lipid aggregates can form when amphipathic lipids are mixed with
water (Fig. 12–4). Micelles are spherical structures containing a few dozen
to a few thousand molecules arranged with their hydrophobic regions aggregated in the interior, excluding water, and their hydrophilic head groups
at the surface, in contact with water. Micelle formation is favored when the
cross-sectional area of the head group is greater than that of the acyl side
chain(s), as in free fatty acids, lysophospholipids (phospholipids lacking one
fatty acid), and detergents such as sodium dodecyl sulfate (SDS; p. 134).
Individual units are
cylindrical (cross-section
of head equals that of side chain)
Bilayer
(b)
Aqueous
cavity
Liposome
(c)
Chapter 12 Biological Membranes and Transport
393
A second type of lipid aggregate in water is the bilayer, in which two
lipid monolayers form a two-dimensional sheet. Bilayer formation occurs
most readily when the cross-sectional areas of the head group and acyl side
chain(s) are similar, as in glycerophospholipids and sphingolipids. The hydrophobic portions in each monolayer, excluded from water, interact with
each other. The hydrophilic head groups interact with water at each surface
of the bilayer. Because the hydrophobic regions at its edges (Fig. 12–4b)
are transiently in contact with water, the bilayer sheet is relatively unstable
and spontaneously forms a third type of aggregate: it folds back on itself to
form a hollow sphere called a vesicle or liposome. By forming vesicles, bilayers lose their hydrophobic edge regions, achieving maximal stability in
their aqueous environment. These bilayer vesicles enclose water, creating a
separate aqueous compartment. It is likely that the first living cells resembled liposomes, their aqueous contents segregated from the rest of the
world by a hydrophobic shell.
All evidence indicates that biological membranes are constructed of
lipid bilayers. The thickness of membranes, measured by electron microscopy, is about that expected for a lipid bilayer 3 nm (30 Å) thick with
proteins protruding on each side. X-ray diffraction studies of membranes
show the distribution of electron density expected for a bilayer structure.
And liposomes formed in the laboratory show the same relative impermeability to polar solutes as is seen in biological membranes (although the latter, as we shall see, are permeable to solutes for which they have specific
transporters).
Membrane lipids are asymmetrically distributed between the two
monolayers of the bilayer, although the asymmetry, unlike that of membrane proteins, is not absolute. In the plasma membrane, for example, certain lipids are typically found primarily in the outer monolayer and others
in the inner (cytosolic) monolayer, but rarely is a lipid found only on one
side, not the other (Fig. 12–5).
Membrane phospholipid
Percent of
total
membrane
phospholipid
Distribution in
membrane
100
Inner
monolayer
0
Outer
monolayer
100
Phosphatidylethanolamine
30
Phosphatidylcholine
27
figure 12–5
Sphingomyelin
23
Phosphatidylserine
15
Asymmetric distribution of phospholipids between the
inner and outer monolayers of the erythrocyte plasma
membrane. The distribution of a specific phospholipid is
determined by treating the intact cell with phospholipase
C, which cannot reach lipids in the inner monolayer but
removes the head groups of lipids in the outer monolayer.
(Other membrane-impermeant reagents could also be
used; see Fig. 12 – 9.) The proportion of each head group
released provides an estimate of the fraction of each lipid
in the outer monolayer.
Phosphatidylinositol
Phosphatidylinositol 4-phosphate
Phosphatidylinositol 4,5-bisphosphate
Phosphatidic acid
5
394
Part II
Structure and Catalysis
Membrane Lipids Are in Constant Motion
Paracrystalline
state (solid)
(a)
Heat produces thermal
motion of acyl side chains
(solid → fluid transition)
Fluid
state
(b)
Lateral diffusion
in plane of bilayer
(c)
Transbilayer diffusion (“flip flop”)
t1/2 = hours to days (uncatalyzed)
= seconds (flippase catalyzed)
Although the lipid bilayer structure itself is stable, the individual phospholipid and sterol molecules have great freedom of motion within the plane of
the membrane (Fig. 12–6). The interior of the bilayer is fluid; individual hydrocarbon chains of fatty acids are in constant motion produced by rotation
about the carbon-carbon bonds of the long acyl side chains. The degree of
fluidity depends on lipid composition and temperature. At low temperature,
relatively little lipid motion occurs and the bilayer exists as a nearly crystalline (paracrystalline) array. Above a certain temperature, lipids can undergo rapid motion. The transition temperature, the temperature above
which the paracrystalline solid changes to fluid, is characteristic for each
membrane and depends on its lipid composition. Saturated fatty acids pack
well into a paracrystalline array, but the kinks in unsaturated fatty acids
(see Fig. 11–1) interfere with this packing, hindering the formation of a
paracrystalline state. The higher the proportion of saturated fatty acids, the
higher is the transition temperature of the membrane.
The sterol content of a membrane is another important determinant of
transition temperature. The rigid planar structure of the steroid nucleus, inserted between fatty acyl side chains, has two effects on fluidity. Below the
membrane’s transition temperature, sterol insertion prevents the highly ordered packing of fatty acyl chains and thus increases membrane fluidity.
Above the thermal transition point, the rigid ring system of the sterol reduces the freedom of neighboring fatty acyl chains to move by rotation
about their carbon-carbon bonds and thus reduces the fluidity in the core
of the bilayer. Sterols therefore tend to moderate the extremes of solidity
and fluidity of membranes.
Cells regulate their lipid composition to achieve a constant membrane
fluidity under various growth conditions. For example, bacteria synthesize
more unsaturated fatty acids and fewer saturated ones when cultured at
low temperatures than when cultured at higher temperatures (Table 12–2).
As a result of this adjustment in lipid composition, membranes of bacteria cultured at high or low temperatures have about the same degree of
fluidity.
A second type of lipid motion involves not merely the flexing of fatty
acyl chains but the movement of an entire lipid molecule relative to its
neighbors (Fig. 12–6b). A molecule in one monolayer, or face, of the lipid
bilayer—the outer face of the erythrocyte plasma membrane, for example—
can diffuse laterally so fast that it circumnavigates the erythrocyte in seconds. This rapid lateral diffusion within the plane of the bilayer randomizes
the positions of individual molecules in a few seconds. The combination of
acyl chain flexing and lateral diffusion produces a membrane bilayer with
the properties of a liquid crystal: a high degree of regularity in one dimension (perpendicular to the bilayer) and great mobility in the other (the
plane of the bilayer).
A third kind of lipid motion, much less probable than conformational
motion or lateral diffusion, is transbilayer or “flip-flop” diffusion of a molecule from one face of the bilayer to the other (Fig. 12–6c). This motion
figure 12–6
Motion of membrane lipids. Lipid motion within a bilayer
includes (a) thermal motion of the fatty acyl groups in the
bilayer interior and (b) lateral diffusion of individual molecules in one face of the bilayer. A single lipid molecule
can diffuse from one end of an E. coli cell to the other in
about one second at 37 C. Because of the thermal
motion, the bilayer is fluid above a certain transition temperature; as temperature decreases, the bilayer becomes
paracrystalline. (c) Movement of individual lipid molecules between the two faces of the bilayer (flip-flop diffusion) is very slow unless catalyzed by a specific lipid
transporter, or flippase. Random distribution of the lipids
in one monolayer between the two monolayers takes
hours if uncatalyzed, but only seconds when a flippase is
present.
Chapter 12 Biological Membranes and Transport
table 12–2
Human
cell
395
Mouse
cell
Fatty Acid Composition of E. coli Cells Cultured
at Different Temperatures
Percentage of total fatty acids*
Myristic acid (14:0)
Palmitic acid (16:0)
Palmitoleic acid (16:1)
Oleic acid (18:1)
Hydroxymyristic acid
Ratio of unsaturated to saturated†
10 C
20 C
30 C
40 C
4
18
26
38
13
2.9
4
25
24
34
10
2.0
4
29
23
30
10
1.6
8
48
9
12
8
0.38
Artificially
induced
cell fusion
Source: Data from Marr, A.G. & Ingraham, J.L. (1962) Effect of temperature on the composition
of fatty acids in Escherichia coli. J. Bacteriol. 84, 1260.
*The exact fatty acid composition depends not only on growth temperature but on growth stage
and growth medium composition.
†
Calculated as the total percentage of 161 plus 181 divided by the total percentage of 140
plus 160. Hydroxymyristic acid was omitted from this calculation.
requires a polar (and possibly charged) head group to leave its aqueous environment and move into the hydrophobic interior of the bilayer, a process
with a large, positive free-energy change. Yet this highly endergonic event
is sometimes necessary, for example during synthesis of the bacterial
plasma membrane, when phospholipids produced on the inside surface of
the membrane must undergo flip-flop diffusion to enter the outer face of the
bilayer. Similar transbilayer diffusion must also occur in eukaryotic cells as
membrane lipids synthesized in one compartment move to other organelles.
A family of proteins (flippases) facilitates flip-flop diffusion, providing a
transmembrane path that is energetically more favorable than uncatalyzed
diffusion.
Membrane Proteins Diffuse Laterally in the Bilayer
Many membrane proteins behave as though they were afloat in a sea of
lipids. Like membrane lipids, these proteins are free to diffuse laterally in
the plane of the bilayer and are in constant motion, as shown by the experiment diagrammed in Figure 12–7. Some membrane proteins associate to
form large aggregates (“patches”) on the surface of a cell or organelle, in
which individual protein molecules do not move relative to one another;
for example, acetylcholine receptors (see Fig. 12–39) form dense patches
on neuron plasma membranes at synapses. Other membrane proteins are
anchored to internal structures that prevent their free diffusion. In the
erythrocyte membrane, both glycophorin and the chloride-bicarbonate
exchanger (p. 413) are tethered to a filamentous cytoskeletal protein,
spectrin (Fig. 12–8).
figure 12–8
Restricted motion of the erythrocyte chloride-bicarbonate exchanger. The protein spans the membrane and
is tethered to the cytoskeletal protein spectrin by another
protein, ankyrin, limiting the exchanger’s lateral mobility.
Ankyrin is anchored in the membrane by a covalently
bound palmitoyl side chain (see Fig. 12–13). Spectrin, a
long, filamentous protein, is cross-linked at junctional
complexes containing actin. A network of cross-linked
spectrin molecules attached to the cytoplasmic face of
the plasma membrane stabilizes the membrane against
deformation.
Lateral diffusion
of membrane
proteins
figure 12–7
Demonstration of lateral diffusion of membrane
proteins. Fusion of a mouse cell with a human cell
results in the randomization of membrane proteins from
the two cells. After fusion, the location of each type of
membrane protein is determined by staining cells with
species-specific antibodies. Anti-mouse and anti-human
antibodies are specifically tagged with molecules that
fluoresce with different colors. Observed with the fluorescence microscope, the colored antibodies are seen to mix
on the surface of the hybrid cell within minutes after
fusion, indicating rapid lateral diffusion of the membrane
proteins throughout the lipid bilayer.
Chloride-bicarbonate
exchange proteins
Plasma
membrane
Ankyrin
Spectrin
Junctional complex
(actin)
Inside
Outside
396
Part II
box 12–1
Structure and Catalysis
Looking at Membranes
Electron microscopy combined with techniques
for preparing and staining tissues has been essential to studies of membrane structure. When
thin sections of tissue are stained with osmium
tetroxide, which accentuates differences in electron density, transmission electron microscopy shows the trilaminar structure (see Fig.
12–1). This level of resolution does not reveal individual membrane proteins, however. When a
tissue sample is quick-frozen (to avoid the distortion that results when ice crystals form within
cells) then cut with a fine knife, fracture lines
frequently run along the surface of a membrane
or through its center, splitting the bilayer into
two monolayers (Fig. 1). The surfaces exposed
by this freeze-fracture technique can be
coated with a thin layer of carbon (evaporated
from a carbon electrode under vacuum). Scanning electron microscopy of the resulting carbon “replica” shows details in bas relief of the
membrane surface and the inside face of one
monolayer exposed by splitting of the bilayer
(Fig. 2). A variety of particles with the dimensions of single protein molecules or protein complexes protrude from a featureless background
(the lipid bilayer). In many membranes, the particles are randomly positioned, but in specialized
regions such as the postsynaptic membrane,
they are clustered and ordered. In the myelin
sheath, the particles are sparsely distributed. In
membranes rich in enzyme complexes—the thylakoid membranes of chloroplasts, for instance—
the surface is virtually filled with particles.
500 nm
75 nm
figure 2
figure 1
Splitting of a membrane bilayer by
the freeze-fracture technique.
An erythrocyte membrane is frozen in water, etched (by
sublimation of some water), and a carbon replica is
viewed in the electron microscope. The replica shows
integral membrane proteins as bumps of various sizes
and shapes, which are more readily visible at higher
magnification (inset).
Some Membrane Proteins Span the Lipid Bilayer
The individual protein molecules and multiprotein complexes of biological
membranes can be visualized by electron microscopy of the freezefractured membranes (Box 12–1). Some proteins appear on only one face
of the membrane; others, which span the full thickness of the bilayer, protrude from both inner and outer membrane surfaces. Among the latter are
proteins that conduct solutes or signals across the membrane.
Membrane protein localization has also been investigated with reagents
that react with protein side chains but cannot cross membranes (Fig. 12–9).
The human erythrocyte is convenient for such studies because it has no
Chapter 12 Biological Membranes and Transport
Erythrocyte membrane
containing three proteins
P1
EA
(permeant)
C
P3
14
14
C-labeled
IEA
HOCH2CH3
NH
CH3
P2
C-labeled
EA
OCH2CH3
NH
H2N
Protein
CH3
C
N
H
Protein
EA labels intracellular and
extracellular domains of proteins
NH
CH3
C
OCH2CH2SO
3
HOCH2CH2SO
3
IEA
(impermeant)
(a)
IEA labels only extracellular
domains of proteins
figure 12–9
Experiments to determine the transmembrane arrangement of membrane proteins. (a) Both ethylacetimidate
(EA) and isoethionylacetimidate (IEA) react with free
amino groups in proteins, but only the ethyl derivative
(EA) diffuses freely through the membrane. (b) Comparison of the labeling patterns after treatment of erythrocytes with the two reagents reveals whether a given
protein is exposed only on the outer surface or only on
the inner surface. A protein (P2) labeled by both
reagents, but more heavily by the permeant reagent (EA),
is exposed on both sides and thus spans the membrane.
Proteins (P1) not labeled by the impermeant reagent
(IEA) are exposed on the inner surface only.
membrane-bounded organelles; the plasma membrane is the only membrane present. If a membrane protein in an intact erythrocyte reacts with a
membrane-impermeant reagent (IEA in Fig. 12–9), it must have at least
one domain exposed on the outer (extracellular) face of the membrane.
Proteases such as trypsin are unable to cross membranes and are commonly
used to explore membrane protein topology, the disposition of protein domains relative to the lipid bilayer. Trypsin cleaves extracellular domains but
does not affect domains buried within the bilayer or exposed on the inner
surface only.
Experiments like those described in Figure 12–9 show that the glycoprotein glycophorin spans the erythrocyte membrane. Its amino-terminal
domain (bearing the carbohydrate) is on the outer surface and can be
cleaved by trypsin. The carboxyl terminus protrudes on the inside of the
IEA
Excess IEA, EA
removed;
membranes isolated,
dissolved in SDS;
proteins separated by
SDS polyacrylamide
gel electrophoresis
EA
P2
Radioactive proteins
detected by exposing
x-ray film to gel
(autoradiography)
P1
P3
(b)
397
398
Part II
Structure and Catalysis
1
figure 12–10
Transbilayer disposition of glycophorin in the
erythrocyte. One hydrophilic domain, containing all
the sugar residues, is on the outer surface, and another
hydrophilic domain protrudes from the inner face of the
membrane. The red hexagons represent a tetrasaccharide (containing two Neu5Ac (sialic acid), Gal, and
GalNAc) O-linked to a Ser or Thr residue; the blue
hexagon represents an oligosaccharide chain N-linked to
an Asn residue. The relative size of the oligosaccharide
units is larger than shown here. A segment of 19
hydrophobic residues (residues 75 to 93) forms an
a helix that traverses the membrane bilayer (see
Fig. 12–17a). The segment from residue 64 to 74 has
some hydrophobic residues and probably penetrates into
the outer face of the lipid bilayer.
Amino
terminus
Tyr
Ala
Ala
Thr Thr Thr Ser
Glu Val Ala Met His
Leu
Ser
Thr
Thr
Ser
Ser
Val
Ser
Thr Asp Arg Lys His Thr Asp
Asn Thr
Lys
Gln Ser Ser Ile
Tyr Ser
Ser Val
Ser Glu Ile
Thr Pro Arg Ala His Glu Val
60
74
Outside
Arg Thr Val
Tyr
Pro
Arg Glu
Glu Thr Glu Glu
Pro
Glu
Val
Thr
Gln
Leu
Ile Leu
Ile
Ile
Ala
Glu
Phe
His
Pro
Gly
Glu
His
Val
Met
Ser Phe
Ala
Gly
Val
Ile
Gly
Thr
Ile
Leu
Leu
Ile
Ser
Tyr
Gly
Ile
Ser
Pro
Ser Lys Lys
Arg
Arg
95
Inside
Ile Leu
Asp
Val
Lys
Pro Leu Pro
Ser
Pro Leu Ser Ser Val
Thr Asp Val
Pro Asp
Glu
Ile
Glu
Gln Asp Ser Thr Glu
Pro Asn
Carboxyl
terminus
131
cell. Both the amino-terminal and carboxyl-terminal domains contain many
polar or charged amino acid residues and are therefore quite hydrophilic.
However, a long segment in the center of the protein contains mainly hydrophobic amino acid residues. These findings suggest that glycophorin has
a transmembrane segment arranged as shown in Figure 12–10.
One further fact may be deduced from the results of the experiments
with glycophorin: its disposition in the membrane is asymmetric. Similar
studies of other membrane proteins show that each has a specific orientation in the bilayer and that proteins reorient by flip-flop diffusion very
slowly, if at all. Furthermore, glycoproteins of the plasma membrane are invariably situated with their sugar residues on the outer surface of the cell.
As we shall see, the asymmetric arrangement of membrane proteins results
in functional asymmetry. All the molecules of a given ion pump, for example, have the same orientation in the membrane and therefore pump in the
same direction.
Peripheral Membrane Proteins Are Easily Solubilized
Membrane proteins may be divided into two operational groups (Fig.
12–11). Integral (intrinsic) proteins are very firmly associated with the
membrane, removable only by agents that interfere with hydrophobic interactions, such as detergents, organic solvents, or denaturants. Peripheral
(extrinsic) proteins associate with the membrane through electrostatic interactions and hydrogen bonding with the hydrophilic domains of integral
proteins and with the polar head groups of membrane lipids. They can
Chapter 12 Biological Membranes and Transport
figure 12–11
Peripheral
protein
Peripheral and integral proteins. Membrane proteins
can be operationally distinguished by the conditions
required to release them from the membrane. Most
peripheral proteins can be released by changes in pH or
ionic strength, removal of Ca2 by a chelating agent, or
addition of urea or carbonate. Peripheral proteins covalently attached to a membrane lipid, such as a glycosyl
phosphatidylinositol (see Fig. 12–13), can be released by
treatment with phospholipase C. Integral proteins can be
extracted with detergents, which disrupt the hydrophobic
interactions with the lipid bilayer and form micelle-like
clusters around individual protein molecules.
change in pH;
chelating agent;
urea; CO32
399
Ca2
detergent
phospholipase C
Lipid-linked
protein
Protein–glycan
Integral protein
(hydrophobic domain
coated with detergent)
be released by relatively mild treatments that interfere with electrostatic
interactions or break hydrogen bonds. Peripheral proteins may serve as
regulators of membrane-bound enzymes or may limit the mobility of integral proteins by tethering them to intracellular structures.
Annexins, for example, are a family of peripheral membrane proteins
that bind reversibly to acidic membrane phospholipids, such as those that
enrich the cytosolic (inner) surface of plasma membranes and the cytosolic (outer) face of organelles. Ca2 ions, which interact simultaneously
with negatively charged groups on annexin and the negatively charged head
group of a phospholipid, are essential to the annexin-membrane interaction
(Fig. 12–12). Roles for various annexins have been suggested in vesicle aggregation, membrane fusion, and interactions between membranes and cytoskeletal elements. A number of other proteins that act at a membrane–
aqueous solution interface (blood-clotting proteins, for example) also
undergo reversible Ca2-dependent membrane binding, perhaps by a similar mechanism.
figure 12–12
Proposed structure of annexin V in association with the
lipid head groups of a membrane. In this image, the
protein backbone of annexin V (derived from crystallographic studies) is seen through a transparent mesh of
the surface contour. The membrane modeled here is
composed entirely of phosphocholine, with the choline
moiety shown in blue, the phosphate groups red, and the
fatty acid tails yellow. The bridging Ca2 ions are purple.
Electrostatic interactions hold annexin to the membrane.
400
Part II
Structure and Catalysis
Covalently Attached Lipids Anchor Some
Peripheral Membrane Proteins
figure 12–13
Lipid-linked membrane proteins. Covalently attached
lipids anchor membrane proteins to the lipid bilayer. A
palmitoyl group is shown attached by thioester linkage to
a Cys residue; an N-myristoyl group is generally attached
to an amino-terminal Gly; the farnesyl and geranylgeranyl
groups attached to carboxyl-terminal Cys residues are isoprenoids of 15 and 20 carbons, respectively. These
three lipid-protein assemblies are found only on the
inside surface of the plasma membrane. Glycosyl
phosphatidylinositol (GPI) anchors are derivatives of
phosphatidylinositol in which the inositol bears a short
oligosaccharide covalently joined to the carboxyl-terminal
residue of a protein through phosphoethanolamine. GPIlinked proteins are always on the extracellular surface of
the plasma membrane.
Some membrane proteins contain one or more covalently linked lipids of
several types: long-chain fatty acids, isoprenoids, or glycosylated derivatives of phosphatidylinositol, GPI (Fig. 12–13). The attached lipid provides
a hydrophobic anchor, which inserts into the lipid bilayer and holds the protein at the membrane surface. The strength of the hydrophobic interaction
between a bilayer and a single hydrocarbon chain linked to a protein is
barely enough to anchor the protein securely. Other interactions, such as
ionic attractions between positively charged Lys residues in the protein and
negatively charged lipid head groups, probably stabilize the attachment.
The association of these lipid-linked proteins with the membrane is certainly weaker than that for integral membrane proteins and is, in at least
some cases, reversible.
Beyond merely anchoring a protein to the membrane, the attached lipid
may have a more specific role. In the plasma membrane, proteins with GPI
anchors are exclusively on the outer (extracellular) face, whereas other
types of lipid-linked proteins (Fig. 12–13) are found exclusively on the inner (cytosolic) face. In epithelial cells, GPI-linked membrane proteins are
far more common on the apical surface (the surface facing the external environment or interior cavity) than on the basal surface (the adjoining lowerlayer epithelium). The attachment of a specific lipid to a membrane protein
may therefore have a targeting function, directing the newly synthesized
protein to its correct membrane location. The association of lipid-linked
proteins with the membrane may be mediated by specific membrane
receptors.
NH3
O
C
Cys
CH2
S
COO
Palmitoyl group on
internal Cys (or Ser)
O
O
C
C
CH2
N
H
Integral Proteins Are Held in the Membrane
by Hydrophobic Interactions with Lipids
COO
The firm attachment of integral proteins to membranes is the result of hydrophobic interactions between membrane lipids and hydrophobic domains
of the protein. Some proteins have a single hydrophobic sequence in the middle (glycophorin, for example) or at the amino or carboxyl terminus. Others
have multiple hydrophobic sequences, each of which, when in the a-helical
conformation, is long enough to span the lipid bilayer (Fig. 12–14). The same
techniques that have allowed us to determine the three-dimensional
N-Myristoyl group on
amino-terminal Gly
O
CH3O
C
CH
CH2
S
NH
NH
3
O
Farnesyl (or geranylgeranyl)
group on
carboxyl-terminal Cys
O
P
O
Inositol
O
GlcNAc
O
Man
O
CH2
Man
C
O
CH2
C
O
CH2
Man
Man
NH
3
O
O
P
O
O
GPI anchor on
carboxyl terminus
Inside
Outside
CH2
CH2
NH
C
O
Chapter 12 Biological Membranes and Transport
401
figure 12–14
Integral membrane proteins. For known proteins of the
plasma membrane, the spatial relationships of protein
domains to the lipid bilayer fall into six categories. Types I
and II have only one transmembrane helix; the aminoterminal domain is outside the cell in type I proteins and
inside in type II. Type III proteins have multiple transmembrane helices in a single polypeptide. In type IV proteins, transmembrane domains of several different
polypeptides assemble to form a channel through the
membrane. Type V proteins are held to the bilayer primarily by covalently linked lipids (see Fig. 12–13), and
type VI proteins have both transmembrane helices and
lipid (GPI) anchors.
In this figure, and in other figures throughout the
book, we represent transmembrane protein segments in
their most likely conformations: as a helices of six to
seven turns. Sometimes these helices will be shown
simply as cylinders. As relatively few membrane protein
structures have been deduced by x-ray crystallography,
our representation of the extra-membrane domains is
arbitrary and not necessarily to scale, unless otherwise
noted.
structures of many soluble proteins can in principle be applied to membrane
proteins. However, generally useful techniques for crystallizing membrane
proteins have only recently been developed, and relatively few membrane
protein structures have been solved crystallographically.
One of the best-studied membrane-spanning proteins, bacteriorhodopsin, has seven very hydrophobic internal sequences and crosses the lipid
bilayer seven times. Bacteriorhodopsin is a light-driven proton pump densely
packed in regular arrays in the purple membrane of the bacterium Halobacterium halobium. When these arrays are viewed with the electron microscope from several angles, the resulting images allow a three-dimensional
reconstruction of the bacteriorhodopsin molecule. X-ray crystallography
yields a structure similar in all essential features (Fig. 12–15). Seven ahelical segments, each traversing the lipid bilayer, are connected by nonhelical loops at the inner and outer face of the membrane. In the amino acid
sequence of bacteriorhodopsin, seven segments of about 20 hydrophobic
residues can be identified, each segment just long enough to make an a helix that spans the bilayer. Hydrophobic interactions between the nonpolar
amino acids and the fatty acyl groups of the membrane lipids firmly anchor
the protein in the membrane. The seven helices are clustered together and
oriented roughly perpendicular to the bilayer plane, providing a transmembrane pathway for proton movement. As we shall see in Chapter 13, this pattern of seven hydrophobic membrane-spanning helices is a common motif in
membrane proteins involved in signal reception.
+
NH3
Type I
–OOC
COO–
+
Type II
H3N
Type III
Type IV
Type V
Type VI
Inside
Outside
Outside
Amino
terminus
figure 12–15
Bacteriorhodopsin, a membrane-spanning protein. The
single polypeptide chain folds into seven hydrophobic
a helices, each of which traverses the lipid bilayer roughly
perpendicular to the plane of the membrane. The seven
transmembrane helices are clustered, and the space
around and between them is filled with the acyl chains of
membrane lipids. The light-absorbing pigment retinal
(see Fig. 11–19) is buried deep in the membrane in
contact with several of the helical segments (not shown).
The helices are colored to correspond with the
hydropathy plot in Figure 12–17b.
Inside
Carboxyl
terminus
402
Part II
Structure and Catalysis
The photosynthetic reaction center from a purple bacterium was the
first membrane protein structure solved by crystallography. Although a
more complex membrane protein than bacteriorhodopsin, it is constructed
on the same structural principles. The reaction center has four protein subunits, three of which contain a-helical segments that span the membrane
(Fig. 12–16). These segments are rich in nonpolar amino acids, with their
hydrophobic side chains oriented toward the outside of the molecule where
they interact with membrane lipids. The architecture of the reaction center
protein is therefore the inverse of that seen in most water-soluble proteins,
in which hydrophobic residues are buried within the protein core and hydrophilic residues are on the surface (recall the structures of myoglobin and
hemoglobin, for example). In Chapter 19 we will encounter several complex
membrane proteins having multiple transmembrane helical segments in
which hydrophobic chains are positioned to interact with the lipid bilayer.
figure 12–16
Outside
Inside
Three-dimensional structure of the photosynthetic
reaction center of Rhodopseudomonas viridis, a purple
bacterium. This was the first integral membrane protein
to have its atomic structure determined by x-ray diffraction methods. Eleven a-helical segments from three of the
four subunits span the lipid bilayer, forming a cylinder
45 Å (4.5 nm) long; hydrophobic residues on the exterior
of the cylinder interact with lipids of the bilayer. In this
ribbon representation, residues that are part of the transmembrane helices are shown in yellow. The prosthetic
groups (light-absorbing pigments and electron carriers;
see Fig. 19– 45) are red.
The Topology of an Integral Membrane Protein Can
Sometimes Be Predicted from Its Sequence
Determining the three-dimensional structure of a membrane protein or its
topology is generally much more difficult than determining its amino acid
sequence, which can be accomplished by sequencing the protein or its
gene. More than a thousand sequences are known for membrane proteins,
but relatively few three-dimensional structures. The presence of long hydrophobic sequences in a membrane protein is commonly taken as evidence
that these sequences traverse the lipid bilayer, acting as hydrophobic anchors or forming transmembrane channels. Virtually all integral proteins
have at least one such sequence.
What can we predict about the secondary structure of the membranespanning portions of integral proteins? An a-helical sequence of 20 to 25
residues is just long enough to span the thickness (30 Å) of the lipid bilayer
(recall that the length of an a helix is 1.5 Å (0.15 nm) per amino acid
residue). A polypeptide chain surrounded by lipids, having no water molecules with which to form hydrogen bonds, will tend to form a helices or b
sheets, in which intrachain hydrogen bonding is maximized. If the side
chains of all amino acids in a helix are nonpolar, hydrophobic interactions
with the surrounding lipids further stabilize the helix.
Several simple methods of analyzing amino acid sequences yield reasonably accurate predictions of secondary structure for transmembrane
proteins. The relative polarity of each amino acid has been determined experimentally by measuring the free-energy change accompanying the
movement of that amino acid’s side chain from a hydrophobic solvent into
water. This free energy of transfer ranges from very exergonic for charged
or polar residues to very endergonic for amino acids with aromatic or
Chapter 12 Biological Membranes and Transport
50
100
130
Hydrophobic
Hydrophilic
0
3
0
50
100
Residue number
130
Glycophorin
(a)
Hydropathy index
Hydropathy index
0
3
10
3
50
100
3
2
1
403
150
200
5
4
6
250
7
Hydrophobic
Hydrophilic
0
3
10
50
100
150
200
250
Residue number
Bacteriorhodopsin
(b)
aliphatic hydrocarbon side chains. The overall hydrophobicity of a sequence
of amino acids is estimated by summing the free energies of transfer for the
residues in the sequence, which yields a hydropathy index for that region
(see Table 5–1). To scan a polypeptide sequence for potential membranespanning segments, one calculates the hydropathy index for successive segments (called windows) of a given size, which may be from seven to 20
residues. For a window of seven residues, for example, the indices for
residues 1 to 7, 2 to 8, 3 to 9, and so on, are plotted as in Figure 12–17 (plotted for the middle residue in each window—residue 4 for the window 1 to 7,
for example). A region with more than 20 residues of high hydropathy index is presumed to be a transmembrane segment. When the sequences of
membrane proteins of known three-dimensional structure are scanned in
this way, we find a reasonably good correspondence between predicted and
known membrane-spanning segments. Hydropathy analysis predicts a single hydrophobic helix for glycophorin (Fig. 12–17a) and seven transmembrane segments for bacteriorhodopsin (Fig. 12–17b)—in agreement with
experimental studies.
Many of the transport proteins described in this chapter are believed,
on the basis of their amino acid sequences and hydropathy plots, to have
multiple membrane-spanning helical regions—that is, they are type III or
type IV integral proteins (Fig. 12–14). When predictions are consistent with
chemical studies of protein localization (such as those described above for
glycophorin and bacteriorhodopsin), the assumption that hydrophobic regions correspond to membrane-spanning domains is better justified.
Not all integral membrane proteins are composed of transmembrane a
helices. Another structural motif common in membrane proteins is the b
barrel (see Fig. 6–20d), in which 20 or more transmembrane segments form
b sheets that line a cylinder (Fig. 12–18). The same factors that favor ahelix formation in the hydrophobic interior of a lipid bilayer also stabilize b
barrels. When no water molecules are available to hydrogen bond with the
carbonyl oxygen and nitrogen of the peptide bond, maximal intrachain hydrogen bonding gives the most stable conformation. Porins, proteins that allow certain polar solutes to cross the outer membrane of gram-negative
bacteria such as E. coli, have many-stranded b barrels lining the polar
transmembrane passage.
figure 12–18
Porin FhuA, an integral membrane protein with b-barrel
structure. This protein of the E. coli outer membrane
includes a b barrel composed of 22 antiparallel b strands
(gray), forming a transmembrane channel through which
iron ion bound to the carrier ferrichrome enters from the
surrounding medium. The residues on the outside surface
of the barrel are hydrophobic and interact with the lipids
and lipopolysaccharides in the outer membrane.
figure 12–17
Hydropathy plots. Hydropathy index (see Table 5–1) is
plotted against residue number for two integral membrane proteins. The hydropathy index for each amino
acid residue in a sequence of defined length (called the
window) is used to calculate the average hydropathy for
the residues in that window. The horizontal axis shows the
residue number for the middle of the window. (a) Glycophorin from human erythrocytes has a single
hydrophobic sequence between residues 75 and 93
(yellow); compare this with Figure 12–10. (b) Bacteriorhodopsin, known from independent physical studies
to have seven transmembrane helices (see Fig. 12–15),
has seven hydrophobic regions. Note, however, that the
hydropathy plot is ambiguous in the region of segments
6 and 7. Physical studies have confirmed that this region
has two transmembrane segments.
404
Part II
Structure and Catalysis
A polypeptide is more extended in the b conformation than in an a helix; just seven to nine residues of b conformation are needed to span a membrane. Recall that in the b conformation, alternating side chains project
above and below the sheet (see Fig. 6–7). In b strands of membrane proteins, every second residue in the membrane-spanning segment is hydrophobic and interacts with the lipid bilayer; aromatic side chains are commonly found at the lipid-protein interface. The other residues may or may
not be hydrophilic. The hydropathy plot is not useful in predicting transmembrane segments for proteins with b barrel motifs, but as the database
of known b barrel motifs increases, sequence-based predictions of transmembrane b sheets become more feasible.
Integral Proteins Mediate Cell-Cell Interactions and Adhesion
Several families of integral proteins in the plasma membrane provide specific points of attachment between cells, or between a cell and extracellular
matrix proteins. Integrins are heterodimeric proteins (with two unlike
subunits, a and b) anchored to the plasma membrane by a single hydrophobic transmembrane helix in each subunit (Fig. 12–19). The large extracellular domains of the a and b subunits combine to form a specific binding site for extracellular proteins such as collagen and fibronectin. As there
are at least 14 different a subunits and at least eight different b subunits, a
wide variety of specificities may be generated from various combinations of
a and b. One common determinant of integrin binding present in several extracellular partners of integrins is the sequence Arg–Gly–Asp (RGD).
Integrins are not merely adhesives; they serve as receptors and signal
transducers, carrying information across the plasma membrane in both directions. Integrins regulate many processes, including platelet aggregation
at the site of a wound, tissue repair, the activity of immune cells, and the
invasion of tissue by a tumor (see Fig. 9–27). Mutation in an integrin gene
encoding the b subunit known as CD18 is the cause of leukocyte adhesion
deficiency in humans, a rare genetic disease in which leukocytes fail to pass
out of blood vessels to reach sites of infection. Infants with a severe defect
in CD18 commonly die of infections before the age of two.
Immunoglobulin-like
domains
Ligand-binding
region
Adhesive
domain
S
S
S
S
Ca2
figure 12–19
Four examples of integral protein types that function in
cell-cell interactions. Integrins consist of a and b transmembrane polypeptides; their extracellular domains
combine to form binding sites for divalent metal ions and
proteins of the extracellular matrix (such as collagen and
fibronectin) or specific surface proteins of other cells.
Cadherin has four extracellular Ca2-binding domains, the
most distal of which contains the site that binds to cadherin on another cell surface. N-CAM (neuronal cell
adhesion molecule) is one of a family of immunoglobulinlike proteins that mediate Ca2-independent interactions
with surface proteins of nearby cells. Selectins bind tightly
to carbohydrate moieties in neighboring cells; the binding
is Ca2-dependent.
b
a Ca
2
Ca2
Ca
Outside
Lectin domain
(binds carbohydrates)
Ca2
S
S
S
S
2
Ca2
S
S
Plasma
membrane
Inside
Integrin
Cadherin
N-CAM
Selectins
Chapter 12
Biological Membranes and Transport
At least three other families of plasma membrane proteins are also involved in surface adhesion (Fig. 12–19). Cadherins undergo homophilic
(“with same kind”) interactions with identical cadherins in an adjacent cell.
Immunoglobulin-like proteins can undergo either homophilic interactions with their identical counterparts on another cell or heterophilic interactions with an integrin on a neighboring cell. Selectins have extracellular
domains that, in the presence of Ca2, bind specific polysaccharides on the
surface of an adjacent cell. Selectins are present primarily in the various
types of blood cells and in the endothelial cells that line blood vessels. They
are an essential part of the blood-clotting process.
Integral proteins play a role in many other cellular processes. They
serve as transporters and ion channels (discussed later in this chapter) and
as receptors for hormones, neurotransmitters, and growth factors (Chapter
13). They are central to oxidative phosphorylation and photosynthesis
(Chapter 19) and to cell-cell and antigen-cell recognition in the immune
system (Chapter 7). Integral proteins are also major players in the membrane fusion that accompanies exocytosis, endocytosis, and the entry of
many types of viruses into host cells.
Membrane Fusion Is Central to Many Biological Processes
A remarkable feature of the biological membrane is its ability to undergo fusion with another membrane without losing its integrity. Although membranes are stable, they are by no means static. Within the endomembrane
system (see Fig. 2–9), the membranous compartments constantly reorganize as small vesicles bud from the Golgi complex to carry newly synthesized lipids and proteins to other organelles and to the plasma membrane.
Exocytosis, endocytosis, cell division, fusion of egg and sperm cells, and entry of a membrane-enveloped virus into its host cell all involve membrane
reorganization in which the fundamental operation is fusion of two membrane segments without loss of continuity (Fig. 12–20).
Specific fusion of two membranes requires that (1) they recognize each
other; (2) their surfaces become closely apposed, which requires the removal of water molecules normally associated with the polar head groups of
lipids; (3) their bilayer structures become locally disrupted; and (4) the two
bilayers fuse to form a single continuous bilayer. Receptor-mediated endocytosis or regulated secretion also requires that (5) the fusion process is
triggered at the appropriate time or in response to a specific signal. Integral
proteins called fusion proteins mediate these events, bringing about specific recognition and a transient, local distortion of the bilayer structure that
favors membrane fusion. (Note that these fusion proteins are unrelated to
the products of two fused genes, also called fusion proteins, as discussed in
Chapter 29).
One of the best-studied cases of membrane fusion occurs during infection of a host cell by a membrane-enveloped virus, such as the influenza
virus or HIV. Infection by influenza virus begins when an integral protein of
Budding of vesicles
from Golgi complex
Exocytosis
Endocytosis
Fusion of endosome
and lysosome
Viral infection
Fusion
of sperm and egg
Fusion of small
vacuoles (plants)
figure 12–20
Membrane fusion. The fusion of two membranes is
central to a variety of cellular processes involving both
organelles and the plasma membrane.
Separation of two
plasma membranes
at cell division
405
406
Part II
Structure and Catalysis
the virus envelope, the hemagglutination protein, binds to sialic acid
(Neu5Ac) residues of glycoproteins or glycolipids in the host cell’s plasma
membrane (Fig. 12–21a). Endocytosis then conveys the virus into the cell
within an endosome. The low pH in the endosome triggers a major alteration in the conformation of the hemagglutination protein, exposing a short
hydrophobic peptide called the fusion peptide. Insertion of the fusion peptide into the lipid bilayer of the endosomal membrane produces a bridge between viral and endosomal membranes. This presumably brings about local
distortion of both bilayers, leading to membrane fusion and release of the
viral contents into the host cell.
In HIV infection, a pair of glycoproteins in the HIV envelope (gp41 and
gp120) bind to two “receptors”—CD4 and a coreceptor protein—on the
surface of the host cell (Fig. 12–21b). CD4 alone is sufficient to allow binding of HIV to the cell, but for fusion (and thus infection) to occur the host
cell also must have a coreceptor such as CCR5. (CCR5 normally functions
as a receptor for growth factors called chemokines that direct leukocytes to
sites of inflammation.) The requirement for both CD4 and a coreceptor in
HIV infection explains the specificity of HIV for T lymphocytes and phagocytes of the immune system; they are the only cell types that have both proteins in their plasma membranes. Presumably, these “receptors” evolved to
serve functions useful to the cell, but the virus has evolved to exploit them.
Agents that interfere with the interaction of HIV with CD4 or the coreceptor may prove useful in the treatment of HIV infection or in the prevention
of progression of HIV infection to AIDS.
According to a current model for HIV entry by fusion with the plasma
membrane, as shown in Figure 12–21b, the gp41-gp120 complex is normally
anchored to the viral membrane by a hydrophobic sequence near the carboxyl terminus of gp41. Contact with CD4 and the coreceptor in the host
cell’s surface triggers a large conformational change in gp41, which
stretches into an extended structure and exposes the fusion peptide near
its amino terminus. This fusion peptide is inserted into the plasma membrane of the host cell; gp41 thus forms a bridge between virus and cell. A
second dramatic structural change now occurs: gp41 bends sharply at a
hinge region near its center, forming a hairpin-like structure that brings its
amino terminus (in the plasma membrane) close to its carboxyl terminus
(in the viral membrane). According to this model, the insertion of the fusion peptide into the plasma membrane somehow disorganizes the lipid bilayer locally. Realignment of lipid molecules in this disorganized region results in coalescence of the two membranes, and fusion occurs.
Membrane fusion is central to other cellular processes, too, such as the
movement of newly synthesized membrane components through the endomembrane system from the endoplasmic reticulum through the Golgi
complex to the plasma membrane via membrane vesicles, and the release of
proteins, hormones, or neurotransmitters by exocytosis. The proteins required for these membrane fusions, called SNARES, resemble the viral fusion proteins in several respects. SNAREs (the family name derives from
synaptosome-associated protein receptors) form hairpin-shaped rods that
bring together protein domains bound to the plasma membrane and the
vesicle membrane. SNAREs in the cytosolic face of the intracellular vesicles
are called v-SNAREs; those in the target membranes with which the vesicles fuse are t-SNAREs. The extended forms of SNAREs, like those of the
viral fusion proteins, have coiled coils, long regions in which several a helices are coiled about each other. These structural similarities very probably
reflect a fundamental similarity in fusion mechanisms in viruses and intracellular vesicles.
Chapter 12
Biological Membranes and Transport
407
figure 12–21
Membrane fusion during viral entry into a host cell. (a)
Influenza virus adheres to carbohydrate moieties in the
host cell’s surface (see Fig. 9–29b) and triggers endocytosis, which encloses the virus in an endosome. Proton
pumps (p. 416) acidify the endosome contents, causing a
pH-dependent change in the structure of the hemagglutination (HA) protein and thus exposing a hydrophobic
fusion peptide. The fusion peptide penetrates the endosomal membrane and brings about fusion of the viral and
endosomal membranes, releasing the viral genome into
the host cell. (b) When HIV encounters a T lymphocyte
with CD4 and a second coreceptor (cytokine receptor)
protein in its plasma membrane, the virus adheres to the
cell through two glycoproteins (gp41 and gp120) in its
own membrane, releasing the viral contents into the host
cell. HIV infects only those cells (lymphocytes) with CD4
in their plasma membrane.
Sialic acid
HIV
Influenza virus
HA protein
CCR5
HIV attaches to host
lymphocyte by interaction
between gp120 in HIV
envelope and CD4 in host
cell plasma membrane.
gp41
gp120
Viral membrane
Host cell
plasma
membrane
Interaction of HA
protein with sialic acid
in host cell membrane
triggers endocytosis.
Low pH in endosome
causes extension
of HA protein’s fusion
peptide, which
penetrates the
endosome membrane.
CD4
Host cell
plasma
membrane
Cytokine receptor (CCR5) in
host cell plasma membrane
binds CD4 and gp120,
allowing amino-terminal
membrane fusion domain
of gp41 to insert into host
cell membrane.
Amino
terminus
Conformational change
in gp41 yields hairpin
structure with coiled-coil
domain, bringing the two
membranes into contact.
Viral and endosomal
membranes fuse,
releasing viral
contents into host cell.
Fusion of HIV and host
cell membranes allows
HIV contents (RNA
genome and enzymes)
to enter host cell.
(a)
(b)
408
Part II
Structure and Catalysis
Solute Transport across Membranes
Every living cell must acquire from its surroundings the raw materials for
biosynthesis and for energy production, and must release to its environment the byproducts of metabolism. The plasma membrane contains proteins that specifically recognize and carry into the cell such necessities as
sugars, amino acids, and inorganic ions. In some cases, these components
are imported against a gradient of concentration, electrical charge, or
both—they are “pumped” in. Certain other materials are pumped out to
keep their concentrations in the cytosol lower than in the surrounding
medium. With few exceptions, the traffic of small molecules across the
plasma membrane is mediated by proteins such as transmembrane channels, carriers, or pumps. Within the eukaryotic cell, different compartments
have different concentrations of metabolic intermediates and products, and
these, too, must move across intracellular membranes in tightly regulated,
protein-mediated processes.
Passive Transport Is Facilitated by Membrane Proteins
figure 12–22
Movement of solutes across a permeable membrane.
(a) Net movement of electrically neutral solutes is toward
the side of lower solute concentration until equilibrium is
achieved. The solute concentrations on the left and right
sides of the membrane are designated C1 and C2. The
rate of transmembrane movement is proportional to the
concentration gradient, C1/C2, as indicated by the large
arrows. (b) Net movement of electrically charged solutes
is dictated by a combination of the electrical potential
(Vm) and the chemical concentration difference across
the membrane; net ion movement continues until this
electrochemical potential reaches zero.
C1
C1
C2
When two aqueous compartments containing unequal concentrations of a
soluble compound or ion are separated by a permeable divider (membrane), the solute moves by simple diffusion from the region of higher
concentration, through the membrane, to the region of lower concentration, until the two compartments have equal solute concentrations (Fig.
12–22a). When ions of opposite charge are separated by a permeable membrane, there is a transmembrane electrical gradient, the membrane potential, Vm (expressed in volts or millivolts). This membrane potential produces a force opposing ion movements that increase Vm and driving ion
movements that reduce Vm (Fig. 12–22b). Thus, the direction in which a
charged solute tends to move spontaneously across a membrane depends
on both the chemical gradient (the difference in solute concentration) and
the electrical gradient (Vm) across the membrane. Together, these two factors are referred to as the electrochemical gradient or the electrochemical potential. This behavior of solutes is in accord with the second
law of thermodynamics: molecules tend spontaneously to assume the distribution of greatest randomness—that is, entropy will increase and the energy of the system will be minimized.
In living organisms, simple diffusion of most solutes is impeded by selectively permeable barriers—membranes that separate intracellular
C2
C1 >> C2
C1 = C2
Vm > 0
Vm = 0
Before equilibrium
Net flux
At equilibrium
No net flux
Before equilibrium
At equilibrium
(a)
(b)
Biological Membranes and Transport
compartments and surround cells. To pass through a lipid bilayer, a polar or
charged solute must first give up its interactions with the water molecules
in its hydration shell, then diffuse about 3 nm (30 Å) through a solvent
(lipid) in which it is poorly soluble (Fig. 12–23). The energy used to strip
away the hydration shell and to move the polar compound from water into
and through lipid is regained as the compound leaves the membrane on the
other side and is rehydrated. However, the intermediate stage of transmembrane passage is a high-energy state comparable to the transition state in an
enzyme-catalyzed chemical reaction. In both cases, an activation barrier must
be overcome to reach the intermediate stage (Fig. 12–23; compare with Fig.
8–3). The energy of activation (DG‡) for translocation of a polar solute across
the bilayer is so large that pure lipid bilayers are virtually impermeable to polar and charged species over periods of time relevant to cells.
A few biologically important gases can cross membranes by simple diffusion: molecular oxygen, nitrogen, and methane, all of which are relatively
nonpolar. Despite its polarity, water crosses some biological membranes
slowly by simple diffusion, presumably as a result of its very high concentration (about 55 M). However, for tissues in which rapid transmembrane
water movement is essential (in the kidney, for example), water diffuses
through channels formed by specific integral proteins, the aquaporins, described below.
Transmembrane passage of polar compounds and ions is made possible
by membrane proteins that lower the activation energy for transport by providing an alternative path for specific solutes through the lipid bilayer. Proteins that bring about this facilitated diffusion or passive transport are
not enzymes in the usual sense; their “substrates” are moved from one compartment to another, but are not chemically altered. Membrane proteins
that speed the movement of a solute across a membrane by facilitating diffusion are called transporters or permeases.
The kind of detailed structural information obtained for many soluble
proteins by x-ray crystallography is not yet available for most membrane
transporters; as a group, these integral membrane proteins are both difficult
to purify and difficult to crystallize. However, from studies of the specificity
and kinetics of transporters we have learned that their action is closely
analogous to that of enzymes. Like enzymes, transporters bind their substrates with stereochemical specificity through many weak, noncovalent interactions. The negative free-energy change associated with these weak interactions, DGbinding, counterbalances the positive free-energy change that
accompanies loss of the water of hydration from the substrate, DGdehydration,
thereby lowering DG‡ for transmembrane passage (Fig. 12–23). Transporters span the lipid bilayer at least once, and usually several times, forming a transmembrane channel lined with hydrophilic amino acid side chains.
The channel provides an alternative path for a specific substrate to move
across the lipid bilayer without its having to dissolve in the bilayer, further
lowering DG‡ for transmembrane diffusion. The result is an increase of orders of magnitude in the rate of transmembrane passage of the substrate.
(a)
Simple diffusion
without transporter
Diffusion
with transporter
∆G‡transport
figure 12–23
Energy changes accompanying passage of a hydrophilic
solute through the lipid bilayer of a biological membrane.
(a) In simple diffusion, removal of the hydration shell is
highly endergonic, and the energy of activation (DG ‡) for
diffusion through the bilayer is very high. (b) A transporter
protein reduces the DG‡ for transmembrane diffusion of
the solute. It does this by forming noncovalent interactions with the dehydrated solute to replace the hydrogen
bonding with water, and by providing a hydrophilic transmembrane passageway.
409
Hydrated
solute
Free energy, G
Chapter 12
(b)
Transporter
‡
∆Gsimple
diffusion
410
Part II
Structure and Catalysis
table 12–3
Aquaporins
Aquaporin
Roles and location
AQP-1
Fluid reabsorption in proximal renal tubule;
secretion of aqueous humor in eye and cerebrospinal fluid in
central nervous system; water homeostasis in lung
Water permeability in renal collecting duct
(mutations produce nephrogenic diabetes insipidus)
Water retention in renal collecting duct
Reabsorption of cerebrospinal fluid in central nervous system;
regulation of brain edema
Fluid secretion in salivary glands, lachrymal glands, and alveolar
epithelium of lung
Water uptake by plant vacuole, regulating turgor pressure
AQP-2
AQP-3
AQP-4
AQP-5
-TIP
Source: King, L.S. & Agre, P. (1996) Pathophysiology of the aquaporin water channels.
Annu. Rev. Physiol. 58, 619–648.
Aquaporins Form Hydrophilic Transmembrane Channels
for the Passage of Water
figure 12–24
Likely transmembrane topology of an aquaporin, AQP-1.
This protein is also called CHIP-28. (a) Each monomer
consists of six transmembrane helices. (b) In a proposed
structure for the aquaporin channel (viewed perpendicular
to, or through, the plane of the membrane), four AQP-1
monomers associate side by side, with their 24 transmembrane helices surrounding a central channel (shaded
blue). The channel, through which water molecules
diffuse one by one, is lined with hydrophilic side chains.
A family of integral proteins, the aquaporins (AQPs), provide channels for
rapid movement of water molecules across plasma membranes in a variety
of specialized tissues. Erythrocytes, which swell or shrink rapidly in response to abrupt changes in extracellular osmolarity as blood travels
through the renal medulla, have a high density of aquaporin in their plasma
membranes (2 105 copies of AQP-1 per cell). Also rich in aquaporins are
the plasma membranes of proximal renal tubule cells, which reabsorb water during urine formation, and the vacuolar membrane of plant cells, which
require osmotic movement of water into the vacuole in order to maintain
turgor pressure (see Fig. 2–10). Table 12–3 provides some examples of
aquaporins.
All aquaporins are type III integral proteins (Fig. 12–14) with six transmembrane helical segments (Fig. 12–24a). In AQP-1, four monomers of Mr
28,000 form a tetrameric transmembrane channel lined with hydrophilic
side chains and having a sufficient diameter (3 Å) to allow passage of water molecules in single file (Fig. 12–24b). Water flows through an AQP-1
channel at the rate of about 5 108 molecules per second. For comparison,
the highest known turnover number for an enzyme is that for catalase, 4 107 s1, and many enzymes have turnover numbers between 1 s1 and
104 s1 (see Table 8–7). The low activation energy for passage of water
through aquaporin channels (DG‡ 15 kJ/mol) suggests that water moves
through the channels in a continuous stream, flowing in the direction dictated
6
Outside
+
H3N
(a)
1
Inside
1
2
3
4
5
6
COO–
(b)
5
3
2
4
Chapter 12
Biological Membranes and Transport
411
by the osmotic gradient. (For a discussion of osmosis, see p. 93.) Like most
aquaporins, AQP-1 does not allow the passage of ions or other small solutes.
The Glucose Transporter of Erythrocytes Mediates Passive Transport
Energy-yielding metabolism in erythrocytes depends on a constant supply
of glucose from the blood plasma, where the glucose concentration is maintained at about 5 mM. Glucose enters the erythrocyte by facilitated diffusion
via a specific glucose transporter at a rate about 50,000 times greater than
the uncatalyzed diffusion rate. This well-studied transporter provides an
excellent example of how passive transport is managed in cells.
The glucose transporter of erythrocytes (called GluT1 to distinguish it
from related glucose transporters in other tissues) is a type III integral protein (Mr 45,000) with 12 hydrophobic segments, each of which is believed
to form a membrane-spanning helix. The detailed structure of GluT1 is not
yet known, but one plausible model suggests that the side-by-side assembly
of several helices produces a transmembrane channel lined with hydrophilic
residues that can hydrogen bond with glucose as it moves through the channel (Fig. 12–25).
Hydrophobic
Polar
Charged
Outside
Inside
+NH
3
COO–
(a)
Ser Leu Val Thr Asn Phe Ile
figure 12–25
Proposed structure of GluT1. (a) Transmembrane
helices are represented as oblique (angled) rows of three
or four amino acid residues, each row depicting one turn
of the a helix. Nine of the 12 helices contain three or
more polar or charged amino acid residues, often separated by several hydrophobic residues. (b) A helical
wheel diagram shows the distribution of polar and nonpolar residues on the surface of a helical segment. The
helix is diagrammed as though observed along its axis
from the amino terminus. Adjacent residues in the linear
sequence are connected with arrows, and each residue is
placed around the wheel in the position it occupies in the
helix; recall that 3.6 residues are required to make one
complete turn of the a helix. In this example, the polar
residues (blue) are on one side of the helix and the
hydrophobic residues (yellow) on the other. This is, by
definition, an amphipathic helix. (c) Side-by-side association of five or six amphipathic helices, each with its polar
face oriented toward the central cavity, can produce a
transmembrane channel lined with polar and charged
residues. This channel provides many opportunities for
hydrogen bonding with glucose moving through the transporter. The three-dimensional structure of GluT1 has not
yet been determined by x-ray crystallography, but
researchers expect that the hydrophilic transmembrane
channels of this and many other transporters and ion
channels will prove to resemble this model.
2
Leu
5
6
Asn
1
Phe
Ser
Glc
Val
3
Ile
Thr
7
4
(b)
(c)
Structure and Catalysis
Vmax
)
Kinetics of glucose transport into erythrocytes. (a) The
initial rate of glucose entry into an erythrocyte, V0,
depends upon the initial concentration of glucose on the
outside, [S]out. (b) Double-reciprocal plot of the data in
(a). The kinetics of facilitated diffusion is analogous to the
kinetics of an enzyme-catalyzed reaction. Compare these
plots with Figure 8–11, and Figure 1 in Box 8–1. Note
that Kt is analogous to Km, the Michaelis constant.
1
2 Vmax
(
figure 12–26
1
1
V0 M/min
Part II
Initial velocity of glucose
entry, V0 (mM/min)
412
Kt
Extracellular glucose
concentration, [S]out (mM)
(a)
1
Kt
1
[S]out
1
Vmax
1
mM
( )
(b)
The process of glucose transport can be described by analogy with an
enzymatic reaction in which the “substrate” is glucose outside the cell
(Sout), the “product” is glucose inside (Sin), and the “enzyme” is the transporter, T. When the rate of glucose uptake is measured as a function of external glucose concentration (Fig. 12–26), the resulting plot is hyperbolic;
at high external glucose concentrations the rate of uptake approaches Vmax.
Formally, such a transport process can be described by the equations
k1
k2
k3
k1
k2
k3
Sout T 3::4 Sout • T 3::4 Sin • T 3::4 Sin T
D-Glucose
T1
in which k1, k1, and so forth, are the forward and reverse rate constants for
each step. The steps are summarized in Figure 12–27.
The rate equations for this process can be derived exactly as for
enzyme-catalyzed reactions (Chapter 8), yielding an expression analogous
to the Michaelis-Menten equation (p. 260):
V0 Step 1
T1
Step 2
T2
Vmax[S]out
Kt [S]out
in which V0 is the initial velocity of accumulation of glucose inside the
cell when its concentration in the surrounding medium is [S]out, and
Kt (Ktransport) is a constant analogous to the Michaelis constant, a combination of rate constants characteristic of each transport system. This equation
describes the initial velocity—the rate observed when [S]in 0. As is the
case for enzyme-catalyzed reactions, the slope-intercept form of the equation describes a linear plot of 1/V0 against 1/[S]out, from which we can obtain
values of Kt and Vmax (Fig. 12–26b). Kt, like Km, is a measure of the affinity
of transporter for substrate; the lower the Kt, the higher the affinity.
Because no chemical bonds are made or broken in the conversion of
Sout to Sin, neither “substrate” nor “product” is intrinsically more stable, and
the process of entry is therefore fully reversible. As [S]in approaches [S]out,
Step 3
figure 12–27
T2
T1
Inside
Step 4
Outside
Model of glucose transport into erythrocytes by GluT1.
The transporter exists in two conformations: T1, with the
glucose-binding site exposed on the outer surface of the
plasma membrane, and T2, with the binding site on the
inner surface. Glucose transport occurs in four steps. In
step 1 , glucose in blood plasma binds to a stereospecific site on T1, thus lowering the activation energy for a
conformational change (step 2 ) from Sout • T1 to Sin • T2,
effecting the transmembrane passage of the glucose. In
step 3 , glucose is released from T2 into the cytosol, and
in step 4 , the transporter returns to the T1 conformation, ready to transport another glucose molecule.
Chapter 12
Biological Membranes and Transport
the rates of entry and exit become equal. Such a system is therefore incapable of accumulating the substrate (glucose) within cells at concentrations
above that in the surrounding medium; it simply achieves equilibration of
glucose on the two sides of the membrane at a much higher rate than would
occur in the absence of a specific transporter. GluT1 is specific for Dglucose, having a measured Kt of 1.5 mM. For the close analogs D-mannose
and D-galactose, which differ only in the position of one hydroxyl group, the
values of Kt are 20 and 30 mM, respectively; and for L-glucose, Kt exceeds
3,000 mM! Thus, GluT1 shows the three hallmarks of passive transport: high
rates of diffusion down a concentration gradient, saturability, and specificity. Because the concentration of blood glucose, 4.5 to 5 mM, is maintained at about three times Kt, GluT1 is nearly saturated with substrate and
operates near Vmax.
In liver, a different glucose transporter, GluT2, transports glucose out
of hepatocytes when liver glycogen is broken down to replenish the blood
glucose. GluT2 has a Kt of about 66 mM and can therefore respond to increased levels of intracellular glucose by increasing outward transport.
Muscle and adipose tissue have yet another glucose transporter, GluT4,
which is distinguished by its stimulation by insulin (Box 12–2).
Chloride and Bicarbonate Are Cotransported
across the Erythrocyte Membrane
The erythrocyte contains another facilitated diffusion system, an anion exchanger, which is essential in CO2 transport to the lungs from tissues such
as muscle and liver. Waste CO2 released from respiring tissues into the
blood plasma enters the erythrocyte, where it is converted into bicarbonate
(HCO3 ) by the enzyme carbonic anhydrase. The HCO3 reenters the blood
plasma for transport to the lungs (Fig. 12–28). Because HCO3 is much
more soluble in blood plasma than is CO2, this roundabout route increases
the blood’s capacity to carry carbon dioxide from the tissues to the lungs.
In the lungs, HCO3 reenters the erythrocyte and is converted to CO2, which
is eventually released into the lung space and exhaled. This shuttle, to be
effective, requires very rapid movement of HCO3 across the erythrocyte
membrane.
The chloride-bicarbonate exchanger, also called the anion exchange protein, increases the permeability of the erythrocyte membrane
to HCO3 by a factor of more than a million. Like the glucose transporter, it
is an integral protein that probably spans the membrane 12 times. This protein mediates the simultaneous movement of two anions; for each HCO3 ion
that moves in one direction, one Cl ion moves in the opposite direction
(Fig. 12–28). The coupling of Cl and HCO3 movement is obligatory; in the
absence of chloride, bicarbonate transport stops. In this respect, the anion
exchanger is typical of all systems, called cotransport systems, that simultaneously carry two solutes across a membrane. When, as in this case,
the two substrates move in opposite directions, the process is antiport. In
symport, two substrates are moved simultaneously in the same direction.
Transporters that carry only one substrate, such as the erythrocyte glucose
transporter, are uniport systems (Fig. 12–29).
413
Carbon dioxide produced
by catabolism enters
erythrocyte
Bicarbonate
dissolves in
blood plasma
Chloride-bicarbonate
exchange protein
CO2
HCO
3
Cl
In respiring tissues
carbonic anhydrase
HCO3 H
CO2 H2O
HCO
3 H
CO2 H2O
Cl
Cl
carbonic anhydrase
In lungs
HCO3
CO2
Carbon dioxide leaves
erythrocyte and is
exhaled
Cl
Bicarbonate enters
erythrocyte from
blood plasma
figure 12–28
Chloride-bicarbonate exchanger of the erythrocyte membrane. This cotransport system allows the entry and exit
of HCO
3 without changes in the transmembrane electrical
potential. Its role is to increase the CO2-carrying capacity
of the blood.
S
S1
S2
S1
figure 12–29
Three general classes of transport systems. Transporters differ in the number of solutes (substrates) transported and the direction in which each is transported.
Examples of all three types of transporters are discussed
in the text. Note that this classification tells us nothing
about whether these are energy-requiring (active transport) or energy-independent (passive transport)
processes.
S2
Uniport
Symport
Antiport
Cotransport
414
Part II
box 12–2
Structure and Catalysis
Defective Glucose and Water Transport in Two Forms of Diabetes
When ingestion of a carbohydrate-rich meal
causes blood glucose to exceed the usual concentration between meals (5 mM), excess glucose is
taken up by cardiac muscle and skeletal muscle
(which store it as glycogen) and by adipocytes
(which convert it to triacylglycerols). Glucose uptake into myocytes and adipocytes is mediated by
the glucose transporter GluT4. Between meals,
the plasma membrane of these cells contains
some GluT4, but most is sequestered in the membranes of small intracellular vesicles (Fig. 1). Insulin released from the pancreas in response to
high blood glucose triggers the movement of
these intracellular vesicles to the plasma membrane, where they fuse, thus exposing GluT4 molecules on the outer surface of the cell. With more
GluT4 molecules in action, the rate of glucose uptake increases 15-fold or more. When blood glucose levels return to normal, insulin release slows
and most GluT4 molecules are removed from the
plasma membranes and stored in vesicles.
In type I (juvenile onset) diabetes mellitus, the
inability to release insulin (and thus to mobilize
glucose transporters) results in low rates of glu-
cose uptake into muscle and adipose tissue. One
consequence is a prolonged period of high blood
glucose after a carbohydrate-rich meal. This condition is the basis for the glucose tolerance test
used to diagnose diabetes (Chapter 23).
The water permeability of epithelial cells lining the renal collecting duct in the kidney is due
to the presence of an aquaporin (AQP-2) in their
apical plasma membranes (facing the lumen of
the duct). Antidiuretic hormone (ADH) regulates the retention of water by mobilizing AQP-2
molecules stored in vesicle membranes within
the epithelial cells, much as insulin mobilizes
GluT4 in muscle and adipose tissue. When the
vesicles fuse with the epithelial cell plasma membrane, water permeability increases dramatically
and more water is reabsorbed from the collecting
duct and returned to the blood. When the ADH
level drops, AQP-2 is resequestered within vesicles, reducing water retention. In the relatively
rare human disease diabetes insipidus, a genetic
defect in AQP-2 leads to impaired water reabsorption by the kidney. The result is excretion of
copious volumes of very dilute urine.
2
Insulin
When insulin interacts with its receptor, vesicles
move to surface and fuse with the plasma
membrane, increasing the number of glucose
transporters in the plasma membrane.
3
When insulin level drops,
glucose transporters are
removed from the plasma
membrane by endocytosis,
forming small vesicles.
Glucose
transporter
Insulin receptor
Plasma
membrane
1
Glucose transporters
“stored” within cell in
membrane vesicles.
5
figure 1
Regulation by insulin of glucose
transport into a myocyte.
Patches of the endosome enriched for
glucose transporters bud off to become
small vesicles, ready to return to the
surface when insulin levels rise again.
4
The smaller
vesicles fuse with
larger endosome.
Chapter 12
Biological Membranes and Transport
415
figure 12–30
S
ADP Pi
ADP Pi
ATP
Two types of active transport. (a) In primary active transport, the energy released by ATP hydrolysis drives solute
movement against an electrochemical gradient. (b) In
secondary active transport, a gradient of an ion X (often
Na) has been established by primary active transport.
Movement of X down its electrochemical gradient then
provides the energy to drive cotransport of a second
solute (S) against its electrochemical gradient.
ATP
X
X
S
X
X
Primary active transport
(a)
Secondary active transport
(b)
Active Transport Results in Solute Movement against
a Concentration or Electrochemical Gradient
In passive transport, the transported species always moves down its electrochemical gradient and is not accumulated above the equilibrium point.
Active transport, by contrast, results in the accumulation of a solute above
the equilibrium point. Active transport is thermodynamically unfavorable
(endergonic) and occurs only when coupled (directly or indirectly) to an
exergonic process such as the absorption of sunlight, an oxidation reaction,
the breakdown of ATP, or the concomitant flow of some other chemical
species down its electrochemical gradient. In primary active transport,
solute accumulation is coupled directly to an exergonic chemical reaction,
such as conversion of ATP to ADP Pi (Fig. 12–30). Secondary active
transport occurs when endergonic (uphill) transport of one solute is coupled to the exergonic (downhill) flow of a different solute that was originally
pumped uphill by primary active transport.
The amount of energy needed for the transport of a solute against a gradient can be calculated from the initial concentration gradient. The general
equation for the free-energy change in the chemical process that converts
S into P is
G G° RT ln [P][S]
(12–1)
where R is the gas constant, 8.315 J/mol
K, and T is the absolute temperature. When the “reaction” is simply transport of a solute from a region
where its concentration is C1 to another region where its concentration is
C2, no bonds are made or broken and the standard free-energy change,
DG, is zero. The free-energy change for transport, DGt, is then
DGt RT ln (C2/C1)
(12–2)
If there is a tenfold difference in concentration between two compartments,
the cost of moving 1 mol of an uncharged solute at 25 C across a membrane
separating the compartments is therefore
DGt (8.315 J/mol • K)(298 K)(ln 10/1) 5,705 J/mol 5.7 kJ/mol
Equation 12–2 holds for all uncharged solutes.
416
Part II
Structure and Catalysis
When the solute is an ion, its movement without an accompanying
counterion results in the endergonic separation of positive and negative
charges, producing an electrical potential; such a transport process is said
to be electrogenic. The energetic cost of moving an ion depends on the
electrochemical potential (p. 408), the sum of the chemical and electrical
gradients:
DGt RT ln (C2/C1) Z
Dw
(12–3)
where Z is the charge on the ion, is the Faraday constant (96,480 J/V
mol),
and Dw is the transmembrane electrical potential (in volts). Eukaryotic cells
typically have electrical potentials across their plasma membranes of the order of 0.05 to 0.2 V (with the interior negative relative to the outside), so
the second term of Equation 12–3 can be a significant contribution to the
total free-energy change for transporting an ion. Most cells maintain more
than tenfold differences in ion concentrations across their plasma or intracellular membranes, and for many cells and tissues, active transport is
therefore a major energy-consuming process.
The mechanism of active transport is of fundamental importance in biology. As we shall see in Chapter 19, the formation of ATP in mitochondria
and chloroplasts occurs by a mechanism that is essentially ATP-driven ion
transport operating in reverse. The energy made available by the spontaneous flow of protons across a membrane is calculable from Equation 12–3;
remember that for flow down an electrochemical gradient, the sign of DG is
opposite to that for transport against the gradient.
There Are at Least Four General Types of Transport ATPases
O
O
P
O
O
OH
Phosphate
O
V
O
OH
Vanadate
In the course of evolution, several distinct types of ATP-dependent active
transporters have arisen, differing in structure, mechanism, and localization
in specific tissues and intracellular compartments (Table 12–4).
P-type ATPases are ATP-driven cation transporters that are reversibly phosphorylated by ATP as part of the transport cycle. All P-type
transport ATPases have similarities in amino acid sequence, especially
near the Asp residue that undergoes phosphorylation, and all are sensitive
to inhibition by the phosphate analog vanadate. Each is an integral protein with multiple membrane-spanning regions in a single polypeptide;
some also have a second subunit (Fig. 12–31a). P-type transporters are
very widely distributed. In animal tissues, the NaK ATPase, an antiporter for Na and K, and the Ca2 ATPase, a uniporter for Ca2, are ubiquitous, well-understood P-type ATPases that maintain disequilibrium in
the ionic composition between the cytosol and the extracellular medium.
Parietal cells in the lining of the mammalian stomach have a
P-type ATPase that pumps H and K across the plasma membrane, thereby
acidifying the stomach contents. In higher plants, a P-type ATPase pumps
protons out of the cell, establishing a difference of as much as 2 pH units
and 250 mV across the plasma membrane. A similar P-type ATPase in the
bread mold Neurospora pumps protons out of cells to establish an insidenegative membrane potential, which is used to drive the uptake of substrates
and ions from the surrounding medium by secondary active transport. Bacteria use P-type ATPases to pump out toxic heavy metal ions such as Cd2
and Cu2.
A distinctly different class of proton-transporting ATPases is responsible for acidifying intracellular compartments in many organisms. The vacuoles of fungi and higher plants maintain a pH between 3 and 6, well
below that of the surrounding cytosol (pH 7.5), by the action of V-type
ATPases—proton pumps. V-type ATPases (V for vacuolar) are also responsible for the acidification of lysosomes, endosomes, the Golgi complex,
A
B
B
A
H
Chapter 12
A
B
V1
a
d
b
b
Biological Membranes and Transport
F1
a
figure 12–31
b
G
C
E
D
a
b
Subunit structure of three types of ion-transporting
ATPases. (a) The P-type ATPases generally have two
types of integral protein subunits. The a subunit, which is
essential, has the Asp residue that is phosphorylated
during transport. (b) V-type ATPases have a peripheral
domain, V1 (blue), composed of seven different types of
subunits including three A and three B subunits, and an
integral domain, Vo (yellow), with three types of subunits
including multiple copies of c. (c) F-type ATPases have a
peripheral domain, F1 (blue), homologous with V1 of the
V-type ATPases. The integral portion of F-type ATPases,
Fo (yellow), also has three types of subunits, with multiple
copies of c. P-type pumps such as the NaK ATPase
move two ions in opposite directions. V-type and F-type
proton pumps move protons in one direction—from top
to bottom in this diagram—into vacuoles for V-type, out of
mitochondria for F-type.
a
d
e
b
P-type
(a)
F
c c c
c
c c
c
a
g
a b
c
Vo
V-type
(b)
417
Fo
F-type
(c)
table 12–4
Four Classes of Transport ATPases
Organism or tissue
Type of membrane
Role of ATPase
NaK
Animal tissues
Plasma
H K
Plasma
H
Acid-secreting (parietal)
cells of mammals
Fungi (Neurospora)
Maintains low [Na], high [K] inside cell; creates
transmembrane electrical potential
Acidifies contents of stomach
H
Ca2
Ca2
Higher plants
Animal tissues
Myocytes of animals
Cd2, Hg2, Cu2
Bacteria
P-type ATPases
Plasma
Plasma
Plasma
Sarcoplasmic reticulum
(endoplasmic reticulum)
Plasma



Create H gradient to drive secondary transport
of extracellular solutes into cell
Maintains low [Ca2] in cytosol
Sequesters intracellular Ca2, keeping cytosolic
[Ca2] low
Pumps heavy metal ions out of cell
V-type ATPases
H
Animals
H
H
Higher plants
Fungi
Lysosomal, endosomal,
secretory vesicles
Vacuolar
Vacuolar





Create low pH in compartment, activating
proteases and other hydrolytic enzymes
Eukaryotes
Higher plants
Prokaryotes
Inner mitochondrial
Thylakoid
Plasma



Catalyze formation of ATP from ADP Pi
Animal tumor cells
Plasma
F-type ATPases
H
H
H
Multidrug transporter
and secretory vesicles in animal cells. Structurally unrelated to P-type
ATPases, the V-type ATPases do not undergo cyclic phosphorylation and
dephosphorylation and are not inhibited by vanadate. All V-type ATPases
have a similar complex structure, with an integral (transmembrane) domain
Removes a wide variety of hydrophobic natural
products and synthetic drugs from cytosol,
including vinblastine, doxorubicin, actinomycin D,
mitomycin, taxol, colchicine, and puromycin
418
Part II
ATP
H
Structure and Catalysis
ADP Pi
H
figure 12–32
Reversibility of F-type ATPases. An ATP-driven proton
transporter also catalyzes ATP synthesis as protons flow
down their electrochemical gradient.
box 12–3
(Vo) that serves as a proton channel and a peripheral domain (V1) that contains the ATP-binding site and the ATPase activity (Fig. 12–31b). The
mechanism by which V-type ATPases couple ATP hydrolysis to the uphill
transport of protons is not understood in detail. The V-type proton pumps
are related in structure, and probably in mechanism, to a third family of proton pumps, the F-type ATPases.
F-type ATPases play a central role in energy-conserving reactions in
bacteria, mitochondria, and chloroplasts; that role will be discussed in detail when we describe oxidative phosphorylation and photophosphorylation
in Chapter 19. (The F in their name originated in their identification as
energy-coupling factors.) They catalyze the uphill transmembrane passage
of protons driven by ATP hydrolysis, as well as the reverse reaction, in
which downhill proton flow drives ATP synthesis (Fig. 12–32). In the second case, the F-type ATPases are more appropriately named ATP synthases. The proton gradient in oxidative phosphorylation and photophosphorylation is established by other types of proton pumps powered by
substrate oxidation or sunlight. The F-type ATPases/ATP synthases are
multisubunit complexes that provide a transmembrane pore (the integral
protein Fo) for protons and a molecular machine (the peripheral protein F1)
A Defective Ion Channel Causes Cystic Fibrosis
Cystic fibrosis (CF) is a serious and relatively
common hereditary disease of humans. About
5% of white Americans are carriers, having one
defective and one normal copy of the gene. Only
individuals with two defective copies show the
severe symptoms of the disease: obstruction of
the gastrointestinal and respiratory tracts, commonly leading to bacterial infection of the airways and death due to respiratory insufficiency
before the age of 30. In CF, the thin layer of mucus that normally coats the internal surfaces of
the lungs is abnormally thick, obstructing air flow
and providing a haven for pathogenic bacteria,
particularly Staphylococcus aureus and
Pseudomonas aeruginosa.
The defective gene in CF patients was discovered in 1989. It encodes a membrane protein
called cystic fibrosis transmembrane conductance regulator, or CFTR. Hydropathy analysis
predicted that CFTR has 12 transmembrane helices and is structurally related to the multidrug
transporters of drug-resistant tumors (Fig. 1).
The normal CFTR protein proved to be an ion
Oligosaccharide
chains of
glycoprotein
Outside
figure 1
Topology of the cystic fibrosis transmembrane conductance regulator, CFTR. There are 12 transmembrane
helices, and three functionally significant domains extend
from the cytosolic surface: NBF1 and NBF2 are
nucleotide-binding folds to which ATP binds, and a regulatory domain (R-domain) is the site of phosphorylation by
cAMP-dependent protein kinase. Oligosaccharide chains
are attached to several residues on the outer surface on
the segment between helices 7 and 8. The most commonly occurring mutation leading to CF is the deletion of
Phe508, in the NBF1 domain. The structure of CFTR is
very similar to that of the multidrug transporter of tumors,
described in the text.
Inside
+
NH3
NBF1
NBF2
Phe508
COO–
R domain
Chapter 12
Biological Membranes and Transport
that uses the energy released by downhill proton flow through Fo to form
the phosphoanhydride bonds of ATP. The ATP-synthesizing and ATPase activities reside in the F1 protein (Fig. 12–31c).
In the mid-1980s it became apparent that some tumors are strikingly resistant to a number of generally effective antitumor compounds. Investigation revealed that the plasma membranes of these tumors contain an ATPdependent transporter that can export many different drugs, preventing
their accumulation within the tumor cells and their growth-inhibitory
effects. The transported drugs are chemically dissimilar but are generally
hydrophobic. The multidrug transporter responsible for removing these
drugs from the tumor cell cytosol is an integral protein (Mr 170,000) with
12 transmembrane segments and two ATP-binding sites. Export of the
drugs is driven by ATP hydrolysis. The multidrug transporter is also an ion
channel that specifically allows transmembrane diffusion of Cl down its
concentration gradient; this activity is independent of ATP. In the genetic
disease cystic fibrosis, the underlying defect is in the gene that encodes a
Cl-transporting protein, the cystic fibrosis conductance transmembrane
regulator (CFTR), which may be related to the multidrug transporter of tumor cells (Box 12–3).
channel specific for Cl. The Cl channel activity
increases greatly when phosphoryl groups are
transferred from ATP to several side chains of
the protein, catalyzed by cAMP-dependent protein kinase (Chapter 13). The mutation responsible for CF in 70% of cases results in deletion of a
Phe residue at position 508, with the effect that
the mutant protein is not correctly folded and inserted in the plasma membrane. Other mutations
yield a protein that is inserted properly but cannot be activated by phosphorylation. In each
case, the fundamental problem is a nonfunctional
Cl channel in the epithelial cells that line the
airways (Fig. 2), the digestive tract, and exocrine
glands (pancreas, sweat glands, bile ducts, and
vas deferens).
figure 2
Mucus lining the surface of the lungs traps bacteria. In
healthy lungs, these bacteria are killed and swept away
by the action of cilia. In CF, the bactericidal activity is
impaired, resulting in recurring infections and progressive
damage to the lungs.
Normally, epithelial cells that line the inner
surface of the lungs secrete a substance that
traps and kills bacteria, and the cilia on the epithelial cells constantly sweep away the resulting
debris. According to one hypothesis, the bactericidal activity requires a relatively low NaCl concentration. In CF patients, the defect in the Cl
channel of CFTR results in surface fluid containing a high concentration of NaCl, and this fluid is
much less effective in killing bacteria. Frequent
infections by bacteria such as S. aureus and P.
aeruginosa progressively damage the lungs and
reduce respiratory efficiency.
419
420
Part II
Structure and Catalysis
3 Na
figure 12–33
Na K ATPase. In animal cells, this active transport
system is primarily responsible for setting and maintaining
the intracellular concentrations of Na and K and for
generating the transmembrane electrical potential. It does
this by moving three Na out of the cell for every two K
it moves in. The electrical potential is central to electrical
signaling in neurons, and the gradient of Na is used to
drive the uphill cotransport of solutes in a variety of cell
types.
Membrane potential 50–70 mV
NaK ATPase
ADP Pi
ATP
2 K
Cytosol
[K] 140 mM
[Na] 12 mM
Extracellular fluid
or blood plasma
[K] 4 mM
[Na] 145 mM
A P-Type ATPase Catalyzes Active Cotransport of Na and K
Jens Skou
In virtually every animal cell, the concentration of Na is lower in the cell
than in the surrounding medium, and the concentration of K is higher
(Fig. 12–33). This imbalance is established and maintained by a primary
active transport system in the plasma membrane. The enzyme NaK
ATPase, discovered by Jens Skou in 1957, couples breakdown of ATP to
the simultaneous movement of both Na and K against their electrochemical gradients. For each molecule of ATP converted to ADP and Pi,
the transporter moves two K ions inward and three Na ions outward
across the plasma membrane. The NaK ATPase is an integral protein
with two subunits (Mr 50,000 and 110,000), both of which span the
membrane.
The detailed mechanism by which ATP hydrolysis is coupled to transport awaits determination of the protein’s three-dimensional structure, but
a current model (Fig. 12–34) supposes that the ATPase cycles between two
forms, a phosphorylated form (designated P–EnzII) with high affinity for K
and low affinity for Na, and a dephosphorylated form (EnzI) with high
affinity for Na and low affinity for K. The conversion of ATP to ADP and
Pi occurs in two steps catalyzed by the enzyme.
(1) Formation of phosphoenzyme:
ATP EnzI 88n ADP P–EnzII
(2) Hydrolysis of phosphoenzyme:
P–EnzII H2O 88n EnzI Pi
The net reaction for these two steps is
ATP H2O 88n ADP Pi
Because three Na ions move outward for every two K ions that move
inward, the process is electrogenic—it creates a net separation of charge
across the membrane. The result is a transmembrane potential of 50 to
70 mV (inside negative relative to outside), which is characteristic of
most animal cells and essential to the conduction of action potentials in
neurons. The central role of the NaK ATPase is reflected in the energy invested in this single reaction: about 25% of the total energy consumption of
Chapter 12
Biological Membranes and Transport
a human at rest! The steroid derivative ouabain (pronounced wah-bane;
from waa bayyo, Somali for “arrow poison”) is a potent and specific inhibitor of the NaK ATPase. Ouabain and another steroid derivative, digitoxigenin, are the active ingredients of digitalis, an extract of the leaves of
the foxglove plant. Digitalis has long been used in human medicine to treat
congestive heart failure. Inhibition of Na efflux by digitalis leads to higher
Na concentration in cells, activating a Na-Ca2 antiporter in cardiac muscle. The increased influx of Ca2 through this antiporter produces elevated
cytosolic Ca2, which strengthens the contractions of heart muscle.
O
C
ATP-Driven Ca Pumps Maintain a Low Concentration
of Calcium in the Cytosol
OH
CH2
O
O
H
H
H
OH
OH
OH
H
Ouabain
Transporter binds 3 Na
from the inside of the
cell.
1
EnzI
3 Na
2
Phosphorylation
favors P–EnzII.
ATP
ADP
P–EnzII
Transporter
releases 3 Na
to the outside
and binds 2 K
from the outside
of the cell.
P
P–EnzII
Postulated mechanism of Na and K transport by the
1 The process begins with the binding
NaK ATPase. of three Na to high-affinity sites on the large subunit of
the transport protein on the inner (cytosolic) surface of
the membrane. This same part of the large subunit also
has the ATP-binding site. Phosphorylation of the transporter changes its conformation 2 and decreases its
3 Na release on the outer
affinity for Na, leading to (extracellular) surface. 4 K on the outside now binds
to high-affinity sites on the extracellular portion of the
large subunit; 5 the enzyme is dephosphorylated,
6 K is discharged on
reducing its affinity for K; and the inside of the cell. The transport protein is now ready
for another cycle of Na and K pumping.
C
H 3C
H
figure 12–34
CH3
HO
HO
CH3
The cytosolic concentration of free Ca2 is generally about 100 nM, far below that in the surrounding medium, whether pond water or blood plasma.
The ubiquitous occurrence of inorganic phosphates (Pi and PPi) at millimolar concentrations in the cytosol necessitates a low cytosolic Ca2 concentration, because inorganic phosphate combines with calcium to form relatively insoluble calcium phosphates. Calcium ions are pumped out of the
cytosol by a P-type ATPase, the plasma membrane Ca2 pump. Another
P-type Ca2 pump in the endoplasmic reticulum moves Ca2 into the ER lumen, a compartment separate from the cytosol. In myocytes, Ca2 is normally sequestered in a specialized form of endoplasmic reticulum called the
sarcoplasmic reticulum. The sarcoplasmic and endoplasmic reticulum calcium (SERCA) pumps are closely related in structure and mechanism, and
both are inhibited by the tumor-promoting agent thapsigargin, which does
not affect the plasma membrane Ca2 pump.
The plasma membrane Ca2 pump and SERCA pumps are integral proteins that cycle between two conformations in a mechanism similar to that
for NaK ATPase (Fig. 12–34). The Ca2 pump of the sarcoplasmic reticulum, which comprises 80% of the protein in that membrane, has been thoroughly characterized and is the prototype for Ca2 pumps of the P-type. It
consists of a single polypeptide (Mr 100,000) that spans the membrane
ten times. A large cytosolic domain includes a site for ATP binding and an
Asp residue that undergoes reversible phosphorylation by ATP. Phosphorylation favors a conformation with a high-affinity Ca2-binding site exposed
on the cytosolic side, and dephosphorylation favors a conformation with a
low-affinity Ca2-binding site on the lumenal side. As a result of the cyclic
changes in conformation, the transporter binds Ca2 on the side of the membrane where Ca2 concentration is low and releases it on the side where the
concentration is high. In this way, the energy released by hydrolysis of ATP
to ADP and Pi during one phosphorylation-dephosphorylation cycle drives
Ca2 across the membrane against a large electrochemical gradient.
O
HC
OH
2
421
3 Na
3
2 K 4
P
Pi
5
6
2 K
EnzI
Dephosphorylation
favors EnzI.
Transporter releases
2 K to the inside.
Inside
Outside
422
Part II
Structure and Catalysis
table 12–5
Cotransport Systems Driven by Gradients of Na or H
Organism or tissue
E. coli
Intestine, kidney of vertebrates
Vertebrate cells (many types)
Higher plants
Fungi (Neurospora)
Transported solute
(moving against its gradient)
Cotransported solute
(moving down its gradient)
Type of
transport
Lactose
Proline
Dicarboxylic acids
Glucose
Amino acids
Ca2
K
K
H
H
H
Na
Na
Na
H
H
Symport
Symport
Symport
Symport
Symport
Antiport
Antiport
Antiport
Ion Gradients Provide the Energy for Secondary Active Transport
Lactose uptake in E. coli. (a) The primary transport of
H out of the cell, driven by the oxidation of a variety of
fuels, establishes both a proton gradient and an electrical
potential (inside negative) across the membrane. Secondary active transport of lactose into the cell involves
symport of H and lactose by the galactoside transporter.
The uptake of lactose against its concentration gradient is
entirely dependent on this inflow of H, driven by the
electrochemical gradient. (b) When the energy-yielding
oxidation reactions of metabolism are blocked by cyanide
(CN), the galactoside transporter allows equilibration of
lactose inside and outside the cell via passive transport.
The dashed line represents the concentration of lactose
in the surrounding medium.
Galactoside
H H
transporter
H
H
H
Lactose
H H
H
(outside)
Proton pump
(inhibited by CN)
Fuel
H
Lactose
(inside)
H
H
(a)
CO2
2Naout glucoseout 88n 2Nain glucosein
The energy required for this process comes from two sources: the greater
concentration of Na outside than inside (the chemical potential) and the
[Lactose]inside
figure 12–35
The ion gradients formed by primary transport of Na or H can themselves
provide the driving force for cotransport of other solutes. Many cells contain transport systems that couple the spontaneous, downhill flow of these
ions to the simultaneous uphill pumping of another ion, sugar, or amino acid
(Table 12–5). The galactoside transporter of E. coli, for example, allows the
accumulation of lactose to levels 100 times higher than the concentration in
the surrounding growth medium (Fig. 12–35). E. coli normally has a gradient of protons and charge across its plasma membrane produced by energyyielding metabolism; protons tend to flow back spontaneously into the cell
down this electrochemical gradient. The lipid bilayer is impermeable to protons, but the galactoside transporter provides a route for proton reentry,
and lactose is simultaneously carried into the cell by symport. The endergonic accumulation of lactose is thereby coupled to the exergonic flow of
protons; the overall free-energy change for the coupled process is negative.
In intestinal epithelial cells, glucose and certain amino acids are accumulated by symport with Na, using the Na gradient established by the
NaK ATPase of the plasma membrane (Fig. 12–36). The apical surface of
the intestinal epithelial cell is covered with microvilli, long, thin projections
of the plasma membrane that greatly increase the surface area exposed to
the intestinal contents. Na-glucose symporters in the apical plasma membrane take up glucose from the intestine in a process driven by the downhill flow of Na:
Active
transport
CN inhibition of
fuel oxidation
Efflux
[Lactose]medium
Time
(b)
Chapter 12
Apical
surface
Biological Membranes and Transport
figure 12–36
Basal
surface
Intestinal
lumen
423
Glucose transport in intestinal epithelial cells. Glucose
is cotransported with Na across the apical plasma membrane into the epithelial cell. It moves through the cell to
the basal surface, where it passes into the blood via
GluT2, a passive glucose transporter. The NaK ATPase
continues to pump Na outward to maintain the gradient
of Na that drives glucose uptake.
Blood
Microvilli
2 K
Epithelial cell
3 Na
2 Na
NaK
ATPase
Glucose
Glucose
Glucose uniporter
GluT2 (facilitates
downhill efflux)
Na glucose
symporter
(driven by high
extracellular [Na])
transmembrane potential (the electrical potential), which is inside-negative
and therefore draws Na inward. The electrochemical potential of Na is
G RT ln
[Na]in
n
[Na]out
DE
where n 2, the number of Na ions cotransported with each glucose molecule. Given the typical membrane potential of 50 mV, an intracellular
[Na] of 12 mM, and an extracellular [Na] of 145 mM, the energy, DG, made
available as two Na ions reenter the cell is 25 kJ, enough to pump glucose against a large concentration gradient.
G 25 kJ RT ln
[glucose]in
[glucose]out
[glucose]in
30,000
[glucose]out
That is, the cotransporter can pump glucose inward until its concentration
within the epithelial cell is about 30,000 times that in the intestine. As glucose is pumped from the intestine into the epithelial cell at the apical surface, it is simultaneously moved from the cell into the blood by passive
transport through a glucose transporter (GluT2) in the basal surface (Fig.
12–36). The crucial role of Na in symport and antiport systems such as
these requires the continued outward pumping of Na to maintain the
transmembrane Na gradient.
Because of the essential role of ion gradients in active transport and
energy conservation, compounds that collapse the ion gradients across cellular membranes are poisons, and those that are specific for infectious microorganisms can serve as antibiotics. Valinomycin is a small, cyclic peptide
that surrounds K and neutralizes its positive charge (Fig. 12–37). The
peptide then acts as a shuttle, carrying K across membranes down its concentration gradient and deflating that gradient. Compounds that shuttle
ions across membranes in this way are called ionophores, literally “ion
bearers.” Both valinomycin and monensin (a Na-carrying ionophore) are
antibiotics; they kill microbial cells by disrupting secondary transport
processes and energy-conserving reactions.
figure 12–37
Valinomycin, a peptide ionophore that binds K. In this
image, the surface contours are shown as a transparent
mesh, through which a stick structure of the peptide and
a K atom (green) are visible. The oxygen atoms (red)
that bind K are part of a central hydrophilic cavity.
Hydrophobic amino acid side chains (yellow) coat the
outside of the molecule. Because the exterior of the
K-valinomycin complex is hydrophobic, the complex
readily diffuses through membranes, carrying K down
its concentration gradient. The resulting dissipation of
the transmembrane ion gradient kills microbial cells,
making valinomycin a potent antibiotic.
424
Part II
Structure and Catalysis
Ion-Selective Channels Allow Rapid Movement
of Ions across Membranes
First recognized in neurons and now known to be present in the plasma
membranes of all cells, as well as in the intracellular membranes of eukaryotes, the ion-selective channel is another mechanism for moving inorganic
ions across membranes. Ion channels determine the plasma membrane’s permeability to specific ions and, together with ion pumps such as the NaK
ATPase, regulate the cytosolic concentration of ions and the membrane potential. In neurons, very rapid changes in the activity of ion channels cause
the changes in membrane potential—the action potentials—that carry signals from one end of a neuron to the other. In myocytes, rapid opening of
Ca2 channels in the sarcoplasmic reticulum releases the Ca2 that triggers
muscle contraction. We discuss the signaling functions of ion channels in
Chapter 13. Here we describe the structural basis for ion channel function,
using as examples a bacterial K channel, the acetylcholine receptor ion
channel, and the voltage-dependent Na channel of neurons.
Ion channels are distinguished from ion transporters in at least three
ways. First, the rate of flux through channels can be orders of magnitude
greater than the turnover number for a transporter—107 to 108 ions per
channel per second, near the theoretical maximum rate for unrestricted diffusion. Second, ion channels are not saturable; their rates do not approach
a maximum at high substrate concentration. Third, they are “gated”—
opened or closed in response to some cellular event. In ligand-gated channels (which are generally oligomeric), binding of some extracellular or intracellular small molecule forces an allosteric transition in the protein,
which opens or closes the channel. In voltage-gated ion channels, a
charged protein domain moves relative to the membrane in response to a
change in transmembrane electrical potential, causing the ion channel to
open or close. Gating by either ligands or membrane potential can be very
fast. A channel typically opens in a fraction of a millisecond, and may remain open for only milliseconds, making these molecular devices effective
for very fast signal transmission in the nervous system.
The Structure of a K Channel Shows the Basis for Its Ion Specificity
The structure of a potassium channel from the bacterium Streptomyces
lividans, determined by x-ray crystallography in 1998, provides much insight into the way ion channels work. This bacterial ion channel is related
in sequence to all other known K channels and serves as the prototype for
such channels, including the voltage-gated K channel of neurons described in the next chapter. Among the members of this protein family, the
similarities in sequence are greatest in the “pore region,” which contains the
ion selectivity filter that allows K (radius 1.33 Å) to pass 10,000 times
more readily than Na (radius 0.95 Å), at a rate (about 108 ions per second)
approaching the theoretical limit for unrestricted diffusion.
The K channel consists of four identical subunits that span the membrane and form a cone within a cone surrounding the ion channel, with the
cone’s wide end facing the extracellular space (Fig. 12–38). Each subunit
has two transmembrane a helices as well as a third, shorter helix that contributes to the pore region. The outer cone is formed by one of the transmembrane helices of each subunit. The inner cone, formed by the other
four transmembrane helices, surrounds the ion channel and cradles the ion
selectivity filter.
Both the ion specificity and the high flux through the channel are understandable from the channel’s structure. At the inner and outer plasma
membrane surfaces, the entryways to the channel have several negatively
charged amino acid residues, which presumably increase the local concen-
Chapter 12
Biological Membranes and Transport
tration of cations such as K and Na. The ion path through the membrane
begins (on the inner surface) as a wide, water-filled channel in which the
ion can retain its hydration sphere. Further stabilization is provided by the
short a helices in the pore region of each subunit, with their carboxyl termini and the associated partial negative charges pointed at K in the channel. About two-thirds of the way through the membrane, this channel narrows in the region of the selectivity filter, forcing the ion to give up its
hydrating water molecules. Carbonyl oxygen atoms in the backbone of the
selectivity filter replace the water molecules in the hydration sphere of K,
forming a series of perfect coordination shells through which the K moves.
This favorable interaction with the filter is not possible with Na, which is
too small to make contact with all the potential oxygen ligands. The preferential stabilization of K is the basis for the ion selectivity of the filter, and
mutations that change residues in this part of the protein eliminate the
channel’s ion selectivity.
K ions pass through the filter in single file. In the crystallographic
structure, two K ions are visible, one at each end of the selectivity filter,
about 7.5 Å apart. Their mutual electrostatic repulsion most likely just balances the interaction of each with the selectivity filter and keeps them moving through it rapidly.
Other K channels are similar in sequence, and presumably in structure
and mechanism, to the S. lividans K channel. For example, the product
of the Shaker gene, which is a K channel in Drosophila, and K channels
from the nematode Caenorhabditis elegans, the ciliated protozoan Paramecium, and the plant Arabidopsis thaliana all have sequences very similar to those in the pore region of the S. lividans K channel. Furthermore,
the amino acid sequences of Na and Ca2 channels suggest that they, too,
share some structural and functional similarities with the bacterial K channel. Determination of this channel’s structure was therefore a landmark in
channel biochemistry.
425
(a)
figure 12–38
Structure of the K channel of Streptomyces lividans.
(a) Viewed in the plane of the membrane, the channel
consists of eight transmembrane helices (two from each
of the four identical subunits), forming a cone with its
wide end toward the extracellular space. The inner
helices of the cone (lighter-colored) line the transmembrane channel, and the outer helices interact with the
lipid bilayer. Short segments of each subunit converge in
the open end of the cone to make a selectivity filter.
(b) This view perpendicular to the plane of the membrane
shows the four subunits arranged around a central
channel just wide enough for a single K ion to pass. Carbonyl oxygens of the peptide backbone protrude into the
channel, interacting with and stabilizing a K ion passing
through. These ligands are perfectly positioned to interact
with K, but not with the smaller Na ion. This preferential interaction with K is the basis for the channel’s ion
selectivity. (c) Cross section showing the water-filled and
relatively broad channel at the cytosolic side (stippled
blue). In this region, a K ion (green) is stabilized by
hydration and by the partial negative charge at the
carboxyl-terminal end of the short helices projecting from
each of the four subunits. The channel narrows at the
selectivity filter, where the water of hydration is stripped
from the K and replaced with carbonyl oxygens of the
selectivity filter.
(b)
Selectivity
filter
Outside
d
d
Inside
(cytosol)
(c)
426
Part II
Structure and Catalysis
The Acetylcholine Receptor Is a Ligand-Gated Ion Channel
O
CH3
CH3
C
O
CH2
CH2
N
CH3
Acetylcholine
CH3
Another very well-studied ion channel is the nicotinic acetylcholine receptor, which is essential in the passage of an electrical signal from a motor neuron to a muscle fiber at the neuromuscular junction (signaling the
muscle to contract). (Nicotinic receptors were originally distinguished from
muscarinic receptors by the sensitivity of the former to nicotine, the latter
to the mushroom alkaloid muscarine.) Acetylcholine released by the motor
neuron diffuses a few micrometers to the plasma membrane of a myocyte,
where it binds to the acetylcholine receptor. This forces a conformational
change in the receptor, causing the ion channel intrinsic to the receptor to
open. The resulting inward movement of positive charges depolarizes the
plasma membrane of the myocyte, triggering contraction. The acetylcholine
receptor allows Na, Ca2, and K to pass through with equal ease, but
other cations and all anions are unable to pass.
Na movement through an acetylcholine receptor ion channel is unsaturable (its rate is linear with respect to extracellular Na concentration)
and very fast—about 2 107 Na ions per second under physiological
conditions.
This receptor channel is typical of many other ion channels that produce or respond to electrical signals: it has a “gate” that opens in response
to stimulation by acetylcholine, and an intrinsic timing mechanism that
closes the gate after a split second. Thus the acetylcholine signal is transient—an essential feature of electrical signal conduction. The structural
changes underlying gating in the acetylcholine receptor are known, but the
mechanism of “desensitization”—of closing the gate even in the continued
presence of acetylcholine—is not well understood.
The nicotinic acetylcholine receptor (which is structurally and functionally distinct from the muscarinic acetylcholine receptors) is composed
of five subunits: single copies of subunits b, g, and d, and two identical a
subunits each with an acetylcholine-binding site. All five subunits are related in sequence and tertiary structure, each having four transmembrane
helical segments (M1 to M4) (Fig. 12–39a). The five subunits surround a
central pore, which is lined with their M2 helices. The pore is about 20 Å
wide in the parts of the channel that protrude on the cytosolic and extracellular surfaces, but narrows as it passes through the lipid bilayer. Near
the center of the bilayer is a ring of bulky hydrophobic Leu side chains
from the M2 helices, positioned so close together that they prevent ions
from passing through the channel. Allosteric conformational changes induced by acetylcholine binding to the two a subunits include a slight twisting of the M2 helices (Fig. 12–39b), which draws these hydrophobic side
chains away from the center of the channel and opens it to the passage of
ions.
Based on similarities between the amino acid sequences of other
ligand-gated ion channels and the acetylcholine receptor, the receptor
channels that respond to the extracellular signals g-aminobutyric acid
(GABA), glycine, and serotonin are classified in an acetylcholine receptor
superfamily and probably share three-dimensional structure and gating
mechanisms. The GABAA and glycine receptors are anion channels specific
for Cl or HCO3 , whereas the serotonin receptor, like the acetylcholine receptor, is cation-specific. The subunits of each of these channels, like those
of the acetylcholine receptor, have four transmembrane helical segments
and form oligomeric channels.
A second class of ligand-gated ion channels responds to intracellular
ligands: 3,5-cyclic guanosine mononucleotide (cGMP) in the vertebrate
eye, cGMP and cAMP in olfactory neurons, and ATP and inositol 1,4,5-
Chapter 12
Biological Membranes and Transport
427
NH3
NH3
COO
COO
M4
M1
M3
Subunit folds into four
transmembrane a helices
M2
M1
M2
M3
Acetylcholine
binding sites
M4
Outside
M1 M4 M2
M3
a Subunit
(b,g,d are homologous)
g
a
a
d
b
Inside
M2 amphipathic helices
surround channel
(a)
Bulky hydrophobic
Leu side chains of
M2 helices close
the channel.
Binding of two acetylcholine
molecules causes twisting
of the M2 helices.
With receptor sites
occupied, the M2
helices have smaller,
polar residues lining
the channel.
2 Acetylcholine
Closed
Open
(b)
trisphosphate (IP3) in many cell types. These channels are composed
of multiple subunits, each with six transmembrane helical domains. We
discuss these channels in the context of their signaling functions in
Chapter 13.
figure 12–39
Structure of the acetylcholine receptor ion channel.
(a) Each of the five subunits (a2bgd) has four transmembrane helices, M1 to M4. The M2 helices are amphipathic; the others have mainly hydrophobic residues. The
five subunits are arranged around a central transmembrane channel, which is lined with the polar sides of the
M2 helices. At the top and bottom of the channel are
rings of negatively charged amino acid residues. Near the
middle of the bilayer, five Leu side chains (one from each
M2 helix) protrude into the channel, constricting it to a
diameter too small to allow passage of ions such as Ca2,
Na, and K. (b) This top view of a cross section through
the center of the M2 helices shows the blocking of the
channel by the bulky Leu side chains. When both acetylcholine receptor sites (one on each a subunit) are occupied, a conformational change occurs. As the M2 helices
twist slightly, the five Leu residues rotate away from the
channel and are replaced by smaller, polar residues. This
gating mechanism opens the channel, allowing the
passage of Ca2, Na, or K.
428
II
I
III
IV
Domain
Selectivity filter
(pore region)
Outside
1
Inside
2345
6
Activation
gate
NH3
COO
Inactivation gate
III
4
1
2
3
5
Voltage sensor
(a)
Voltage
sensor
Selectivity
filter (pore)
6
IV
Activation
gate
Outside
II
Activation
gate
Inside
Membrane polarized,
channel closed
Aqueous ion
channel
Na
Outside
Tether
Inactivation
gate (open)
I
(b)
figure 12–40
Voltage-gated Na channels of neurons. Sodium channels of various tissues and organisms have a variety of
subunits, but only the principal subunit (a) is essential.
(a) The a subunit is a large protein with four homologous
domains (I to IV), each containing six transmembrane
helices. Helix 4 in each domain (blue) is the voltage
sensor; helix 6 (orange) is believed to be the activation
gate. The segments between helices 5 and 6, the pore
region (red), form the selectivity filter, and the segment
connecting domains III and IV (green) is the inactivation
gate. (b) The four domains are wrapped about a central
transmembrane channel lined with polar amino acid
residues. The segments linking helices 5 and 6 (red) in
each domain come together near the extracellular surface
to form the selectivity filter, which is conserved in all Na
channels. The filter gives the channel its ability to discriminate between Na and other ions of similar size. The
inactivation gate (green) closes (dotted lines) soon after
the activation gate opens. (c) The voltage-sensing mechanism involves movement of helix 4 (blue) perpendicular
to the plane of the membrane in response to a change in
transmembrane potential. The strong positive charge on
helix 4 allows it to be pulled inward in response to the
inside-negative membrane potential (Vm). Depolarization
lessens this pull, and helix 4 relaxes by moving outward.
This movement is communicated to the activation gate
(orange), inducing conformational changes that open the
channel in response to depolarization.
Inside
Voltage
sensor
Na
Membrane depolarized,
channel open
(c)
The Neuronal Na Channel Is a Voltage-Gated Ion Channel
Na channels in the plasma membranes of neurons and of myocytes of
heart and skeletal muscle sense electrical gradients across the membrane
and respond by opening or closing. These voltage-gated ion channels are
typically very selective for Na over other monovalent or divalent cations
(by factors of 100 or more) and have a very high flux rate (107 ions per
second). Normally in the closed conformation, Na channels are activated
(opened) by reduction in the transmembrane electrical potential. They
then undergo very rapid inactivation. Within milliseconds of the opening,
the channel closes and remains inactive for many milliseconds. Activation
followed by inactivation of Na channels is the basis for signaling by neurons (see Fig. 13–5).
The essential component of Na channels is a single, large polypeptide
(1,840 amino acid residues) organized into four domains (Fig. 12–40)
clustered around a central channel, providing a path for Na through the
Chapter 12
Biological Membranes and Transport
429
membrane. That path is made Na-specific by a “pore region” composed of
the segments between transmembrane helices 5 and 6 of each domain, which
fold into the channel. Helix 4 of each domain has a high density of positively
charged residues. This segment is believed to move within the membrane in response to changes in the transmembrane voltage, from the “resting” potential
of about 60 mV (inside negative) to about 30 mV. The movement of helix
4 triggers opening of the channel, and this is the basis for voltage gating.
Inactivation of the channel is believed to occur by a ball-and-chain
mechanism. A protein domain on the cytosolic surface of the Na channel,
called the inactivation gate (the ball), is tethered to the channel by a short
segment of the polypeptide (the chain) (Fig. 12–40b). This domain is free to
move about when the channel is closed, but when it opens, a site on the inner face of the channel becomes available for the tethered ball to bind, blocking the channel. The length of the tether appears to determine how long an
ion channel stays open; the longer the tether, the longer the open period. Inactivation of other ion channels may occur by a similar mechanism.
Ion-Channel Function Is Measured Electrically
figure 12–41
A single ion channel typically remains open for only a few milliseconds, beyond the limit of most biochemical measurements. Ion fluxes must therefore be measured electrically, either as changes in Vm (in the millivolt
range) or as electrical currents I (in the microampere or picoampere
range), using microelectrodes and appropriate amplifiers (Fig. 12–41).
Patch-clamping, a technique in which very small currents are measured
through a tiny region of the membrane surface containing only one or a few
ion channel molecules, reveals that as many as 104 ions can move through
a single ion channel in one millisecond. This represents a huge amplification
of the initial signal; for the acetylcholine receptor, for example, the signal
may have been only a few molecules of acetylcholine.
Electrical measurements of ion channel function. The
“activity” of an ion channel is estimated by measuring the
flow of ions through it, using the patch-clamping technique. A finely drawn-out pipette (micropipette) is
pressed against a cell’s surface, and negative pressure in
the pipette is used to form a pressure seal between
pipette and membrane. When the pipette is pulled away
from the cell, it pulls off a tiny patch of membrane (which
may contain one or a few ion channels). When the pipette
and attached patch are placed in an aqueous solution,
channel activity can be measured as the electrical current
that flows between the contents of the pipette and the
aqueous solution. In practice, a circuit is set up that
“clamps” the transmembrane potential at a given value
and measures the current that must flow to maintain this
voltage. With highly sensitive current detectors,
researchers can measure the current flowing through a
single ion channel, typically a few picoamperes. The trace
showing the current as a function of time (in milliseconds) reveals how fast the channel opens and closes,
how frequently it opens, and how long it stays open.
Clamping the Vm at different values permits a determination of the effect of membrane potential on these parameters of channel function.
Channel
Micropipette applied tightly
to plasma membrane
Patch of membrane
pulled from cell
Patch of membrane
placed in aqueous
solution
Time
50ms
Inward 10pA
current
Micropipette
Electrodes
Electronics to hold transmembrane
potential (Vm) constant and measure
current flowing across membrane
430
Part II
Structure and Catalysis
Defective Ion Channels Can Have Striking Physiological Consequences
O
HO
O
O
H
H
H
N
N
H
H2N
OH
H
H
HO
CH2OH
H
H
OH
Tetrodotoxin
O
H2N
H H
N
O
HN
H2N
NH2
N
H
N
HO
OH
Saxitoxin
H3C
CH3
N
CH2
H
2Cl
OH
H
O
H3CO
OCH3
O
OH
D-Tubocurarine
CH2
H
chloride
N
CH3
The importance of ion channels to physiological processes is clear from the
effects of mutations in specific ion channel proteins. Genetic defects in the
voltage-gated Na channel of the myocyte plasma membrane result in diseases in which muscles are periodically either paralyzed (as in hyperkalemic periodic paralysis) or stiff (as in paramyotonia congenita). As noted
earlier, cystic fibrosis is the result of a mutation that changes one amino
acid in the protein CFTR, a chloride ion channel. The defective process
here is not neurotransmission, but secretion by various exocrine gland cells
whose activities are tied to Cl ion fluxes.
Naturally occurring toxins often act on ion channels, and the potency of
these toxins further illustrates the importance of normal ion channel function. Tetrodotoxin (produced by the puffer fish, Spheroides rubripes)
and saxitoxin (produced by the marine dinoflagellate Gonyaulax, which
causes the occasional phenomenon of “red tides”) are poisons that act by
binding to the voltage-gated Na channels of neurons and preventing normal action potentials. Puffer fish is an ingredient of the Japanese delicacy
fugu, which may only be prepared by chefs specially trained to separate
succulent morsel from deadly poison. Eating shellfish that have fed on
Gonyaulax can also be fatal; shellfish are not sensitive to saxitoxin, but
they concentrate it in their muscles, which become highly poisonous to organisms farther up the food chain. The venom of the black mamba snake
contains dendrotoxin, which interferes with voltage-gated K channels.
Tubocurarine, the active component of curare (used as an arrow poison in
the Amazon), and two other toxins from snake venoms, cobrotoxin and
bungarotoxin, block the acetylcholine receptor or prevent the opening of
its ion channel. By blocking signals from nerves to muscles, all of these toxins cause paralysis and possibly death. On the positive side, the extremely
high affinity of bungarotoxin for the acetylcholine receptor (Kd 1015 M)
proved useful when radiolabeled toxin was used to quantify the receptor
during its purification.
Porins Are Transmembrane Channels for Small Molecules
In the outer membrane of gram-negative bacteria such as E. coli, protein
channels called porins allow the passage of molecules much larger than
ions, but by a mechanism more like a gated channel than a transporter. The
porin FhuA (Fig. 12–18) serves in E. coli to bring iron (in the form of a
chelate of ferrichrome) from the extracellular medium across the outer
membrane and into the periplasmic space. (In gram-negative bacteria, the
periplasm is the material between the inner (plasma) and outer membranes.) FhuA is composed of a large 22-stranded b-barrel domain of about
560 residues and an amino-terminal “cork” domain of 160 residues, which
normally obstructs the barrel and keeps the channel closed (Fig. 12–42).
Binding of the ferrichrome complex to a specific site on the outer surface of
the cell triggers allosteric changes that move the ferrichrome into the barrel and allow interaction of the FhuA protein with proteins of the inner
membrane and periplasm. This interaction moves the cork out of the barrel
and allows passage of the ferrichrome through the channel. With the emptying of the ferrichrome binding site, the changes are reversed and the
channel closes.
The transport systems discussed in this chapter are summarized in Figure
12–43.
Chapter 12
(a)
Biological Membranes and Transport
(b)
figure 12–42
Structure of FhuA, an iron transporter from E. coli. This
protein of the E. coli outer membrane transports iron ion
into the cell in the form of a ferrichrome-iron chelate.
Figure 12–18 shows a ribbon representation of the
protein viewed parallel to the membrane. Here we see
(a) ribbon and (b) space-filling models, viewed perpendicular to the membrane. The 22-stranded b barrel (gray)
is seen as a hollow pipe, plugged at one end by the cork
domain (red). The binding of a ferrichrome-iron molecule
at a specific site on the outer surface of FhuA drives a
conformational change that helps to move the plug out of
the channel; the ferrichrome-iron then moves through
the channel, and the transporter resumes its closed
conformation.
Sout
Facilitated diffusion
(down electrochemical
gradient)
Simple diffusion
(nonpolar compounds only,
down concentration
gradient)
Sout
Primary active
transport (against
electrochemical
gradient)
Sin
Sout
ATP
Sin
Sin
Ionophoremediated
ion transport
(down
electrochemical
gradient)
ADP + Pi
Ion
Ion
Sin
Ion
Ion
Sout
figure 12–43
Summary of transport types.
Ion channel
(down
electrochemical
gradient; may
be gated by a
ligand or ion)
Ion
Ion
Secondary
active transport
(against electrochemical
gradient, driven by ion
moving down its gradient)
431
432
Part II
Structure and Catalysis
summary
Biological membranes are central to life. They
define cellular boundaries, divide cells into discrete compartments, organize complex reaction
sequences, and act in signal reception and energy transformations. Membranes are composed
of lipids and proteins in varying combinations
particular to each species, cell type, and organelle. The fluid mosaic model describes features common to all biological membranes. The
lipid bilayer is the basic structural unit. Fatty
acyl chains of phospholipids and the steroid nucleus of sterols are oriented toward the interior
of the bilayer; their hydrophobic interactions stabilize the bilayer but allow the structure to be
flexible. Lipids and most proteins are free to diffuse laterally within the membrane, and the hydrophobic moieties of the lipids undergo rapid
thermal motion, making the interior of the bilayer fluid. Fluidity is affected by temperature,
fatty acid composition, and sterol content. Cells
regulate their lipid compositions to maintain a
constant fluidity when external circumstances
change.
Peripheral proteins are loosely associated
with the membrane through electrostatic interactions and hydrogen bonds or by covalently attached lipid anchors. Integral proteins associate
firmly with membranes by hydrophobic interactions between the lipid bilayer and their nonpolar amino acid side chains, which are oriented toward the outside of the protein molecule. Some
membrane proteins span the lipid bilayer several
times, with hydrophobic sequences of about 20
amino acid residues forming transmembrane a
helices. Such hydrophobic sequences detected
in the primary structures of proteins can be used
to predict the secondary structure and transmembrane disposition of these molecules. Multistranded b barrels are also common in integral
membrane proteins.
The lipids and proteins of membranes are inserted into the bilayer with specific sidedness;
thus, membranes are structurally and functionally asymmetric. Many membrane proteins contain covalently attached oligosaccharides of various degrees of complexity. Plasma membrane
glycoproteins are always oriented with the
carbohydrate-bearing domain on the extracellular surface. A variety of proteins mediate the
fusion of two membranes, which accompanies
processes such as endocytosis and exocytosis.
Fusion requires close apposition of two mem-
branes and local disruption of the lipid bilayers,
both processes attributed to fusion proteins
called SNAREs.
The lipid bilayer is impermeable to polar substances. Water is an important exception; it can
diffuse passively across the bilayer. Cells that require large fluxes of water have water-specific
channels called aquaporins in their plasma membranes. Other polar species cross biological
membranes only by way of specific membrane
proteins—transporters and ion channels.
Transporters (transport proteins), like enzymes, show saturation and substrate specificity.
Transport via these systems may be passive
(down the electrochemical gradient, hence independent of metabolic energy) or active (against
the gradient and thus dependent on metabolic
energy). Glucose transport into erythrocytes and
water transport through aquaporins is passive.
The energy input for active transport may come
from light, oxidation reactions, ATP hydrolysis,
or cotransport of some other solute. Some transporters carry out symport, the simultaneous passage of two substances in the same direction;
others mediate antiport, in which two substances
move simultaneously in opposite directions. The
lactose transporter of E. coli and the glucose
transporter of intestinal epithelial cells are symporters, both cotransporting Na along with the
sugar. The chloride-bicarbonate exchanger of
erythrocytes and the ubiquitous NaK ATPase
are antiporters. In animal cells, the differences in
cytosolic and extracellular concentrations of Na
and K are established and maintained by active
transport through the NaK ATPase. The resulting Na gradient across the plasma membrane is used as an energy source for a variety of
secondary active transport processes, both symport and antiport.
In primary active transport, ATP serves directly as the energy source. There are four general types of ATP-driven active transporters.
P-type ATPases undergo reversible phosphorylation during their catalytic cycle and are inhibited
by the phosphate analog vanadate; examples are
the NaK ATPase of the plasma membrane and
the Ca2 transporters of the sarcoplasmic and
endoplasmic reticulum (the SERCA pumps).
V-type ATPases produce gradients of protons
across a variety of intracellular membranes, including plant vacuolar membranes. F-type proton pumps (ATP synthases) are central to
Chapter 12
energy-conserving mechanisms in mitochondria
and chloroplasts. A multidrug transporter in tumor cells uses ATP to drive the export of a variety of drugs. Ionophores are lipid-soluble molecules that bind specific ions and carry them
passively across membranes, dissipating the energy of electrochemical ion gradients.
Ion channels provide hydrophilic pores
through which select ions can diffuse, moving
down their electrical or chemical concentration
gradients. They are characteristically unsaturable and have very high flux rates. Many are
highly specific for one ion, and most are gated by
either voltage or a ligand. The structure of the
Biological Membranes and Transport
433
K channel is known and serves as the prototype
for other channels such as the acetylcholine
receptor ion channel and the voltage-gated Na
channel. Several a-helical transmembrane domains cluster about a central aqueous channel.
Ion passage is restricted by a selectivity filter and
by a gate that obstructs the ion channel when it
is not activated by voltage or a bound ligand.
Porins are integral membrane proteins that
consist of a b barrel with a central transmembrane opening. A globular protein domain hinders passage of ion, but the binding of a specific
substrate transiently opens the central pore, allowing uptake of that substrate.
further reading
Membrane Architecture
Membrane Fusion
Devaux, P.F. (1991) Static and dynamic lipid asymmetry in cell membranes. Biochemistry 30, 1163–
1173.
Nichols, B.J. & Pelham, H.R. (1998) SNAREs and
membrane fusion in the Golgi. Biochim. Biophys.
Acta 1404, 9–31.
Frye, L.D. & Ediden, M. (1970) The rapid intermixing of cell-surface antigens after formation of
mouse-human heterokaryons. J. Cell Sci. 7, 319–335.
The classic demonstration of membrane protein mobility.
Haltia, T. & Freire, E. (1995) Forces and factors
that contribute to the structural stability of membrane
proteins. Biochim. Biophys. Acta 1241, 295–322.
Good discussion of the secondary and tertiary structures of membrane proteins and the factors that stabilize them.
Jacobson, K., Sheets, E.D., & Simson, R. (1995)
Revisiting the fluid mosaic model of membranes. Science 268, 1441–1442.
Discussion of the restricted mobility of membrane
proteins, updating the model in paper by Singer and
Nicolson (below).
Singer, S.J. & Nicolson, G.L. (1972) The fluid mosaic model of the structure of cell membranes. Science 175, 720–731.
The classic statement of the model.
von Heijne, G. (1994) Membrane proteins: from sequence to structure. Annu. Rev. Biophys. Biomol.
Struct. 23, 167–192.
A review of the steps required to predict the structure of an integral protein from its sequence.
White, S.H. & Wimley, W.C. (1999) Membrane protein folding and stability: Physical principles. Annu.
Rev. Biophys. Biomol. Struct. 28, 319–365.
Transporters
Lienhard, F.E., Slot, J.W., James, D.E., &
Mueckler, M.M. (1992) How cells absorb glucose.
Sci. Am. 266 (January), 86–91.
Introduction to the glucose transporter and its regulation by insulin.
Lingrel, J.B. & Kuntzweiler, T. (1994) Na, KATPase. J. Biol. Chem. 269, 19,659–19,662.
Møller, J.V., Juul, B., & LeMaire, M. (1996)
Structural organization, ion transport, and energy
transduction of P-type ATPases. Biochim. Biophys.
Acta 1286, 1–51.
Mueckler, M. (1994) Facilitative glucose transporters. Eur. J. Biochem. 219, 713–725.
Postle, K. (1999) Active transport by customized
b-barrels. Nat. Struct. Biol. 6, 3–6.
Introductory description of the structure of the E.
coli porins and their coupling to metabolic energy.
Sheppard, D.N. & Welsh, M.J. (1999) Structure
and function of the CFTR chloride channel. Physiolog. Rev. 79, S23–S46.
This issue of the journal has 11 reviews of the CFTR
chloride channel, covering its structure, activity, regulation, biosynthesis, and pathophysiology.
434
Part II
Structure and Catalysis
Ion Channels
Aidley, D.J. & Stanfield, P.R. (1996) Ion Channels:
Molecules in Action, Cambridge University Press.
Excellent chapters on molecular structure, gating,
and drug interactions of ion channels, and human
diseases that result from defective channels.
Changeux, J.P. (1993) Chemical signaling in the
brain. Sci. Am. 269 (November), 58–62.
Structure and function of the acetylcholine receptor
channel.
Doyle, D.A., Cabral, K.M., Pfuetzner, R.A., Kuo,
A., Gulbis, J.M., Cohen, S.L., Chait, B.T., &
MacKinnon, R. (1998) The structure of the potassium channel: Molecular basis of K conduction and
selectivity. Science 280, 69–77.
The first crystal structure of an ion channel.
Edelstein, S.J. & Changeux, J.P. (1998) Allosteric
transitions of the acetylcholine receptor. Adv. Prot.
Chem. 51, 121–184.
Advanced discussion of the conformational changes
induced by acetylcholine.
Marban, E., Yamagishi, T., & Tomaselli, G.F.
(1998) Structure and function of voltage-gated
sodium channels. J. Physiol. 508, 647–657.
Review of evolution, structure, gating, drug interactions, and synthesis of voltage-gated Na channels.
Neher, E. & Sakmann, B. (1992) The patch clamp
technique. Sci. Am. (March) 266, 44–51.
Clear description of the electrophysiological techniques used to measure the activity of single ion
channels, by their Nobel Prize-winning developers.
Perozo, E., Cortes, D.M., & Cuello, L.G. (1999)
Structural rearrangements underlying K channel activation gating. Science 285, 73–78.
Physical studies of the mechanism by which the K
channel opens and closes.
Unwin, N. (1998) The nicotinic acetylcholine receptor of the Torpedo electric ray. J. Struct. Biol. 121,
181–190.
Evidence for the structural change that accompanies
channel closing.
Hille, B. (1992) Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, Massachusetts.
Emphasizes the function of ion channels; good complement to Aidley and Stanfield (above).
problems
Force applied here
to compress
monolayer
(a)
40
Force (dyne/cm)
1. Determining the Cross-Sectional Area of a
Lipid Molecule When phospholipids are layered
gently onto the surface of water, they orient at the airwater interface with their head groups in the water
and their hydrophobic tails in the air. An experimental
apparatus (a) has been devised that reduces the surface area available to a layer of lipids. By measuring
the force necessary to push the lipids together, it is
possible to determine when the molecules are packed
tightly in a continuous monolayer; when that area is
approached, the force needed to further reduce the
surface area increases sharply (b). How would you
use this apparatus to determine the average area occupied by a single lipid molecule in the monolayer?
30
20
10
0.2
0.6
1.0
Area (nm2/molecule)
1.4
(b)
2. Evidence for Lipid Bilayer In 1925, E. Gorter
and F. Grendel used an apparatus like that described
in Problem 1 to determine the surface area of a lipid
monolayer formed by lipids extracted from erythrocytes of several animal species. They used a microscope to measure the dimensions of individual cells,
from which they calculated the average surface area
of one erythrocyte. They obtained the data shown on
the next page. Were these investigators justified in
Chapter 12
Biological Membranes and Transport
concluding that “chromocytes [erythrocytes] are covered by a layer of fatty substances that is two molecules thick” (i.e., a lipid bilayer)?
Animal
Volume of
packed
cells (mL)
Number
of cells
(per mm3)
Total surface
area of lipid
monolayer
from cells (m2)
Total surface
area of one
cell (mm2)
Dog
Sheep
Human
40
10
1
8,000,000
9,900,000
4,740,000
62
6.0
0.92
98
29.8
99.4
Source: Data from Gorter, E. & Grendel, F. (1925) On bimolecular layers of lipoids on the
chromocytes of the blood. J. Exp. Med. 41, 439–443.
3. Number of Detergent Molecules per Micelle
When a small amount of sodium dodecyl sulfate
(NaCH3(CH2)11OSO3 ) is dissolved in water, the detergent ions enter the solution as monomeric species.
As more detergent is added, a concentration is
reached (the critical micelle concentration) at which
the monomers associate to form micelles. The critical
micelle concentration of SDS is 8.2 mM. The micelles
have an average particle weight (the sum of the molecular weights of the constituent monomers) of 18,000.
Calculate the number of detergent molecules in the
average micelle.
free-energy change for ATP hydrolysis under cellular
conditions is about 58 kJ/mol (as we will explain in
Chapter 14). Ignore the effects of the transmembrane electric potential.
4. Properties of Lipids and Lipid Bilayers Lipid
bilayers formed between two aqueous phases have
this important property: they form two-dimensional
sheets, the edges of which close upon each other and
undergo self-sealing to form liposomes.
9. Action of Ouabain on Kidney Tissue Ouabain
specifically inhibits the NaK ATPase activity of animal tissues but is not known to inhibit any other enzyme. When ouabain is added to thin slices of living
kidney tissue, it inhibits oxygen consumption by 66%.
Why? What does this observation tell us about the use
of respiratory energy by kidney tissue?
(a) What properties of lipids are responsible for
this property of bilayers? Explain.
(b) What are the consequences of this property
with regard to the structure of biological membranes?
5. Length of a Fatty Acid Molecule The carbon–
carbon bond distance for single-bonded carbons such
as those in a saturated fatty acyl chain is about 1.5 Å.
Estimate the length of a single molecule of palmitate
in its fully extended form. If two molecules of palmitate were placed end to end, how would their total
length compare with the thickness of the lipid bilayer
in a biological membrane?
6. Temperature Dependence of Lateral Diffusion The experiment described in Figure 12–7 was
performed at 37 C. If the experiment were carried out
at 10 C, what effect would you expect on the rate of
cell-cell fusion and the rate of membrane protein mixing? Why?
7. Synthesis of Gastric Juice: Energetics Gastric juice (pH 1.5) is produced by pumping HCl from
blood plasma (pH 7.4) into the stomach. Calculate
the amount of free energy required to concentrate
the H in 1 L of gastric juice at 37 C. Under cellular
conditions, how many moles of ATP must be hydrolyzed to provide this amount of free energy? The
8. Energetics of the NaK ATPase The concentration of Na inside a vertebrate cell is about 12 mM,
and that in blood plasma is about 145 mM. For a typical cell with a transmembrane potential of –0.07 V
(inside negative relative to outside), what is the freeenergy change for transporting 1 mol of Na out of the
cell and into the blood at 37 C?
10. Energetics of Symport Suppose that you determined experimentally that a cellular transport system
for glucose, driven by symport of Na, could accumulate glucose to concentrations 25 times greater than in
the external medium, while the external [Na] was
only ten times greater than the intracellular [Na].
Would this violate the laws of thermodynamics? If not,
how could you explain this observation?
11. Location of a Membrane Protein The following observations are made on an unknown membrane
protein, X. It can be extracted from disrupted erythrocyte membranes into a concentrated salt solution,
and the isolated X can be cleaved into fragments by
proteolytic enzymes. Treatment of erythrocytes with
proteolytic enzymes followed by disruption and extraction of membrane components yields intact X.
However, treatment of erythrocyte “ghosts” (which
consist of only membranes, produced by disrupting
the cells and washing out the hemoglobin) with proteolytic enzymes followed by disruption and extraction
yields extensively fragmented X. What do these observations indicate about the location of X in the plasma
membrane? Do the properties of X resemble those of
an integral or peripheral membrane protein?
435
436
Part II
Structure and Catalysis
12. Membrane Self-Sealing Cellular membranes are
self-sealing—if they are punctured or disrupted mechanically, they quickly and automatically reseal. What
properties of membranes are responsible for this important feature?
13. Lipid Melting Temperatures Membrane lipids
in tissue samples obtained from different parts of the
leg of a reindeer have different fatty acid compositions. Membrane lipids from tissue near the hooves
contain a larger proportion of unsaturated fatty acids
than those from tissue in the upper leg. What is the
significance of this observation?
14. Flip-Flop Diffusion The inner face (monolayer)
of the human erythrocyte membrane consists predominantly of phosphatidylethanolamine and phosphatidylserine. The outer face consists predominantly
of phosphatidylcholine and sphingomyelin. Although
the phospholipid components of the membrane can
diffuse in the fluid bilayer, this sidedness is preserved
at all times. How?
15. Membrane Permeability At pH 7, tryptophan
crosses a lipid bilayer about 1,000 times more slowly
than does the closely related substance indole:
N
H
Suggest an explanation for this observation.
16. Water Flow through an Aquaporin Each human erythrocyte has about 2 105 AQP-1 monomers.
If water flows through the plasma membrane at a rate
of 5 108 H2O molecules per AQP-1 tetramer per second, and the volume of an erythrocyte is 5 1011 mL,
how rapidly could an erythrocyte halve its volume as it
encounters the high osmolarity (1 M) in the interstitial
fluid of the renal medulla?
17. Labeling the Galactoside Transporter The
galactoside transporter of a bacterium, which is highly
specific for its substrate lactose, contains a Cys residue
that is essential to its transport activity. Covalent reaction of N-ethylmaleimide (NEM) with this Cys residue
irreversibly inactivates the transporter. A high concentration of lactose in the medium prevents inactivation
by NEM, presumably by sterically protecting the Cys
residue, which is in or near the lactose binding site. You
know nothing else about the transporter protein. Suggest an experiment that might allow you to determine
the Mr of the Cys-containing transporter polypeptide.
18. Predicting Membrane Protein Topology from
Sequence You have cloned the gene for a human erythrocyte protein, which you suspect is a membrane
protein. From the nucleotide sequence of the gene, you
know the amino acid sequence. From this sequence
alone, how would you evaluate the possibility that the
protein is an integral protein? Suppose the protein
proves to be an integral protein, either type I or type II.
Suggest biochemical or chemical experiments that
might allow you to determine which type it is.
19. Intestinal Uptake of Leucine You are studying
the uptake of L-leucine by epithelial cells of the mouse
intestine. Measurements of the rate of uptake of Lleucine and several of its analogs, with and without
Na in the assay buffer, yield the following results.
What can you conclude about the properties and
mechanism of the leucine transporter? Would you expect L-leucine uptake to be inhibited by ouabain?
Substrate
L-Leucine
D-Leucine
L-Valine
Uptake in
presence of Na
Uptake in
absence of Na
Vmax
K t (mM)
Vmax
K t (mM)
420
310
225
0.24
4.7
0.31
23
5
19
0.24
4.7
0.31
20. Effect of an Ionophore on Active Transport
Consider the leucine transporter described in Problem
19. Would Vmax and/or Kt change if you added a Na
ionophore to the assay solution containing Na?
Explain.
21. Surface Density of a Membrane Protein
E. coli can be induced to make about 10,000 copies of
the galactoside transporter (Mr 31,000) per cell. Assume that E. coli is a cylinder 1 mm in diameter and
2 mm long. What fraction of the plasma membrane surface is occupied by the galactoside transporter molecules? Explain how you arrived at this conclusion.
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22. Membrane Protein Topology The receptor for
the hormone epinephrine in animal cells is an integral
membrane protein (Mr 64,000) that is believed to have
seven membrane-spanning regions.
(a) Show that a protein of this size is capable of
spanning the membrane seven times.
(b) Given the amino acid sequence of this protein,
how would you predict which regions of the protein
form the membrane-spanning helices?
(c) Go to the Protein Data Bank Web site. Use the
PDB identifier 1DEP to retrieve the data page for a
portion of the b-adrenergic receptor (one type of epinephrine receptor) from turkey. Using Chime to explore the structure, predict where this portion of the
receptor is located: within the membrane or at the
membrane surface. Explain.
(d) Retrieve the data for a portion of another receptor, the acetylcholine receptor of neurons and myocytes, using the PDB identifier 1A11. As in (c), predict where this portion of the receptor is located and
explain your answer.
If you have not used the PDB or Chemscape Chime,
you can find instructions at
http://www.worthpublishers.com/lehninger.