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Problems 743

1. Explain the following observations: (a) Thyroidectomized

rats, when deprived of food, survive for 20 days while normal rats

starve to death within 7 days. (b) Cushing’s syndrome, which re-

sults from excessive secretion of adrenocortical steroids, can be

caused by a pituitary tumor. (c) Diabetes insipidus, which is char-

acterized by unceasing urination and unquenchable thirst, results

from an injury to the pituitary. (d) The growth of malignant tu-

mors derived from sex organs may be slowed or even reversed by

the surgical removal of the gonads and the adrenal glands.

2. How does the presence of the nonhydrolyzable GTP analog

GMPPNP affect cAMP-dependent receptor systems?

3. Explain why individuals who regularly handle dynamite

(which is nitroglycerin soaked into an absorbant such as wood

pulp) as part of their jobs have an unusually high incidence of

heart attacks on weekends.

4. A dose-dependent side effect of sildenafil (Viagra) is the

transient impairment of blue/green color discrimination. What is

the biochemical basis for this phenomenon?

5. Retroviruses bearing oncogenes will infect cells from their

corresponding host animal but will usually not transform them.

Yet these retroviruses will readily transform immortalized cells

derived from the same organism. Explain.

6. Explain why mutations of the Arg residue in G

s

that is

ADP-ribosylated by cholera toxin are oncogenic mutations. Why

doesn’t cholera toxin cause cancer?

7. Would the following alterations to Src be oncogenic? Ex-

plain. (a) The deletion or inactivation of the SH3 domain. (b) The

mutation of Tyr 416 to Phe. (c) The mutation of Tyr 527 to

Phe. (d) The replacement of Src residues 249 to 253 with the

sequence APTMP.

8. JIP-1 was originally so named because,when it was first char-

acterized by overexpression in mammalian cells, it appeared to act

as a “JNK inhibitor protein.”What is the basis of this observation?

9. Why does pertussis toxin appear to inhibit certain isozymes

of PLC? Identify these isozymes.

10. Phosphatidylethanolamine and PIP

containing identical

fatty acyl residues can be hydrolyzed with the same efficiency by a

certain phospholipase C. Will the hydrolysis products of the two

lipids have the same effect on protein kinase C? Explain.

11. PKC’s autoinhibitory pseudosubstrate occurs at its N-

terminus, whereas that of MLCK occurs at its C-terminus (Fig.

18-20). To further investigate this phenomenon, a colleague pro-

poses to construct a PKC with its pseudosubstrate attached to the

protein’s C-terminus with a sufficiently long linker so that the

pseudosubstrate could bind in the enzyme’s active site. Would you

expect this variant PKC to be activatable? Explain.

PROBLEMS

JWCL281_c19_671-743.qxd 3/16/10 7:17 PM Page 743

744

CHAPTER 20

Transport through

Membranes

1 Thermodynamics of Transport

2 Kinetics and Mechanisms of Transport

A. Nonmediated Transport

B. Kinetics of Mediated Transport: Glucose Transport

Into Erythrocytes

C. Ionophores

D. Maltoporin: The Structural Basis of Sugar Discrimination

E. Passive-Mediated Glucose Transport

F. K

⫹

Channels: Ion Discrimination

G. Cl

⫺

Channels

H. Aquaporins

3 ATP-Driven Active Transport

A. (Na

⫹

–K

⫹

)–ATPase of Plasma Membranes

B. Ca

2⫹

–ATPase

C. (H

⫹

–K

⫹

)–ATPase of Gastric Mucosa

D. Group Translocation

E. ABC Transporters

4 Ion Gradient–Driven Active Transport

A. Na

⫹

–Glucose Symport

B. Lactose Permease

C. ATP–ADP Translocator

5 Neurotransmission

A. Voltage-Gated Ion Channels

B. Action Potentials

C. Neurotransmitters and Their Receptors

Metabolism occurs within cells that are separated from

their environments by plasma membranes. Eukaryotic

cells, in addition, are compartmentalized by intracellular

membranes that form the boundaries and internal struc-

tures of their various organelles. The nonpolar cores of bi-

ological membranes make them highly impermeable to

most ionic and polar substances, so that these substances

can traverse membranes only through the action of specific

transport proteins. Such proteins are therefore required to

mediate all transmembrane movements of ions, such as

⫹

,Ca

2⫹

, and Cl

⫺

, as well as metabolites such as pyru-

vate, amino acids, sugars, and nucleotides, and even water

(despite its relatively high permeability in bilayers; Section

12-2B). Transport proteins are also responsible for all bio-

logical electrochemical phenomena such as neurotransmis-

sion. In this chapter, we discuss the thermodynamics, kinet-

ics, and chemical mechanisms of these membrane transport

systems and end with a discussion of the mechanism of

neurotransmission.

1 THERMODYNAMICS OF TRANSPORT

As we saw in Section 3-4A, the free energy of a solute, A,

varies with its concentration:

[20.1]

where is the chemical potential (partial molar free en-

ergy) of A (the bar indicates quantity per mole) and is

the chemical potential of its standard state. Strictly speak-

ing, this equation applies only to ideal solutions; for non-

ideal (real) solutions, molar concentrations must be re-

placed by activities (Appendix to Chapter 3). In the dilute

(millimolar) solutions that are characteristic of laboratory

conditions, the activity of a substance closely approaches

its molar concentration in value. However, this is not the

case in the highly concentrated cellular milieu (Appendix

to Chapter 3). Yet it is difficult to determine the activity of

a substance in a cellular compartment. Hence, in the fol-

lowing derivations, we shall make the simplifying assump-

tion that activities are equal to molar concentrations.

The diffusion of a substance between two sides of a

membrane

thermodynamically resembles a chemical equilibration.

A difference in the concentrations of the substance on

two sides of a membrane generates a chemical potential

difference:

[20.2]

Consequently, if the concentration of A outside the mem-

brane is greater than that inside, for the transfer of A

from outside to inside will be negative and the spontaneous

net flow of A will be inward. If, however, [A] is greater inside

than outside, is positive and an inward net flow of A can

only occur if an exergonic process, such as ATP hydrolysis, is

coupled to it to make the overall free energy change negative.

¢G

⫽ G

(in) ⫺ G

(out) ⫽ RT lna

[A]

out

A(out) Δ A(in)

°¿

⫺ G

°¿ ⫽ RT ln[A]

JWCL281_c20_744-788.qxd 3/17/10 1:47 PM Page 744

a. Membrane Potentials Arise from Transmembrane

Concentration Differences of Ionic Substances

The permeabilities of biological membranes to ions

such as H

⫹

,Na

⫹

,Cl

⫺

, and Ca

2⫹

are controlled by spe-

cific membrane-embedded transport systems that we shall

discuss in later sections. The resulting charge differences

across a biological membrane generate an electric potential

difference, ⌬⌿ ⫽ ⌿(in) ⫺⌿(out), where ⌬⌿ is termed the

membrane potential. Consequently, if A is ionic, Eq. [20.2]

must be amended to include the electrical work required to

transfer a mole of A across the membrane from outside to

inside:

[20.3]

where Z

is the ionic charge of A; f, the Faraday constant,

is the charge of one mole of electrons (96,485 C ⴢ mol

⫺1

);

and is now termed the electrochemical potential of A.

Membrane potentials in living cells can be measured di-

rectly with microelectrodes. ⌬⌿ values of ⫺100 mV (inside

negative) are not uncommon (note that 1 V ⫽ 1 J ⴢ C

⫺1

Thus the last term of Eq. [20.3] is often significant for ionic

substances.

2 KINETICS AND MECHANISMS

OF TRANSPORT

Thermodynamics indicates whether a given transport

process will be spontaneous but, as we saw for chemical and

enzymatic reactions, provides no indication of the rates of

these processes. Kinetic analyses of transport processes to-

gether with mechanistic studies have nevertheless permit-

ted these processes to be characterized. There are two

types of transport processes: nonmediated transport and

mediated transport. Nonmediated transport occurs through

simple diffusion. In contrast, mediated transport occurs

through the action of specific carrier proteins that are vari-

ously called carriers, permeases, porters, translocases,

translocators, and transporters. Mediated transport is fur-

ther classified into two categories depending on the ther-

modynamics of the system:

1. Passive-mediated transport or facilitated diffusion in

which specific molecules flow from high concentration to

low concentration so as to equilibrate their concentration

gradients.

2. Active transport in which specific molecules are

transported from low concentration to high concentration,

that is, against their concentration gradients. Such an en-

dergonic process must be coupled to a sufficiently exer-

gonic process to make it favorable.

In this section, we consider the nature of nonmediated

transport and then compare it to passive-mediated trans-

port as exemplified by ionophores, porins, glucose trans-

porters, K

⫹

channels, Cl

⫺

channels, and aquaporins. Active

transport is examined in succeeding sections.

¢G

⫽ RT lna

[A]

out

b⫹ Z

f ¢␺

A. Nonmediated Transport

The driving force for the nonmediated flow of a substance A

through a medium is A’s electrochemical potential gradient.

This relationship is expressed by the Nernst–Planck

equation:

[20.4]

where J

is the flux (rate of passage per unit area) of A, x is

distance, is the electrochemical potential gradient

of A, and U

is its mobility (velocity per unit force) in the

medium. If we assume, for simplicity, that A is an un-

charged molecule so that is given by Eq. [20.1], the

Nernst–Planck equation reduces to

[20.5]

where D

K RTU

is the diffusion coefficient of A in the

medium of interest. This is Fick’s first law of diffusion,

which states that a substance diffuses in the direction that

eliminates its concentration gradient, d[A]/dx, at a rate pro-

portional to the magnitude of this gradient.

For a membrane of thickness x, Eq. [20.5] is approxi-

mated by

[20.6]

where D

is the diffusion coefficient of A inside the mem-

brane and P

⫽ D

/x is termed the membrane’s perme-

ability coefficient for A. The permeability coefficient is in-

dicative of the solute’s tendency to transfer from the

aqueous solvent to the membrane’s nonpolar core. It

should therefore vary with the ratio of the solute’s solubil-

ity in a nonpolar solvent resembling the membrane’s core

(e.g., olive oil) to that in water, a quantity known as the

solute’s partition coefficient between the two solvents. In-

deed, the fluxes of many nonelectrolytes across erythrocyte

membranes vary linearly with their concentration differences

across the membrane as predicted by Eq. [20.6] (Fig. 20-1).

Moreover,their permeability coefficients, as obtained from

the slopes of plots such as Fig. 20-1, correlate rather well

⫽

([A]

out

⫺ [A]

) ⫽ P

([A]

out

⫺ [A]

)

⫽⫺D

(d[A])>dx

>dx

⫽⫺[A]U

(dG

>dx)

Section 20-2. Kinetics and Mechanisms of Transport 745

[ A ]

out

– [ A ]

Slope = P

Figure 20-1 Linear relationship between diffusional flux (J

)

and ([A]

out

⫺ [A]

) across a semipermeable membrane. See

Eq. [20.6].

JWCL281_c20_744-788.qxd 6/4/10 12:13 PM Page 745

with their measured partition coefficients between nonpo-

lar solvents and water (Fig. 20-2).

B. Kinetics of Mediated Transport: Glucose

Transport Into Erythrocytes

Despite the success of the foregoing model in predicting

the rates at which many molecules pass through mem-

branes, there are numerous combinations of solutes and

membranes that do not obey Eq. [20.6]. The flux in such a

system is not linear with the solute concentration differ-

ence across the corresponding membrane (Fig. 20-3) and,

furthermore, the solute’s permeability coefficient is much

larger than is expected on the basis of its partition coeffi-

cient. Such behavior indicates that these solutes are con-

veyed across membranes in complex with carrier molecules;

that is, they undergo mediated transport.

The system that transports glucose across the erythro-

cyte membrane provides a well-characterized example of

passive-mediated transport: It invariably transports glu-

cose down its concentration gradient but not at the rate

predicted by Eq. [20.6]. Indeed, the erythrocyte glucose

transporter exhibits four characteristics that differentiate

mediated from nonmediated transport: (1) speed and

746 Chapter 20. Transport Through Membranes

Figure 20-2 Permeability correlates with membrane solubility.

The permeability coefficients of various organic molecules in

plasma membranes from the alga Nitella mucronata versus their

partition coefficients between olive oil and water (a measure of a

molecule’s polarity).This more or less linear log–log plot

Figure 20-3 Variation of glucose flux into human erythrocytes

with the external glucose concentration at 5°C. The black dots

are experimentally determined data points, and the solid green

line is computed from Eq. [20.7] with J

max

⫽ 1.0 ⫻ 10

⫺6

mM ⴢ

cm ⴢ s

⫺1

and K

⫽ 0.5 mM.The nonmediated glucose flux

increases linearly with [glucose] (Fig. 20-1) but would not visibly

depart from the baseline on the scale of this drawing. [Based on

data from Stein,W.D., Movement of Molecules across

Membranes, p. 134, Academic Press (1967).]

Permeability coefficient (cm

–1

)

0.001 0.010.0001

Oil–water partition coefficient

Ethylurea

Urea

Ethylene glycol

Acetamide

Succinimide

N,N'-Diethylurea

n-Butyramide

Propionamide

Formamide

–7

–6

–5

HO OH

NHH

NCH

NHHN

indicates that the rate-limiting step for the nonmediated entry of

a molecule into a cell is its passage through the membrane’s

hydrophobic core. [Based on data from Collander, R., Physiol.

Plant. 7, 433–434 (1954).]

glucose

(mM

–1

× 10

)

1.0

0.5

2468

100

[Glucose] mM

max

= 1.0 × 10

–6

–1

max

JWCL281_c20_744-788.qxd 3/17/10 1:47 PM Page 746

specificity, (2) saturation kinetics, (3) susceptibility to com-

petitive inhibition, and (4) susceptibility to chemical inacti-

vation. In the following paragraphs we shall see how the

erythrocyte glucose transporter exhibits these qualities.

a. Speed and Specificity

Table 20-1 indicates that the permeability coefficients of

D-glucose and D-mannitol in synthetic bilayers, and that of

D-mannitol in the erythrocyte membrane, are in reasonable

agreement with the values calculated from the diffusion

and partition coefficients of these sugars between water

and olive oil. However, the experimentally determined

permeability coefficient for

D-glucose in the erythrocyte

membrane is four orders of magnitude greater than its pre-

dicted value. The erythrocyte membrane must therefore

contain a system that rapidly transports glucose and that can

distinguish

D-glucose from D-mannitol.

b. Saturation Kinetics

The concentration dependence of glucose transport in-

dicates that its flux obeys the relationship

[20.7]

This saturation function has a familiar hyperbolic form

(Fig. 20-3). We have seen it in the equation describing the

binding of O

to myoglobin (Eq. [10.4]) and in the

Michaelis–Menten equation describing the rates of enzy-

matic reactions (Eq. [14.24]). Here, as before, K

may be

defined operationally as the concentration of glucose

when the transport flux is half of its maximal rate, J

max

/2.

This observation of saturation kinetics for glucose trans-

port was the first evidence that a specific, saturatable

⫽

max

[A]

⫹ [A]

number of sites on the membrane were involved in the

transport of any substance.

The transport process can be described by a simple four-

step kinetic scheme involving binding, transport, dissocia-

tion, and recovery (Fig. 20-4). Its binding and dissociation

steps are analogous to the recognition of a substrate and

the release of product by an enzyme. The mechanisms of

transport and recovery are discussed in Section 20-2D.

c. Susceptibility to Competitive Inhibition

Many compounds structurally similar to D-glucose in-

hibit glucose transport. A double-reciprocal plot (Section

14-2B) for the flux of glucose into erythrocytes in the pres-

ence or absence of 6-O-benzyl-

D-galactose (Fig. 20-5)

shows behavior typical of competitive inhibition of glucose

transport (competitive inhibition of enzymes is discussed

in Section 14-3A). Susceptibility to competitive inhibition

indicates that there is a limited number of sites available for

mediated transport.

Section 20-2. Kinetics and Mechanisms of Transport 747

Table 20-1 Permeability Coefficients of Natural and Synthetic

Membranes to

D-Glucose and D-Mannitol at 25°C

Permeability Coefficient

(cm ⴢ s

⫺1

)

Membrane Preparation

D-Glucose D-Mannitol

Synthetic lipid bilayer 2.4 ⫻ 10

⫺10

4.4 ⫻ 10

⫺11

Calculated nonmediated

diffusion 4 ⫻ 10

⫺9

3 ⫻ 10

⫺9

Intact human erythrocyte 2.0 ⫻ 10

⫺4

5 ⫻ 10

⫺9

Source: Jung, C.Y., in Surgenor, D. (Ed.), The Red Blood Cell, Vol. 2,

p. 709,Academic Press (1975).

Figure 20-4 General kinetic scheme for membrane transport.

The scheme involves four steps: binding, transport, dissociation,

and recovery. T is the transport protein whose binding site for

solute A is located on either the inner or the outer side of the

membrane at any one time.

out

• A (out)

•

A (in)

1. Binding

3. Dissociation

TransportRecovery4. 2.

A (out)

A (in)

1/J

max

– 1/K

1/[Glucose]

glucose

Glucose + 10 mM

6-O-benzyl-

D-galactose

Glucose alone

Figure 20-5 Double-reciprocal plots for the net flux of glucose

into erythrocytes in the presence and absence of 6-O-benzyl-

D-

galactose. The pattern is that of competitive inhibition. [After

Barnett, J.E.G., Holman, G.D., Chalkley, R.A., and Munday,

K.A., Biochem. J. 145, 422 (1975).]

JWCL281_c20_744-788.qxd 3/17/10 1:47 PM Page 747

d. Susceptibility to Chemical Inactivation

Treatment of erythrocytes with HgCl

, which reacts with

protein sulfhydryl groups

and thus inactivates many enzymes, causes the rapid, saturat-

able flux of glucose to disappear so that its permeability con-

stant approaches that of mannitol. The erythrocyte glucose

transport system’s susceptibility to such protein-modifying

agents indicates that it, in fact, is a protein.

All of the above observations indicate that glucose

transport across the erythrocyte membrane is mediated by a

limited number of protein carriers. Before we discuss the

mechanism of this transport system, however, we shall ex-

amine some simpler models of facilitated diffusion.

C. Ionophores

Our understanding of mediated transport has been en-

hanced by the study of ionophores (Greek: phoros, bearer),

substances that vastly increase the permeability of mem-

branes to particular ions.

a. Ionophores May Be Carriers or Channel Formers

Ionophores are organic molecules of diverse types,

many of which are antibiotics of bacterial origin. Cells and

organelles actively maintain concentration gradients of

various ions across their membranes (Section 20-3A). The

antibiotic properties of ionophores arise from their ten-

dency to discharge these vital concentration gradients.

There are two types of ionophores:

1. Carriers, which increase the permeabilities of mem-

branes to their selected ion by binding it, diffusing through the

membrane,and releasing the ion on the other side (Fig. 20-6a).

For net transport to occur, the uncomplexed ionophore

must then return to the original side of the membrane

ready to repeat the process. Carriers therefore share the

common property that their ionic complexes are soluble in

nonpolar solvents.

2. Channel formers, which form transmembrane chan-

nels or pores through which their selected ions can diffuse

(Fig. 20-6b).

Both types of ionophores transport ions at a remarkable

rate. For example,a single molecule of the carrier antibiotic

valinomycin transports up to 10

⫹

ions per second across

a membrane. Channel formers have an even greater ion

throughput; for example, each membrane channel com-

posed of the antibiotic gramicidin A permits the passage of

over 10

⫹

ions ⴢ s

⫺1

. Clearly, the presence of either type

of ionophore, even in small amounts, greatly increases the

permeability of a membrane toward the specific ions trans-

ported. However, since ionophores passively permit ions to

diffuse across a membrane in either direction, their effect

can only be to equilibrate the concentrations of their selected

ions across the membrane.

Carriers and channel formers are easily distinguished

experimentally through differences in the temperature

RSH ⫹ HgCl

RS¬Hg¬Cl ⫹ HCl

dependence of their action. Carriers depend on their abil-

ity to diffuse freely across the membrane. Consequently,

cooling a membrane below its transition temperature (the

temperature below which it becomes a gel-like solid; Sec-

tion 12-2Cb) essentially eliminates its ionic permeability in

the presence of carriers. In contrast, membrane permeabil-

ity in the presence of channel formers is rather insensitive

to temperature because, once in place, channel formers

need not move to mediate ion transport.

b. The K

⫹

–Valinomycin Complex Has a Polar Interior

and a Hydrophobic Exterior

Valinomycin, a product of several strains of Strepto-

myces bacteria that specifically binds K

⫹

(and the biologi-

cally unimportant Rb

⫹

), is perhaps the best characterized

carrier ionophore. It is a cyclic depsipeptide that contains

both

D- and L-amino acid residues (Fig. 20-7a; a depsipep-

tide contains ester linkages as well as peptide bonds). The

X-ray structure of valinomycin’s K

⫹

complex (Fig. 20-7b)

indicates that the K

⫹

is octahedrally coordinated by the car-

bonyl groups of its six Val residues, which also form its ester

linkages. The cyclic, intramolecularly hydrogen bonded

valinomycin backbone follows a zigzag path that surrounds

the K

⫹

coordination shell with a sinuous molecular bracelet.

Its methyl and isopropyl side chains project outward from

the bracelet to provide the spheroidal complex with a

hydrophobic exterior that makes it soluble in nonpolar

solvents and in the hydrophobic cores of lipid bilayers.

Uncomplexed valinomycin has a more open conformation

than its K

⫹

complex, which presumably facilitates the rapid

binding of K

⫹

(ionic radius, r ⫽ 1.33 Å) and Rb

⫹

(r ⫽ 1.49 Å) fit

snugly into valinomycin’s coordination site. However, the

rigidity of the valinomycin complex makes this site too

large to accommodate Na

⫹

(r ⫽ 0.95 Å) or Li

⫹

(r ⫽ 0.60 Å)

properly; that is, valinomycin’s six carbonyl oxygen atoms

cannot simultaneously coordinate these ions. Complexes of

these ions with water are therefore energetically more

favorable than their complexes with valinomycin. This

748 Chapter 20. Transport Through Membranes

Figure 20-6 Ion transport modes of ionophores. (a) Carrier

ionophores transport ions by diffusing through the lipid bilayer.

(b) Channel-forming ionophores span the membrane with a

channel through which ions can diffuse.

(a) Carrier ionophore (b) Channel-forming

ionophore

JWCL281_c20_744-788.qxd 3/17/10 1:47 PM Page 748

accounts for valinomycin’s 10,000-fold greater binding

affinity for K

⫹

over Na

⫹

. No other substance discriminates

more acutely between Na

⫹

and K

⫹

. A variety of carrier

ionophores with similar characteristics but with different

chemical structures and metal ion specificities are known.

D. Maltoporin: The Structural Basis

of Sugar Discrimination

The porins are homotrimeric transmembrane proteins

that facilitate the transport of small molecules and ions

across the outer membranes of gram-negative bacteria

and mitochondria. Each subunit consists mainly of a 16- to

22-stranded antiparallel ␤ barrel that forms a solvent-

accessible channel along the barrel axis (Section 12-3Ad). In

the E.coli OmpF porin (Fig.12-27), this ⬃50-Å-long channel

is constricted near its center to an elliptical pore that has a

minimum cross section of 7 ⫻ 11 Å. Consequently, solutes

of more than ⬃600 D are too large to pass through this

channel.

Maltoporin is a bacterial porin that facilitates the diffu-

sion of maltodextrins [the ␣(1 S 4)-linked glucose oligosac-

charide degradation products of starch; e.g., maltose (Fig.

11-13)].The X-ray structure of E. coli maltoporin (Fig. 20-8),

determined by Tilman Schirmer, reveals that maltoporin is

structurally similar to OmpF porin (Fig. 12-27), but with an

18-stranded rather than a 16-stranded antiparallel ␤ barrel

Section 20-2. Kinetics and Mechanisms of Transport 749

Figure 20-7 Valinomycin. (a) This cyclic depsipeptide (has

both ester and amide bonds) contains both

D- and L-amino acids.

(b) The X-ray structure of valinomycin in complex with a K

⫹

ion

shown in stick form colored according to atom type (C green, H

white, N blue, O red, and K

⫹

purple) and embedded in its

molecular surface. Note that the K

⫹

ion is octahedrally

coordinated by the carbonyl atoms of valinomycin’s six Val

residues and that the surface of the complex is largely covered

with methyl groups. [Based on an X-ray structure by Max

Dobler, ETH, Zürich, Switzerland.]

Valinomycin

L-Val D-Val L-Lactic

acid

D-Hydroxy-

isovaleric

acid

(a)

()

Figure 20-8 X-ray structure of a subunit of E. coli maltoporin

in complex with a maltodextrin of six glucosyl units (Glc

). The

structure is viewed from within the bacterial outer membrane

with its extracellular surface above. The polypeptide backbone is

represented by a multithreaded ribbon (cyan).The Glc

(only

five of whose glucosyl units are observed) and the aromatic side

chains lining the constricted region of the protein’s centrally

located transport channel are shown in space-filling form colored

according to atom type (protein side chain C gold, glucosyl

C green, N blue, and O red). Note the pronounced left-handed

helical twist of the Glc

unit.The so-called greasy slide, which

consists of the aromatic side chains of six residues (W74¿ is

contributed by an overhanging loop from an adjacent subunit),

conforms closely to this shape. The side chain of Y118 protrudes

into the channel opposite the greasy slide so as to allow only the

transit of near planar groups such as glucosyl residues.The

maltodextrin’s hydroxyl groups are arranged in two strips flank-

ing the greasy slide (only one of which is seen here) that form an

extensive hydrogen bonded network with mainly charged side

chains (not shown). [Based on an X-ray structure by Tilman

Schirmer, University of Basel, Switzerland. PDBid 1MPO.]

JWCL281_c20_744-788.qxd 6/4/10 12:13 PM Page 749

enclosing each subunit’s transport channel.Three long loops

from the extracellular face of each maltoporin subunit fold

inward into the barrel, thereby constricting the channel near

the center of the membrane to a diameter of ⬃5 Å (which is

considerably smaller than OmpF’s aperture) and giving the

channel an hourglasslike cross section. The channel is lined

on one side with a series of six contiguous aromatic side

chains arranged in a left-handed helical path that matches

the left-handed helical curvature of ␣-amylose (Fig. 11-18).

This “greasy slide” extends from the channel’s vestibule

floor, through its constriction, to its periplasmic outlet.

The way in which oligosaccharides interact with malto-

porin was investigated by determining the X-ray structures

of maltoporin in its complexes with the maltodextrins Glc

(maltose), Glc

, Glc

, and sucrose (a glucose–fructose

disaccharide; Fig. 11-13).Two Glc

molecules, one Glc

mole-

cule, and a Glc

segment of Glc

occupied the maltoporin

channel in contact and conformity with the greasy slide.

Thus the hydrophobic faces of the maltodextrin’s glycosyl

residues stack on aromatic side chains, as is often observed

in complexes of sugars with proteins. The glucose hydroxyl

groups, which are arranged in two strips along opposite

edges of the maltodextrins, form numerous hydrogen

bonds with polar side chains that line these strips. Six of

these seven polar side chains are charged, which probably

strengthens their hydrogen bonds, as has also been ob-

served in complexes of sugars with proteins. Tyr 118, which

protrudes into the channel opposite the greasy slide, appar-

ently functions as a steric barrier that permits only the pas-

sage of near-planar groups such as glucosyl residues. Thus

the hook-shaped sucrose, which maltoporin transports

quite slowly, binds to maltoporin with only its glucose

residue inserted into the constricted part of the channel

and its bulky fructose residue extending into the extracel-

lular vestibule.

The above structures suggest a model for the selective

transport of maltodextrins by maltoporin. At the start of

the translocation process, the entering glucosyl residue in-

teracts with the readily accessible end of the greasy slide in

the extracellular vestibule of the channel. Further translo-

cation along the helical channel requires the maltodextrin

to follow a screwlike path that maintains the helical struc-

ture of the oligosaccharide, much like the movement of a

bolt through a nut, thereby excluding molecules of compa-

rable size that have different shapes. The translocation

process is unlikely to encounter any large energy barrier

due to the smooth surface of the greasy slide and the mul-

tiple polar groups at the channel constriction that would

permit the essentially continuous exchange of hydrogen

bonds as a maltodextrin moves through the constriction.

Thus, maltoporin can be regarded as an enzyme that cat-

alyzes the translocation of its substrate from one compart-

ment to another.

E. Passive-Mediated Glucose Transport

The human erythrocyte glucose transporter is a 492-residue

glycoprotein which, according to sequence hydropathy

analysis (Sections 8-4C and 12-3Aa), has 12 membrane-

spanning ␣ helices (Fig. 20-9) that are thought to form a

hydrophobic cylinder. Five of these helices (3, 5, 7, 8, and 11)

are amphipathic and hence most likely form a hydrophilic

channel through which glucose is transported. A highly

charged 66-residue domain located between helices 6 and

7, together with the 43-residue C-terminal domain, occupy

the cytoplasm, whereas a 34-residue carbohydrate-bearing

domain located between helices 1 and 2 is externally lo-

cated. The glucose transporter accounts for 2% of erythro-

cyte membrane proteins and runs as band 4.5 in SDS–PAGE

gels of erythrocyte membranes (Section 12-3Da; it is not

visible on the gel depicted in Fig. 12-37 because the het-

erogeneity of its oligosaccharides makes the protein

band diffuse).

a. Glucose Transport Occurs via a Gated

Pore Mechanism

The erythrocyte glucose transporter has glucose binding

sites on each side of the erythrocyte membrane but these

have different steric requirements. Thus, John Barnett

showed that 1-propylglucose will not bind to the extracellular

surface of the glucose transporter but will bind to its cytoplas-

mic surface, whereas the converse is true of 6-propylglucose.

He therefore proposed that the glucose transporter has

two alternate conformations: one with the glucose binding

site facing the external cell surface, requiring O1 contact

and leaving O6 free, and the other with the glucose binding

site facing the cytoplasm, requiring O6 contact and leaving

O1 free (Fig. 20-10). Transport apparently takes place by

binding glucose to the protein on one face of the membrane,

followed by a conformational change that closes the first site

while exposing the other. Glucose can then dissociate from

the protein, having been translocated across the membrane.

The transport cycle of this so-called gated pore is completed

by the reversion of the glucose transporter to its initial con-

formation in the absence of bound glucose. Since this cycle

can occur in either direction, the direction of net glucose

transport is from high to low glucose concentrations. The

glucose transporter thereby provides a means of equili-

brating the glucose concentration across the erythrocyte

750 Chapter 20. Transport Through Membranes

Figure 20-9 Predicted secondary structure and membrane

orientation of the glucose transporter.

Inside

–

Outside

Glycosylation site

121110987654321

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