voet-biochemistrychap20.pdf

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CHAPTER 20

Transport throughMembranes

1 Thermodynamics of Transport2 Kinetics and Mechanisms of Transport

A. Nonmediated TransportB. Kinetics of Mediated Transport: Glucose Transport

Into ErythrocytesC. IonophoresD. Maltoporin: The Structural Basis of Sugar DiscriminationE. Passive-Mediated Glucose TransportF. K Channels: Ion DiscriminationG. Cl ChannelsH. Aquaporins

3 ATP-Driven Active TransportA. (NaK)ATPase of Plasma MembranesB. Ca2ATPaseC. (HK)ATPase of Gastric MucosaD. Group TranslocationE. ABC Transporters

4 Ion GradientDriven Active TransportA. NaGlucose SymportB. Lactose PermeaseC. ATPADP Translocator

5 NeurotransmissionA. Voltage-Gated Ion ChannelsB. Action PotentialsC. Neurotransmitters and Their Receptors

Metabolism occurs within cells that are separated fromtheir environments by plasma membranes. Eukaryoticcells, in addition, are compartmentalized by intracellularmembranes that form the boundaries and internal struc-tures of their various organelles. The nonpolar cores of bi-ological membranes make them highly impermeable tomost ionic and polar substances, so that these substancescan traverse membranes only through the action of specifictransport proteins. Such proteins are therefore required tomediate all transmembrane movements of ions, such asNa, K, Ca2, 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; Section12-2B). 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 ofneurotransmission.

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 isthe 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 laboratoryconditions, the activity of a substance closely approachesits molar concentration in value. However, this is not thecase in the highly concentrated cellular milieu (Appendixto Chapter 3). Yet it is difficult to determine the activity ofa 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 amembrane

thermodynamically resembles a chemical equilibration.A difference in the concentrations of the substance ontwo sides of a membrane generates a chemical potentialdifference:

[20.2]

Consequently, if the concentration of A outside the mem-brane is greater than that inside, for the transfer of Afrom outside to inside will be negative and the spontaneousnet flow of A will be inward. If, however, [A] is greater insidethan outside, is positive and an inward net flow of A canonly occur if an exergonic process, such as ATP hydrolysis, iscoupled to it to make the overall free energy change negative.

GA

GA

GA GA(in) GA(out) RT lna [A] in[A]outb

A(out) A(in)

GAGA

GA GA RT ln[A]

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a. Membrane Potentials Arise from TransmembraneConcentration Differences of Ionic SubstancesThe permeabilities of biological membranes to ions

such as H, Na, K, Cl, and Ca2 are controlled by spe-cific membrane-embedded transport systems that we shalldiscuss in later sections. The resulting charge differencesacross a biological membrane generate an electric potentialdifference, (in) (out), where is termed themembrane potential. Consequently, if A is ionic, Eq. [20.2]must be amended to include the electrical work required totransfer a mole of A across the membrane from outside toinside:

[20.3]

where ZA is the ionic charge of A;f, the Faraday constant,is the charge of one mole of electrons (96,485 C mol1);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 (insidenegative) are not uncommon (note that 1 V 1 J C1).Thus the last term of Eq. [20.3] is often significant for ionicsubstances.

2 KINETICS AND MECHANISMS OF TRANSPORT

Thermodynamics indicates whether a given transportprocess will be spontaneous but, as we saw for chemical andenzymatic reactions, provides no indication of the rates ofthese processes. Kinetic analyses of transport processes to-gether with mechanistic studies have nevertheless permit-ted these processes to be characterized. There are twotypes of transport processes: nonmediated transport andmediated transport. Nonmediated transport occurs throughsimple diffusion. In contrast, mediated transport occursthrough 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 inwhich specific molecules flow from high concentration tolow concentration so as to equilibrate their concentrationgradients.

2. Active transport in which specific molecules aretransported 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 nonmediatedtransport and then compare it to passive-mediated trans-port as exemplified by ionophores, porins, glucose trans-porters, K channels, Clchannels, and aquaporins. Activetransport is examined in succeeding sections.

GA

GA RT lna [A] in[A]outb ZAf

A. Nonmediated Transport

The driving force for the nonmediated flow of a substance Athrough a medium is As electrochemical potential gradient.This relationship is expressed by the NernstPlanckequation:

[20.4]

where JA is the flux (rate of passage per unit area) of A, x isdistance, is the electrochemical potential gradientof A, and UA is its mobility (velocity per unit force) in themedium. If we assume, for simplicity, that A is an un-charged molecule so that is given by Eq. [20.1], theNernstPlanck equation reduces to

[20.5]

where DA K RTUA is the diffusion coefficient of A in themedium of interest. This is Ficks first law of diffusion,which states that a substance diffuses in the direction thateliminates 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 DA is the diffusion coefficient of A inside the mem-brane and PA DA/x is termed the membranes perme-ability coefficient for A. The permeability coefficient is in-dicative of the solutes tendency to transfer from theaqueous solvent to the membranes nonpolar core. Itshould therefore vary with the ratio of the solutes solubil-ity in a nonpolar solvent resembling the membranes core(e.g., olive oil) to that in water, a quantity known as thesolutes partition coefficient between the two solvents. In-deed, the fluxes of many nonelectrolytes across erythrocytemembranes vary linearly with their concentration differencesacross the membrane as predicted by Eq. [20.6] (Fig. 20-1).Moreover, their permeability coefficients, as obtained fromthe slopes of plots such as Fig. 20-1, correlate rather well

JA DAx

([A]out [A]in) PA([A]out [A]in)

JA DA(d [A] )>dx

GA

dGA>dx

JA [A]UA(dGA>dx)

Section 20-2. Kinetics and Mechanisms of Transport 745

[ A ]out [ A ] in

JA

Slope = PA

Figure 20-1 Linear relationship between diffusional flux (JA)and ([A]out [A]in) across a semipermeable membrane. SeeEq. [20.6].

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with their measured partition coefficients between nonpo-lar solvents and water (Fig. 20-2).

B. Kinetics of Mediated Transport: GlucoseTransport Into Erythrocytes

Despite the success of the foregoing model in predictingthe rates at which many molecules pass through mem-branes, there are numerous combinations of solutes andmembranes that do not obey Eq. [20.6]. The flux in such asystem is not linear with the solute concentration differ-ence across the corresponding membrane (Fig. 20-3) and,furthermore, the solutes permeability coefficient is muchlarger 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 ofpassive-mediated transport: It invariably transports glu-cose down its concentration gradient but not at the ratepredicted by Eq. [20.6]. Indeed, the erythrocyte glucosetransporter exhibits four characteristics that differentiatemediated 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 inplasma membranes from the alga Nitella mucronata versus theirpartition coefficients between olive oil and water (a measure of amolecules polarity). This more or less linear loglog plot

Figure 20-3 Variation of glucose flux into human erythrocyteswith the external glucose concentration at 5C. The black dotsare experimentally determined data points, and the solid greenline is computed from Eq. [20.7] with Jmax 1.0 10

6 mM cm s1 and KM 0.5 mM. The nonmediated glucose flux increases linearly with [glucose] (Fig. 20-1) but would not visiblydepart from the baseline on the scale of this drawing. [Based ondata from Stein, W.D., Movement of Molecules across Membranes, p. 134, Academic Press (1967).]

Perm

eabi

lity

coef

ficie

nt (

cm .

s1)

0.001 0.010.0001

Oilwater partition coefficient

Ethylurea

Urea

Ethylene glycol

Acetamide Succinimide

N,N'-Diethylurea

n-Butyramide

PropionamideFormamide

107

106

105

NH2

CH2CH2

H2N C

O

HO OH

NHH2N CH2CH3C

O

NHHN

NH

CH2CH3CH3CH2

CH3CH2C

C

C

H2C

H2C

C

O

O

O

NH2CH3 C

O

NH2

HC

O

NH2

OCH3CH2CH2C

NH2

O

indicates that the rate-limiting step for the nonmediated entry ofa molecule into a cell is its passage through the membraneshydrophobic core. [Based on data from Collander, R., Physiol.Plant. 7, 433434 (1954).]

Jgl

ucos

e (m

M. c

m. s

1

10

6)

1.0

0.5

02 4 6 8 100

[Glucose] mM

Jmax = 1.0 106 mM.cm.s1

KM

1/2 Jmax

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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 theerythrocyte glucose transporter exhibits these qualities.

a. Speed and SpecificityTable 20-1 indicates that the permeability coefficients of

D-glucose and D-mannitol in synthetic bilayers, and that ofD-mannitol in the erythrocyte membrane, are in reasonableagreement with the values calculated from the diffusionand partition coefficients of these sugars between waterand olive oil. However, the experimentally determinedpermeability coefficient for D-glucose in the erythrocytemembrane is four orders of magnitude greater than its pre-dicted value. The erythrocyte membrane must thereforecontain a system that rapidly transports glucose and that candistinguish D-glucose from D-mannitol.

b. Saturation KineticsThe 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 thebinding of O2 to myoglobin (Eq. [10.4]) and in theMichaelisMenten equation describing the rates of enzy-matic reactions (Eq. [14.24]). Here, as before, KM may bedefined operationally as the concentration of glucosewhen the transport flux is half of its maximal rate, Jmax /2.This observation of saturation kinetics for glucose trans-port was the first evidence that a specific, saturatable

JA Jmax[A]

KM [A]

number of sites on the membrane were involved in thetransport 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 dissociationsteps are analogous to the recognition of a substrate andthe release of product by an enzyme. The mechanisms oftransport and recovery are discussed in Section 20-2D.

c. Susceptibility to Competitive InhibitionMany compounds structurally similar to D-glucose in-

hibit glucose transport. A double-reciprocal plot (Section14-2B) 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 glucosetransport (competitive inhibition of enzymes is discussedin Section 14-3A). Susceptibility to competitive inhibitionindicates that there is a limited number of sites available formediated transport.


Table 20-1 Permeability Coefficients of Natural and SyntheticMembranes to D-Glucose and D-Mannitol at 25C

Permeability Coefficient(cm s1)

Membrane Preparation D-Glucose D-Mannitol

Synthetic lipid bilayer 2.4 1010 4.4 1011

Calculated nonmediateddiffusion 4 109 3 109

Intact human erythrocyte 2.0 104 5 109

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 forsolute A is located on either the inner or the outer side of themembrane at any one time.

Tout Tout A (out)

Tin Tin A (in)

1. Binding

3. Dissociation

TransportRecovery4. 2.

A (out)

A (in)

1/Jmax

1/KM 1/[Glucose]

1/JglucoseGlucose + 10 mM6-O-benzyl-D-galactose

Glucose alone

Figure 20-5 Double-reciprocal plots for the net flux of glucoseinto erythrocytes in the presence and absence of 6-O-benzyl-D-galactose. The pattern is that of competitive inhibition. [AfterBarnett, J.E.G., Holman, G.D., Chalkley, R.A., and Munday,K.A., Biochem. J. 145, 422 (1975).]

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d. Susceptibility to Chemical InactivationTreatment of erythrocytes with HgCl2, 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 glucosetransport systems susceptibility to such protein-modifyingagents indicates that it, in fact, is a protein.

All of the above observations indicate that glucosetransport across the erythrocyte membrane is mediated by alimited number of protein carriers. Before we discuss themechanism 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 FormersIonophores are organic molecules of diverse types,

many of which are antibiotics of bacterial origin. Cells andorganelles actively maintain concentration gradients ofvarious ions across their membranes (Section 20-3A). Theantibiotic 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 themembrane, and releasing the ion on the other side (Fig. 20-6a).For net transport to occur, the uncomplexed ionophoremust then return to the original side of the membraneready to repeat the process. Carriers therefore share thecommon property that their ionic complexes are soluble innonpolar 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 remarkablerate. For example, a single molecule of the carrier antibioticvalinomycin transports up to 104 K ions per second acrossa membrane. Channel formers have an even greater ionthroughput; for example, each membrane channel com-posed of the antibiotic gramicidin A permits the passage ofover 107 K ions s1. Clearly, the presence of either typeof ionophore, even in small amounts, greatly increases thepermeability of a membrane toward the specific ions trans-ported. However, since ionophores passively permit ions todiffuse across a membrane in either direction, their effectcan only be to equilibrate the concentrations of their selectedions across the membrane.

Carriers and channel formers are easily distinguishedexperimentally through differences in the temperature

RSH HgCl2 RSHgCl 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 (thetemperature below which it becomes a gel-like solid; Sec-tion 12-2Cb) essentially eliminates its ionic permeability inthe presence of carriers. In contrast, membrane permeabil-ity in the presence of channel formers is rather insensitiveto temperature because, once in place, channel formersneed not move to mediate ion transport.

b. The KValinomycin Complex Has a Polar Interiorand a Hydrophobic ExteriorValinomycin, a product of several strains of Strepto-

myces bacteria that specifically binds K (and the biologi-cally unimportant Rb), is perhaps the best characterizedcarrier ionophore. It is a cyclic depsipeptide that containsboth D- and L-amino acid residues (Fig. 20-7a; a depsipep-tide contains ester linkages as well as peptide bonds). TheX-ray structure of valinomycins 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 esterlinkages. The cyclic, intramolecularly hydrogen bondedvalinomycin backbone follows a zigzag path that surroundsthe K coordination shell with a sinuous molecular bracelet.Its methyl and isopropyl side chains project outward fromthe bracelet to provide the spheroidal complex with ahydrophobic exterior that makes it soluble in nonpolarsolvents and in the hydrophobic cores of lipid bilayers.Uncomplexed valinomycin has a more open conformationthan its K complex, which presumably facilitates the rapidbinding of K.

K (ionic radius, r 1.33 ) and Rb (r 1.49 ) fitsnugly into valinomycins coordination site. However, therigidity of the valinomycin complex makes this site toolarge to accommodate Na (r 0.95 ) or Li (r 0.60 )properly; that is, valinomycins six carbonyl oxygen atomscannot simultaneously coordinate these ions. Complexes ofthese ions with water are therefore energetically morefavorable than their complexes with valinomycin. This


Figure 20-6 Ion transport modes of ionophores. (a) Carrierionophores transport ions by diffusing through the lipid bilayer.(b) Channel-forming ionophores span the membrane with achannel through which ions can diffuse.

(a) Carrier ionophore (b) Channel-forming ionophore

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accounts for valinomycins 10,000-fold greater bindingaffinity for K over Na. No other substance discriminatesmore acutely between Na and K. A variety of carrierionophores with similar characteristics but with differentchemical structures and metal ion specificities are known.

D. Maltoporin: The Structural Basis of Sugar Discrimination

The porins are homotrimeric transmembrane proteinsthat facilitate the transport of small molecules and ionsacross the outer membranes of gram-negative bacteria

and mitochondria. Each subunit consists mainly of a 16- to22-stranded antiparallel barrel that forms a solvent-accessible channel along the barrel axis (Section 12-3Ad). Inthe E. coli OmpF porin (Fig. 12-27), this 50--long channelis constricted near its center to an elliptical pore that has aminimum cross section of 7 11 . Consequently, solutesof more than 600 D are too large to pass through thischannel.

Maltoporin is a bacterial porin that facilitates the diffu-sion of maltodextrins [the (1S 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 isstructurally similar to OmpF porin (Fig. 12-27), but with an18-stranded rather than a 16-stranded antiparallel barrel


Figure 20-7 Valinomycin. (a) This cyclic depsipeptide (hasboth ester and amide bonds) contains both D- and L-amino acids.(b) The X-ray structure of valinomycin in complex with a K ionshown in stick form colored according to atom type (C green, Hwhite, 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 valinomycins six Valresidues and that the surface of the complex is largely coveredwith methyl groups. [Based on an X-ray structure by MaxDobler, ETH, Zrich, Switzerland.]

CCHN

Valinomycin

CH3

H3C

CO NC CC CO C

OOOO H H H3C

CH CH HH

CH

H3C

CH3 H3C CH3 3

H

L-Val D-Val L-Lacticacid

D-Hydroxy-isovaleric

acid

(a)

( )

Figure 20-8 X-ray structure of a subunit of E. coli maltoporinin complex with a maltodextrin of six glucosyl units (Glc6). Thestructure is viewed from within the bacterial outer membranewith its extracellular surface above. The polypeptide backbone isrepresented by a multithreaded ribbon (cyan). The Glc6 (onlyfive of whose glucosyl units are observed) and the aromatic sidechains lining the constricted region of the proteins centrallylocated transport channel are shown in space-filling form coloredaccording to atom type (protein side chain C gold, glucosylC green, N blue, and O red). Note the pronounced left-handedhelical twist of the Glc6 unit. The so-called greasy slide, whichconsists of the aromatic side chains of six residues (W74 iscontributed by an overhanging loop from an adjacent subunit),conforms closely to this shape. The side chain of Y118 protrudesinto the channel opposite the greasy slide so as to allow only thetransit of near planar groups such as glucosyl residues. Themaltodextrins hydroxyl groups are arranged in two strips flank-ing the greasy slide (only one of which is seen here) that form anextensive hydrogen bonded network with mainly charged sidechains (not shown). [Based on an X-ray structure by TilmanSchirmer, University of Basel, Switzerland. PDBid 1MPO.]

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enclosing each subunits transport channel.Three long loopsfrom the extracellular face of each maltoporin subunit foldinward into the barrel, thereby constricting the channel nearthe center of the membrane to a diameter of 5 (which isconsiderably smaller than OmpFs aperture) and giving thechannel an hourglasslike cross section. The channel is linedon one side with a series of six contiguous aromatic sidechains arranged in a left-handed helical path that matchesthe left-handed helical curvature of -amylose (Fig. 11-18).This greasy slide extends from the channels vestibulefloor, through its constriction, to its periplasmic outlet.

The way in which oligosaccharides interact with malto-porin was investigated by determining the X-ray structuresof maltoporin in its complexes with the maltodextrins Glc2(maltose), Glc3, Glc6, and sucrose (a glucosefructosedisaccharide; Fig. 11-13).Two Glc2 molecules, one Glc3 mole-cule, and a Glc5 segment of Glc6 occupied the maltoporinchannel in contact and conformity with the greasy slide.Thus the hydrophobic faces of the maltodextrins glycosylresidues stack on aromatic side chains, as is often observedin complexes of sugars with proteins. The glucose hydroxylgroups, which are arranged in two strips along oppositeedges of the maltodextrins, form numerous hydrogenbonds with polar side chains that line these strips. Six ofthese seven polar side chains are charged, which probablystrengthens their hydrogen bonds, as has also been ob-served in complexes of sugars with proteins. Tyr 118, whichprotrudes 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. Thusthe hook-shaped sucrose, which maltoporin transportsquite slowly, binds to maltoporin with only its glucoseresidue inserted into the constricted part of the channeland its bulky fructose residue extending into the extracel-lular vestibule.

The above structures suggest a model for the selectivetransport of maltodextrins by maltoporin. At the start ofthe translocation process, the entering glucosyl residue in-teracts with the readily accessible end of the greasy slide inthe extracellular vestibule of the channel. Further translo-cation along the helical channel requires the maltodextrinto follow a screwlike path that maintains the helical struc-ture of the oligosaccharide, much like the movement of abolt through a nut, thereby excluding molecules of compa-rable size that have different shapes. The translocationprocess is unlikely to encounter any large energy barrierdue to the smooth surface of the greasy slide and the mul-tiple polar groups at the channel constriction that wouldpermit the essentially continuous exchange of hydrogenbonds 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-residueglycoprotein which, according to sequence hydropathyanalysis (Sections 8-4C and 12-3Aa), has 12 membrane-

spanning helices (Fig. 20-9) that are thought to form ahydrophobic cylinder. Five of these helices (3, 5, 7, 8, and 11)are amphipathic and hence most likely form a hydrophilicchannel through which glucose is transported. A highlycharged 66-residue domain located between helices 6 and7, together with the 43-residue C-terminal domain, occupythe cytoplasm, whereas a 34-residue carbohydrate-bearingdomain 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 SDSPAGEgels of erythrocyte membranes (Section 12-3Da; it is notvisible on the gel depicted in Fig. 12-37 because the het-erogeneity of its oligosaccharides makes the proteinband diffuse).

a. Glucose Transport Occurs via a Gated Pore MechanismThe erythrocyte glucose transporter has glucose binding

sites on each side of the erythrocyte membrane but thesehave different steric requirements. Thus, John Barnettshowed that 1-propylglucose will not bind to the extracellularsurface 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 hastwo alternate conformations: one with the glucose bindingsite facing the external cell surface, requiring O1 contactand leaving O6 free, and the other with the glucose bindingsite facing the cytoplasm, requiring O6 contact and leavingO1 free (Fig. 20-10). Transport apparently takes place bybinding glucose to the protein on one face of the membrane,followed by a conformational change that closes the first sitewhile exposing the other. Glucose can then dissociate fromthe protein, having been translocated across the membrane.The transport cycle of this so-called gated pore is completedby the reversion of the glucose transporter to its initial con-formation in the absence of bound glucose. Since this cyclecan occur in either direction, the direction of net glucosetransport is from high to low glucose concentrations. Theglucose transporter thereby provides a means of equili-brating the glucose concentration across the erythrocyte


Figure 20-9 Predicted secondary structure and membraneorientation of the glucose transporter.

CInside

+

+

+

+

Outside

Glycosylation site

N

121110987654321

JWCL281_c20_744-788.qxd 3/18/10 12:20 PM Page 750

membrane without any accompanying leakage of smallmolecules or ions.

b. Eukaryotes Express a Variety ofGlucose TransportersThe erythrocyte glucose transporter, known also as

GLUT1 (for glucose transporter 1) has a highly conservedamino acid sequence (98% sequence identity between hu-mans and rats), which suggests that all segments of this pro-tein are functionally significant. GLUT1 is expressed inmost tissues, although in liver and muscle, tissues that arehighly active in glucose transport, it is present in only tinyamounts. Three other glucose transporters, GLUT2,GLUT3, and GLUT4, have been well characterized(GLUT5 was originally thought to be a glucose transporterbut was later shown to be a fructose transporter). They are40 to 65% identical to GLUT1 but have different tissuedistributions. For example, GLUT2 is prominent in pancre-atic cells (which secrete insulin in response to increased[glucose] in blood; Section 18-3F), liver (where its defectsresult in symptoms resembling Type I glycogen storage dis-ease; Section 18-4), and the intestine (which absorbs di-etary glucose; Section 20-4A); GLUT3 is expressed in neu-rons and the placenta, and GLUT4 occurs mainly in muscleand fat cells. Note that the tissue distributions of these glu-cose transporters correlate with the response of these tis-sues to insulin: Liver is unresponsive to insulin (liver func-tions, in part, to maintain the level of blood glucose; Section18-3Fb), whereas muscle and fat cells take up glucose whenstimulated by insulin. Analysis of the human genome hasidentified eight other members of the GLUT family,GLUT6 through GLUT12 and HMIT (for H-coupled

myo-inositol transporter), although they have yet to bewell characterized. All of them are members of the majorfacilitator superfamily (MFS).

c. Cellular Glucose Uptake Is Regulated through the Insulin-Sensitive Exocytosis/Endocytosis of Glucose TransportersInsulin stimulates fat and muscle cells to take up glucose.

Within 2 or 3 min after the administration of insulin to fatcells, the Jmax for passive-mediated glucose transport intothese cells increases 20- to 30-fold, whereas the KM remainsconstant. On withdrawal of the insulin, the rate of glucoseuptake returns to its basal level within 20 min to 2 h de-pending on conditions. Neither the increase nor the de-crease in the rate of glucose transport is affected by thepresence of protein synthesis inhibitors, so that these ob-servations cannot be a consequence of the synthesis of newglucose transporter or of a protein that inhibits it. How,then, does insulin regulate glucose transport?

GLUT4 is the dominant glucose transporter in skeletalmuscle and adipose (fat) cells. In their basal state, thesecells store most of their GLUT4 in specialized GLUT4storage vesicles. On insulin stimulation, these vesicles fusewith the plasma membrane in a process known as exocyto-sis (Fig. 20-11). The consequent increased number of


Figure 20-10 Alternating conformation model for glucosetransport. Such a system is also known as a gated pore. [AfterBaldwin, S.A. and Lienhard, G.E., Trends Biochem. Sci. 6, 210(1981).] See the Animated Figures

Figure 20-11 Regulation of glucose uptake in muscle and fatcells. Regulation is mediated by the insulin-stimulated exocytosis(the opposite of endocytosis; Section 12-5Bc) of membranousvesicles containing GLUT4 glucose transporters (left). On insulinwithdrawal, the process reverses itself through endocytosis(right). See the Animated Figures

Glucose

Binding

Dissociation

Recovery Transport

Glucose

GLUT4

Exocytosis Endocytosis

Membranousvesicle

Stimulationby insulin

Plasmamembrane

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cell-surface glucose transporters results in a proportionalincrease in the cells glucose uptake rate. On insulin with-drawal, the process is reversed through the endocytosisof plasma membrane-embedded glucose transporters.The deletion or mutation of GLUT4s N-terminal eightresidues, particularly Phe 5, causes this transporter to ac-cumulate in the plasma membrane. A Leu-Leu sequenceand an acidic motif near GLUT4s C-terminus are likewiseessential for its sequestration by the cells endocytotic ma-chinery. The way in which insulin controls this system,which accounts for most of insulins effects on muscle andfat cells, is imperfectly understood. However, it is clearthat this mechanism involves a tyrosine phosphorylationcascade that is triggered by the binding of insulin to the in-sulin receptor (Section 19-3Ac and Fig. 19-67) and includesthe activation of a class IA phosphoinositide 3-kinase(PI3K; Section 19-4Da).

F. K Channels: Ion Discrimination

Potassium ions diffuse from the cytoplasm (where [K]

100 mM) to the extracellular space (where [K] 5 mM)through transmembrane proteins known as K channels, aprocess that underlies numerous important biologicalprocesses including maintenance of cellular osmotic bal-ance, neurotransmission (Section 20-5), and signal transduc-tion (Chapter 19). Although there is a large diversity of K

channels, even within single organisms, all of them havesimilar sequences, exhibit comparable permeability charac-teristics, and most importantly, are at least 10,000-foldmore permeable to K than Na. Since this high selectivity(around the same as that of valinomycin; Section 20-2Cb)implies energetically strong interactions between K and theprotein, how can the K channel maintain its observed nearlydiffusion-limited throughput rate of up to 108 ions per second(a 104-fold greater rate than that of valinomycin)?

a. The X-Ray Structure of KcsA Reveals the Basis of K Channel SelectivityKcsA, the K channel from Streptomyces lividans, is a

tetramer of identical 158-residue subunits. The X-raystructure of its N-terminal 125-residue segment, deter-mined by Roderick MacKinnon, reveals that each KcsAsubunit forms two nearly parallel transmembrane helicesthat are inclined 25 from the normal to the membraneplane and which are connected by an 20-residue poreregion (Fig. 20-12a). As is true of all known K channels,four such subunits associate to form a 4-fold rotationallysymmetric assembly surrounding a central pore. The fourinner (C-terminal) helices, which largely form the pore,pack against each other near the cytoplasmic side of themembrane much like the poles of an inverted teepee. Thefour outer helices, which face the lipid bilayer, buttressthe inner helices but do not contact the adjacent outer he-lices. The pore regions, which each consist of a so-calledturret, pore helix, and selectivity filter, occupy the openextracellular end of the teepee, with the pore helicesfitting in between its poles. Several K ions and ordered

water molecules are seen to occupy the central pore (Figs.20-12b and 20-13a).

The 45--long central pore has variable width: It startsat its cytoplasmic side (Fig. 20-12b, bottom) as an 6--diameter and 18--long tunnel, the so-called internalpore, whose entrance is lined with four anionic side chainsthat presumably help exclude anions (red area at the bot-tom of Fig. 20-12b).The internal pore then widens to forma cavity 10 in diameter. These regions of the centralpore are both wide enough so that a K ion could movethrough them in its hydrated state. However, the upperpart of the pore, the so-called selectivity filter, narrows to3 , thereby forcing a transiting K ion to shed its watersof hydration. The walls of the internal pore and the cavityare lined with hydrophobic groups that interact minimallywith diffusing ions (yellow area of the pore in Fig. 20-12b). However, the selectivity filter (red area of the poreat the top of Fig. 20-12b) is lined with closely spaced mainchain carbonyl oxygens of residues (Fig. 20-13a, top) thatare highly conserved in all K channels (their so-calledsignature sequence, TVGYG) and whose mutations dis-rupt the ability of the channel to discriminate between K

and Na ions.What is the function of the cavity? Energy calculations

indicate that an ion moving through a narrow transmem-brane pore must surmount an energy barrier that is maximalat the center of the membrane. The existence of the cavityreduces this electrostatic destabilization by surrounding theion with polarizable water molecules (Fig. 20-12c). In addi-tion, the C-terminal ends of the four pore helices point di-rectly at the center of the cavity, so that their helix dipolesimpose a negative electrostatic potential on the cavity thatlowers the electrostatic barrier facing a cation crossing alipid bilayer.

Remarkably, the K ion occupying the cavity is ligandedby 8 ordered water molecules located at the corners of asquare antiprism (a cube with one face twisted by 45 withrespect to the opposite face) in which the K ion is cen-tered (Fig. 20-13a, bottom; K in aqueous solution wasknown to have such an inner hydration shell but it hadnever before been visualized). The K ion is precisely cen-tered in the cavity but yet its liganding water molecules arenot in van der Waals contact with the walls of the cavity. In-deed, there is room in the cavity for 40 additional watermolecules although they are unseen in the X-ray structurebecause they are disordered. This disorder arises becausethe cavity is lined with hydrophobic groups (mainly theside chains of Ile 100 and Phe 103; Fig. 20-13a) that interactbut weakly with water molecules, thus allowing them to in-teract freely with the K ion so as to form an outer hydra-tion shell. What, then, holds the hydrated K ion in place?Apparently, it is very weak indirect hydrogen bonds involv-ing such protein groups as the hydroxyl group of Thr 107and possibly carbonyl O atoms from the pore and inner he-lices. The absence of such an ordered hydration complexwhen Na rather than K occupies the cavity is indicativeof a precise geometric match between the hydrated K andthe cavity (the ionic radii of Na and K are 0.95 and


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1.33 , respectively).The cavity thereby provides a high ef-fective K concentration (2M) at the center of the mem-brane and positions the K ion on the pore axis ready toenter the selectivity filter.

How does the K channel discriminate so acutely be-tween K and Na ions? The main chain O atoms lining the

selectivity filter form a stack of rings (Fig. 20-13a, top) thatprovide a series of closely spaced sites of appropriate di-mensions for coordinating dehydrated K ions but not thesmaller Na ions. If the observed diameter of the selectiv-ity filter is rigidly maintained, it would make the energy ofa dehydrated Na in the selectivity filter considerably


Figure 20-12 X-ray structure of the KcsA K channel.(a) Ribbon diagram of the tetramer as viewed from within theplane of the membrane with the cytoplasm below and theextracellular region above. The proteins 4-fold axis of rotation isvertical and each of its identical subunits is differently colored.(b) A cutaway diagram viewed similarly to Part a in which theK channel is represented by its solvent-accessible surface. Thesurface is colored according to its physical properties, withnegatively charged areas red, uncharged areas white, positivelycharged areas blue, and hydrophobic areas of the central poreyellow. K ions are represented by green spheres. (c) A schematicdiagram indicating how the K channel stabilizes a cation in thecenter of the membrane.The central pores 10--diameter aqueouscavity (which contains 50 water molecules) stabilizes a K ion(green spheres) in the otherwise hydrophobic membrane interior.In addition, the C-terminal ends of the pore helices (red) all pointtoward the K ion, thereby electrostatically stabilizing it via theirdipole moments (an helix has a strong dipole moment with itsnegative end pointing toward the helixs C-terminal end becausethe bond dipoles of its component carbonyl and NH groups are

(a)

(b)

(c)

all parallel to the helix axis with their negative ends pointingtoward its C-terminal end; Fig. 8-11).This effect is magnified by thelow dielectric constant at the center of the membrane interior.Electrostatic calculations indicate that the cavity is tuned tomaximally stabilize monovalent cations. [Courtesy of RoderickMacKinnon, Rockefeller University. PDBid 1BL8.]

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higher than that of hydrated Na and thus account for theK channels high selectivity for K ions. However, pro-teins are not static structures. In fact, both X-ray evidenceand molecular dynamics simulations (Section 9-4a) indi-cate that, at physiological temperatures, the atoms forming

the KcsA selectivity filter undergo thermal excursions av-eraging 1 , fluctuations sufficient to snugly cradle Na

ions with little energetic cost. Instead, as free energy calcu-lations have demonstrated, it is the electrostatic interac-tions of the carbonyl groups with the cation and with each


(a)

(b)

(c)

Figure 20-13 Portions of the KcsA K channel responsible forits ion selectivity viewed similarly to Fig. 20-12. (a) The X-raystructure of the residues forming the cavity (bottom) and selectivity filter (top) but with the front and back subunits omitted for clarity. Atoms are colored according to type, with C yellow, N blue, O red, and K ions represented by green spheres.The water and protein O atoms that ligand the K ions, includingthose contributed by the front and back subunits, are representedby red spheres. The coordination polyhedra formed by these O atoms are outlined by thin white lines. (b and c) Two alternativeK binding states of the selectivity filter, whose superposition is

presumed to be responsible for the electron density observed inthe X-ray structure of KcsA. Atoms are colored as in Part a.Note that K ions occupying the selectivity filter are interspersedwith water molecules and that the K ion immediately above theselectivity filter in Part b is farther above the protein than that inPart c. Hence these ions maintain a constant spacing while traversing the selectivity filter. [Part a based on an X-ray structure by, and Parts b and c courtesy of, Roderick MacKinnon,Rockefeller University. PDBid 1K4C.] See Interactive Exercise 14

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other that confer specificity for binding K ions. This isconsistent with the observation that no Na-specific pro-tein channels have evolved by refining the structure of aKcsA-like channel.

Since the selectivity filter appears designed to specifi-cally bind K ions, how does it support such a highthroughput of these ions (up to 108 ions s1)? The struc-ture in Fig. 20-13a shows what appear to be 4 K ions in theselectivity filter and two more just outside it on its extracel-lular side. Such closely spaced positive ions would stronglyrepel one another and hence represent a high energy situa-tion. However, a variety of evidence suggests that thisstructure is really a superposition of two sets of K ions,one with K ions at the topmost position in Fig. 20-13a andat positions 1 and 3 in the selectivity filter (Fig. 20-13b) andthe second with K ions at the second position from the topin Fig. 20-13a and at positions 2 and 4 in the selectivity fil-ter (Fig. 20-13c; X-ray structures can show overlappingatoms because they are averages of many unit cells).Within the selectivity filter, the positions not occupied byK ions are instead occupied by water molecules that coor-dinate the neighboring K ions.

The electron density that is represented as the topmost4 water molecules in Fig. 20-13a is highly elongated in thevertical direction in this otherwise high-resolution (2.0 )structure. Hence it is thought to actually arise from 8 watermolecules that ligand the topmost K ion in Fig. 20-13b toform an inner hydration shell similar to that of the K inthe central cavity (Fig. 20-13a, bottom). Moreover, the fourwater molecules liganding the topmost K ion in Fig. 20-13c also contribute to this electron density. This latter ringof 4 waters provides half of the associated K ions 8 lig-anding O atoms. The others are contributed by the car-bonyl O atoms of the 4 Gly 79 residues, which are properlyoriented to do so. It therefore appears that a dehydratedK ion transits the selectivity filter (moves to successivepositions in Figs. 20-13b,c) by exchanging the properlyspaced ligands extending from its walls and then exits into

the extracellular solution by exchanging protein ligands forwater molecules and hence again acquiring a hydrationshell.These ligands are spaced and oriented such that thereis little free energy change (estimated to be 12 kJ mol1)along the reaction coordinate via which a K ion transitsthe selectivity filter and enters the extracellular solution.The rapid dehydration of the K ion entering the selectiv-ity channel from the cavity is, presumably, similarly man-aged. The essentially level free energy landscape through-out this process is, of course, conducive to the rapid transitof K ions through the ion channel and hence must be aproduct of evolutionary fine-tuning. Energy calculationsindicate that mutual electrostatic repulsions between suc-cessive K ions, whose movements are concerted, balancesthe attractive interactions holding these ions in the selec-tivity filter and hence further facilitates their rapid transit.

G. Cl Channels

Cl channels, which occur in all cell types, permit the trans-membrane movement of chloride ions along their concen-tration gradient. In mammals, the extracellular Cl concen-tration is 120 mM and the intracellular concentration is4 mM.

ClC channels form a large family of Clchannels thatoccur widely in all kingdoms of life.The X-ray structures ofClC channels from two species of bacteria, determined byRaimund Dutzler and MacKinnon, reveal, as biophysicalmeasurements had previously suggested, that ClC channelsare homodimers with each 470-residue subunit formingan anion-selective pore (Fig. 20-14). Each subunit consistsmainly of 18 mostly transmembrane helices that are re-markably tilted with respect to the membrane plane andhave variable lengths compared to the transmembrane he-lices in other integral proteins of known structures. The N-and C-terminal halves of each subunit are related by apseudo-2-fold axis parallel to the plane of the membraneand hence these two halves have opposite orientations in


Figure 20-14 X-ray structure of the ClC Cl channel fromE. coli. Each subunit of the homodimer contains 18 helices ofvariable lengths. The subunits are drawn in ribbon form withone colored in rainbow order from its N-terminus (blue) to its C-terminus (red) and the other pink. The two Cl ions bound inthe selectivity filter of each subunit are represented by pale

(a) (b)

green spheres. (a) View from within the membrane with the extracellular surface above and the 2-fold axis relating the twosubunits vertical. (b) View from the extracellular side of themembrane along the molecular 2-fold axis. [Based on an X-raystructure by Raimund Dutzler and Roderick MacKinnon,Rockefeller University. PDBid 1OTS.]

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the membrane. This suggests that the ClC channel arosethrough gene duplication although its two halves exhibitonly weak sequence similarity. Such antiparallel architec-ture occurs in several types of transmembrane transportproteins.

The ClC Clchannel is located at the interface betweenits N- and C-terminal halves. The specificity of the ClCchannel results from an electrostatic field established bybasic amino acids on the protein surface, which helps fun-nel anions toward the pore, and by a selectivity filterformed by the dipoles of several helices oriented withtheir positively charged N-terminal ends pointing towardthe Cl ions (opposite to their orientation in the KcsAchannel; Fig. 20-12c). This feature of the selectivity filterhelps attract Cl ions, which are specifically coordinated bymain chain amide nitrogens and side chain hydroxyls fromSer and Tyr residues. A positively charged residue such asLys or Arg, if it were present in the selectivity filter, wouldprobably bind a Cl ion too tightly to facilitate its rapidtransit through the channel.

Unlike the KcsA channel, which has a central aqueouscavity (Fig. 20-12c), the Cl channel is hourglass-shaped,with its narrowest part in the center of the membrane andflanked by wider aqueous vestibules. A conserved Glu sidechain projects into the pore. This group would repel otheranions, suggesting that rapid Cl flux requires a proteinconformational change in which the Glu side chain movesaside. Another anion could push the Glu away, which ex-plains why some Cl channels appear to be activated byCl ions; that is, they open in response to a certain concen-tration of Cl in the extracellular fluid.

H. Aquaporins

The observed rapid passage of water molecules across bio-logical membranes had long been assumed to occur viasimple diffusion that was made possible by the small sizeand high concentration of water molecules. However, cer-tain cells, such as erythrocytes and those of the kidney, cansustain particularly rapid rates of water transport, whichare reversibly inhibited by mercuric ion.This suggested theexistence of previously unrecognized protein pores thatconduct water through biological membranes. The first ofthese proteins was discovered in 1992 by Peter Agre, whonamed them aquaporins.

Aquaporins occur widely in all kingdoms of life. Plantshave up to 50 different aquaporins, which is indicative ofthe importance of water transport to plant physiology. The13 known mammalian aquaporins, AQP0 through AQP12,are selectively expressed at high levels in tissues that rap-idly transport water, such as kidneys, salivary glands, sweatglands, and lacrimal glands (which produce tears). In fact,kidneys alone employ seven different aquaporins, eachwith specific locations and regulatory properties. There aretwo subfamilies of aquaporins: those that permit only thepassage of water and those that also allow the passage ofsmall neutral molecules such as glycerol and urea andhence are named aquaglyceroporins. Aquaporins permitthe passage of water molecules at extremely high rates (up

to 3 109 per second) but, quite surprisingly, not protons(really hydronium ions; H3O

), whose free passage woulddischarge the cells membrane potential.

All known aquaporins are homotetramers, each ofwhose subunits contain a water-transport channel (unlikeK channels, whose transport channels lie along their 4-foldaxes; Section 20-2Fa). The X-ray structure of the most ex-tensively studied aquaporin, bovine AQP1, reveals thateach of its 271-residue subunits consists mainly of six trans-membrane helices plus two shorter helices that are com-ponents of loops that extend only to the middle of the bi-layer (Fig. 20-15a). Other aquaporins of known structurehave similar structures. The N- and C-terminal halves ofaquaporins are 20% identical in sequence and related bya pseudo-2-fold axis of symmetry that is parallel to theplane of the membrane (Fig. 20-15a). Evidently, these seg-ments arose through gene duplication. ClC channels have asimilar antiparallel architecture (Section 20-2G).

The helices in AQP1 surround an elongated hourglass-shaped channel through the membrane (Fig. 20-16) that atits narrowest point is 2.8 wide, the diameter of a watermolecule. This region is formed by the side chains of thehighly conserved Phe 58, His 182, and Arg 197 (Fig. 20-15b,lower right subunit) and hence is known as the ar/R con-striction (ar for aromatic).The side chain of Cys 191, whichalso forms part of the ar/R constriction, is the site of chan-nel blockage by the binding of mercuric ion. For a watermolecule to pass through the ar/R constriction, it mustshed its shell of associated water molecules. This is facili-tated by the side chains of His 182 and Arg 197. The watermolecules then continue in single file through the 25--long and 4--wide portion of the channel, which is linedwith hydrophobic groups interspersed with several hydro-gen bonding groups. The water molecules lack of interac-tion with the hydrophobic walls of the channel facilitatestheir rapid passage through the channel, whereas the hy-drogen bonding groups reduce the energy barrier to watertransport. It is the balancing of these opposing factors thatis presumably responsible for aquaporins selective perme-ability to water and its rapid transport rate.

If water were to pass through aquaporin as an uninter-rupted chain of hydrogen-bonded molecules, then protonswould pass even more rapidly through the channel via pro-ton jumping (Fig. 2-10; in order for more than one such se-ries of proton jumps to occur, each water molecule in thechain must reorient such that one of its protons forms a hy-drogen bond to the next water molecule in the chain).However, aquaporin interrupts this process by forming hy-drogen bonds from the side chain NH2 groups of the highlyconserved Asn 78 and Asn 194, to a water molecule that iscentrally located in the channel (Fig. 20-16). Consequently,although this central water molecule can readily donatehydrogen bonds to its neighboring water molecules in thehydrogen bonded chain, it cannot accept one from themnor reorient, thereby severing the proton-conductingwire. Both of these Asn residues occur in the sequenceAsn-Pro-Ala (NPA), the signature sequence of aquaporins,in which the Ala is located at the N-terminal end of each ofthe half-spanning helices.


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Figure 20-15 X-ray structure of the aquaporin AQP1 frombovine erythrocytes. (a) Ribbon diagram of an aquaporin subunit colored in rainbow order from its N-terminus (blue) toits C-terminus (red). The view is from within the membrane withits extracellular surface above and along the subunits pseudo-2-fold axis of symmetry. Note that the two helices closest to theviewer (orange and blue-green) are both portions of loops thatextend only to the center of the bilayer. The four water molecules that occupy the central portion of AQP1s water-transport channel are represented by red spheres. (b) View of theaquaporin homotetramer from the extracellular surface along its

Figure 20-16 Schematic drawing of the water-conducting poreof bovine aquaporin AQP1. The pore is viewed from within themembrane with the extracellular surface above. The positions ofresidues critical for preventing the passage of protons, other ions,and small molecule solutes are indicated. [Courtesy of PeterAgre, Johns Hopkins School of Medicine.]

(a)

(b)

4-fold axis. The subunit in the upper right is drawn in space-filling form with C green, N blue, and O red; that in the upper leftis drawn in ribbon form colored in rainbow order from its N-terminus (blue) to its C-terminus (red), that in the lower left isrepresented by its solvent-accessible surface; and that in thelower right displays the side chains forming the ar/R constriction(those of Phe 58, His 182, Cys 191, and Arg 197) in stick form.Each subunit forms a water-transport channel, which is mostclearly visible in the subunit drawn in space-filling form. [Basedon an X-ray structure by Bing Jap, University of California atBerkeley. PDBid 1J4N.]

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3 ATP-DRIVEN ACTIVE TRANSPORT

Mediated transport is categorized according to the stoi-chiometry of the transport process (Fig. 20-17):

1. A uniport involves the movement of a single mole-cule at a time. Maltoporin and GLUT1 are uniports.

2. A symport simultaneously transports two differentmolecules in the same direction.

3. An antiport simultaneously transports two differentmolecules in opposite directions.

The electrical character of ion transport is further spec-ified as:

1. Electroneutral (electrically silent) if there is simulta-neous charge neutralization, either by symport of oppo-sitely charged ions or antiport of similarly charged ions.Aquaporin is electroneutral.

2. Electrogenic if the transport process results in acharge separation across the membrane. KcsA and ClC areelectrogenic.

Since the glucose concentration in blood plasma is gen-erally higher than that in cells, GLUT1 normally transportsglucose into the erythrocyte, where it is metabolized viaglycolysis. Many substances, however, are available on oneside of a membrane in lower concentrations than are re-quired on the other side of the membrane. Such substancesmust be actively and selectively transported across themembrane against their concentration gradients.

Active transport is an endergonic process that is oftencoupled to the hydrolysis of ATP. How is this coupling ac-complished? In endergonic biosynthetic reactions, it oftenoccurs through the direct phosphorylation of a substrate by

ATP; for example, the formation of UTP in the synthesis ofglycogen (Section 18-2B). Membrane transport, however,is usually a physical rather than a chemical process; thetransported molecule is not chemically altered. Determin-ing the mechanism by which the free energy of ATP hy-drolysis is coupled to endergonic physical processes hastherefore been a challenging problem.

Three types of ATP hydrolyzing, transmembrane pro-teins have been identified that actively transport cations:

1. P-type ATPases are located mostly in plasma mem-branes and are so named because they are phosphorylatedby ATP during the transport process. P-type ATPases areknown that transport H, Na, K, Ca2, Cu2, Cd2, andMg2 against their concentration gradients. They are distin-guished from the other types of cation-translocating ATPasesby their inhibition by vanadate ( , a phosphate analog;see Problem 8 in this chapter).

2. F-type ATPases (F1F0) function to translocate pro-tons into mitochondria and bacterial cells, which in turnpowers ATP synthesis.They are discussed in Section 22-3C.

3. V-type ATPases are located in plant vacuolar mem-branes and acidic vesicles, such as animal lysosomes, andare homologous to the F-type ATPases.

Anions are transported by a fourth type of ATPase, the so-called A-type ATPases. In this section, we discuss P-typeATPases. We also examine a bacterial active transportprocess, in which the molecules transported are concomi-tantly phosphorylated, and the ABC transporters, whichtransport a wide variety of substances across membranes.In the next section, we study secondary active transportsystems, so called because they utilize the free energy ofelectrochemical gradients generated by ion-pumpingATPases to transport ions and neutral molecules againsttheir concentration gradients.

A. (NaK)ATPase of Plasma Membranes

One of the most thoroughly studied active transport sys-tems is the (NaK)ATPase of plasma membranes. Thistransmembrane protein, which was first isolated in 1957 byJens Skou, is often called the (NaK) pump because itpumps Na out of and K into the cell with the concomitanthydrolysis of intracellular ATP. Unlike most P-type ATPases,which are monomeric, (NaK)ATPases consist of and subunits. The 1000-residue, nonglycosylated subunitcontains the enzymes ATP and ion binding sites. It ishighly conserved (98% identical among mammals) andhomologous to single-subunit P-type ATPases such asthe Ca2-ATPase (Section 20-3B). The 300-residue, gly-cosylated subunit facilitates the correct insertion of the subunit into the plasma membrane and has been impli-cated in K transport.

The overall stoichiometry of the (NaK)ATPase re-action is

3Na (out) 2K (in) ADP Pi

3Na (in) 2K (out) ATP H2O

VO34


Figure 20-17 Uniport, symport, and antiport translocationsystems.

A (in)B (in)

Symport

A (out)B (out)

A (out)B (out)

A (in)B (in)

Antiport

A (in)

Uniport

A (out)

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The (NaK)ATPase is therefore an electrogenic an-tiport: Three positive charges exit the cell for every twothat enter. This extrusion of Na enables animal cells tocontrol their water content osmotically; without function-ing (NaK) pumps, animal cells, which lack cell walls,would swell and burst (recall that lipid bilayers are perme-able to H2O; Section 12-2Ba). Moreover, the electrochemi-cal potential gradient generated by the (NaK) pump isresponsible for the electrical excitability of nerve cells(Section 20-5Ba) and provides the free energy for the ac-tive transport of glucose and amino acids into some cells(Section 20-4A). In fact, all cells expend a large fraction ofthe ATP they produce (typically 30% and up to 70% innerve cells) to maintain their required cytosolic Na and K

concentrations.

a. ATP Phosphorylates an Essential Asp during theTransport ProcessThe free energy of ATP hydrolysis powers the ender-

gonic transport of Na and K against an electrochemicalgradient. In coupling these two processes, a kinetic barriermust somehow be erected against the downhill transportof Na and K along their ion concentration gradients,while simultaneously facilitating their uphill transport. Inaddition, futile ATP hydrolysis must be prevented in theabsence of uphill transport. How the enzyme does so is byno means well understood, although many of its mechanis-tic aspects have been elucidated.

A key discovery was that the protein is phosphorylatedby ATP in the presence of Na during the transportprocess. The use of chemical trapping techniques demon-strated that this phosphorylation occurs on an Asp residueto form a highly reactive aspartyl phosphate intermediate.For instance, sodium borohydride reduces acyl phosphatesto their corresponding alcohols. In the case of an aspartylphosphate residue, the alcohol is homoserine. By use of[3H]NaBH4 to reduce the phosphorylated enzyme, radioac-tive homoserine was, in fact, isolated from the acid hy-drolysate (Fig. 20-18). The phosphorylated residue, Asp374, begins the highly conserved sequence DKTG that oc-curs in the central region of the polypeptide chain.

b. The (NaK)ATPase Has Two MajorConformational StatesThe observations that ATP phosphorylates the

(NaK)ATPase only in the presence of Na, while theaspartyl phosphate residue is only subject to hydrolysis inthe presence of K, led to the realization that the enzymehas two major conformational states, E1 and E2. Thesestates have different tertiary structures, different catalyticactivities, and different ligand specificities:

1. E1 has an inward-facing high-affinity Na bindingsite (KM 0.2 mM, well below the intracellular [Na

]) andreacts with ATP to form the activated product E1P onlywhen Na is bound.

2. E2P has an outward-facing high-affinity K bind-ing site (KM 0.05M, well below the extracellular [K

])and hydrolyzes to form Pi E2 only when K

is bound.

c. An Ordered Sequential Kinetic ReactionMechanism Accounts for the Coupling of ActiveTransport with ATP HydrolysisThe (NaK)ATPase is thought to operate in accor-

dance with the following ordered sequential reactionscheme (Fig. 20-19):

1. E1 ATP, which acquired its ATP inside the cell,binds 3Na to yield the ternary complex E1 ATP 3Na.

2. The ternary complex reacts to form the high-energyaspartyl phosphate intermediate E1P 3Na.

3. This high-energy intermediate relaxes to its low-energy conformation, E2P 3Na, and releases itsbound Na outside the cell; that is, Na is transportedthrough the membrane.

4. E2P binds 2K from outside the cell to formE2P 2K.

5. The phosphate group is hydrolyzed, yielding E2 2K.

6. E2 2K changes conformation to E1, binds ATP,and releases its 2K inside the cell, thereby completing thetransport cycle.

The enzyme appears to have only one set of cation bindingsites, which apparently changes both its orientation and itsspecificity during the course of the transport cycle.

Section 20-3. ATP-Driven Active Transport 759

Figure 20-18 Reaction of [3H]NaBH4 with phosphorylated(NaK)ATPase. The isolation of [3H]homoserine followingacid hydrolysis of the protein indicates that the original phospho-rylated amino acid residue is Asp.

Aspartyl phosphateresidue

Homoserine

CH CH2 OPO3C

O

NaB

NH

C O

acid hydrolysis

+ 3H4

CH CH2 OHC Pi

NH

C O

+

3H

3H

CH CH2 OHC

NH3

COO

3H

3H

+

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The obligatory order of the reaction requires that ATPcan be hydrolyzed only as Na is transported uphill.Conversely, Na can be transported downhill only if ATPis concomitantly synthesized. Consequently, although eachof the above reaction steps is, in fact, individually re-versible, the cycle, as is diagrammed in Fig. 20-19, circulatesonly in the clockwise direction under normal physiologicalconditions; that is, ATP hydrolysis and ion transport arecoupled processes. Note that the vectorial (unidirectional)nature of the reaction cycle results from the alternation ofthe steps of the exergonic ATP hydrolysis reaction (Step 2,Step 5, and ATP binding in Step 6) with the steps of the en-dergonic ion transport process (Step 1, Steps 3 4, and K

release in Step 6). Thus, neither reaction can go to comple-tion unless the other one also does.

d. Mutual Destabilization Accounts for the Rate of Na and K TransportThe above ordered kinetic mechanism accounts only for

the coupling of active transport with ATP hydrolysis. In or-der to maintain a reasonable rate of transport, the free ener-gies of all its intermediates must be roughly equal. If someintermediates were much more stable than others, the stableintermediates would accumulate, thereby severely reducingthe overall transport rate. For example, in order for Na tobe transported out of the cell, uphill, its binding must bestrong to E1 on the inside and weak to E2 on the outside.Strong binding means greater stability and a potential bot-tleneck. This difficulty is counteracted by the phosphoryla-tion of E1 3Na and its subsequent conformationalchange to yield the low Na affinity E2P (Steps 2 and 3,Fig. 20-19). Likewise, the strong binding of K to E2P onthe outside is attenuated by its dephosphorylation and con-formational change to yield the low K affinity E1 (Steps 5

and 6, Fig. 20-19). It is these mutual destabilizations thatpermit Na and K to be transported at a rapid rate.

e. The X-Ray Structure of the (NaK)ATPaseChikashi Toyoshima determined the X-ray structure of

shark (NaK)ATPase in complex with K ions, anMgF4

2ion (a Pi mimic), and a 74-residue subunit namedFXYD that functions as a tissue-specific regulator. This X-ray structure (Fig. 20-20) is that of the E2P 2K complex(Fig. 20-19). The subunit of this 160--long proteinconsists of a transmembrane domain (M) composed of 10helices (M1M10) of varied lengths and, from top tobottom in Fig. 20-20, three well-separated cytoplasmic do-mains: the nucleotide-binding domain (N), which bindsATP; the actuator domain (A), so named because it partic-ipates in the transmission of major conformational changes(see below); and the phosphorylation domain (P), whichcontains the proteins phosphorylatable Asp residue. The subunits single transmembrane helix is tilted 32 fromthe normal to the plane of the membrane. The FXYDsubunit also has a single transmembrane helix but it isnearly perpendicular to the plane of the membrane. TheMgF4

2 ion marks the ATPases catalytic site and is coordi-nated by conserved residues from both its A and P do-mains. Two K ions are located 4.1 apart in a commonbinding cavity near the center of the subunits transmem-brane domain that is formed, in large part, by the partialunwinding of helices M5 and M7, and where they areeach liganded by several main chain carbonyl and sidechain oxygen atoms. The same cavity is implicated in bind-ing the three Na ions bound to the enzymes E1 form, withtwo of these binding sites probably formed by the sameside chains that coordinate the K ions and the third suchsite formed, in part, by the side chains of the subunits


Figure 20-19 Kinetic scheme for the active transport of Na and K by (NaK)ATPase.Here (in) refers to the cytosol and (out) refers to the exterior of the cell.

Mg2+

Mg2+

Na+ binding

H2O

1 Formation ofhigh-energy aspartylphosphate intermediate

2

Na+ transport3

K+ binding4Phosphatehydrolysis

5

K+ transport andATP binding

6

E1 ATP E1 ATP 3Na+ E1 P 3Na+

ATP Inside

3Na+ (in)

2K+ (in)

Outside3Na+ (out)

ADP

Pi 2K+ (out)

E2 P 2K+E2 2K+ E2 P

JWCL281_c20_744-788.qxd 6/4/10 12:15 PM Page 760

small C-terminal helix. No channel leading from either sideof the membrane to this binding cavity is apparent.

f. Cardiac Glycosides Specifically Inhibit the(NaK)ATPaseStudy of the (NaK)ATPase has been greatly facili-

tated by the use of cardiac glycosides (also called car-diotonic steroids), natural products that increase the inten-sity of heart muscle contraction. Indeed, digitalis, an extractof purple foxglove leaves (Fig. 20-21a), which contains amixture of cardiac glycosides including digitalin (Fig. 20-21b), has been used to treat congestive heart failure for cen-turies. The cardiac glycoside ouabain (pronounced wabane;Fig. 20-21b), a product of the East African ouabio tree, hasbeen long used as an arrow poison. These two steroids,which are still among the most commonly prescribed car-diac drugs, inhibit the (NaK)ATPase by bindingstrongly to an externally exposed portion of the enzyme(the drugs are ineffective when injected inside cells) so as toblock Step 5 in Fig. 20-19.The resultant increase in intracel-lular [Na] stimulates the cardiac (NaCa2) antiport sys-tem, which pumps Na out of and Ca2 into the cell (Sec-tion 22-1Bb). The increased cytosolic [Ca2] boosts the[Ca2] in other cellular organelles, principally the sarcoplas-mic reticulum (SR). Thus, the release of Ca2 to trigger


Figure 20-20 X-ray structure of shark (NaK)ATPase incomplex with K and MgF4

2 ions. The protein is drawn in ribbonform viewed parallel to the plane of the membrane (gray box) withthe cytosol above.The subunit is colored in rainbow order fromits N-terminus (blue) to its C-terminus (red), the subunit ismagenta, and the FXYD subunit is brown.The subunits boundK and MgF4

2 ions are drawn in space-filling form with K lightblue, Mg pink, and F light green. [Based on an X-ray structure byChikashi Toyoshima, University of Tokyo, Japan. PDBid 2ZXE.]

Ouabain

CH3

HOH2C

CH2

O O

H

OH

OH

HO

HO

HO

HCC

C OO

H

H

H H

Digitalin

HOCH3

CH3

H3C

CH2

O O

H

OH

H

OH

H

H

OH

H

H

HCC

C OO

OH

HOH

CH2OHO

H

OH

H

H

HOH

H

H

H3C

CH3

OH

OH

(b)

Figure 20-21 Cardiac glycosides. (a) The leaves of the purplefoxglove plant are the source of the heart muscle stimulant digitalis. [iStockphoto.] (b) Digitalin, a major component of

digitalis, and ouabain, a cardiac glycoside isolated from the EastAfrican ouabio tree, are among the most commonly prescribedcardiac drugs.

JWCL281_c20_744-788.qxd 6/12/10 11:07 AM Page 761

muscle contraction (Section 35-3Cb) produces a larger thannormal increase in cytosolic [Ca2], thereby intensifying theforce of cardiac muscle contraction. Ouabain, which wasonce thought to be produced only by plants, is now knownto also be an animal hormone that is secreted by the adre-nal cortex and functions to regulate cell [Na] and overallbody salt and water balance.


B. Ca2ATPase

Calcium ion often acts as a second messenger in a mannersimilar to cAMP (Section 19-4). Transient increases in cy-tosolic [Ca2] trigger numerous cellular responses, includingmuscle contraction (Section 35-3Ca), release of neurotrans-mitters (Section 20-5Cb), and, as we have seen, glycogen

Figure 20-22 Mechanism of SERCA based on four X-raystructures. (a) The X-ray structures of the SERCA complexes indicated in lightface are models for the conformational statesindicated in boldface. These states roughly correspond to those inthe corners of Fig. 20-19. The structures are represented by theirribbon diagrams embedded in their transparent surfaces (gray)as viewed along the plane of the membrane with the cytosolabove. The A, N, and P domains are colored yellow, red, and blue,the transmembrane helices M12, M34, M56, and M710 arepurple, green, tan, and gray, and bound Ca2 ions are representedby gray spheres. ATP and its mimics are drawn in stick form with C green, N blue, O red, and P orange. A conserved TGES

(a) (b)

sequence is drawn in space-filling form in magenta. In the lowerright drawing, the position of the lipid bilayer is indicated by the yellow rectangle. (b) Schematic diagram of the conformationalchanges made by SERCA during its catalytic cycle. The proteincomponents are colored as in Part a except that helices M510are all tan and Ca2 ions are represented by green spheres. In addition, protons are represented by gray spheres, and theresidues that ligand Ca2 ions in the binding cavity are indicatedby red spheres. [Modified from drawings by Poul Nissen,University of Aarhus, Denmark. PDBids 2C88, 3BA6, 3B9B, and3B9R.]

JWCL281_c20_744-788.qxd 6/12/10 11:07 AM Page 762

phosphorylated [via a high energy bond, which is repre-sented by a squiggle ()] and in complex with the ADPanalog adenosine-5-(-amino)diphosphate (AMPPN;Fig. 22-23c).

2. The A domain rotates toward the phosphorylationsite, thereby contacting the P and N domains. This move-ment pushes down on the M34 segment and pulls up onthe M12 segment, thereby separating the lower portionsof these segments from that of the M56 segment. This ex-poses the Ca2-binding cavity to the lumen of the SR andforces apart the residues that had bound the Ca2 ions inthe cavity, resulting in the release of these ions into the lu-men.A model for this E2P state is provided by the X-raystructure of SERCA in its E2 state in which BeF3

is cova-lently linked to Asp 351 to form a mimic of a phosphategroup.

3. Two or three protons bind in the lumenally exposedbinding cavity. This, together with the exchange of the ADPbound to the N domain for ATP results in movements oftransmembrane helices that again occludes the cavity fromthe lumen. A model for the resulting E2P* ATP state[where the star (*) is indicative of a transition state] is pro-vided by the X-ray structure of SERCA in its E2 state in

breakdown (Section 18-3Ce). Moreover, Ca2 is an impor-tant activator of oxidative metabolism (Section 22-4).

The use of phosphate as a basic energy currency requirescells to maintain a low internal [Ca2] because, for example,Ca3(PO4)2 has a maximum aqueous solubility of 65 M.Thus, the [Ca2] in the cytosol (0.1 M) is four ordersof magnitude less than it is in the extracellular spaces(1500 M). This large concentration gradient is maintainedby the active transport of Ca2 across the plasma mem-brane, the endoplasmic reticulum (ER; SR in muscle), andthe mitochondrial inner membrane. We discuss the mito-chondrial system in Section 22-1Bb.The plasma membraneand sarco(endo)plasmic reticulum each contain a P-typeCa2ATPase (Ca2 pump) that actively pumps Ca2 outof the cytosol at the expense of ATP hydrolysis. Their ki-netic mechanisms match that of the (NaK)ATPase(Fig. 20-19) except that two Ca2 ions replace the three Na

ions, two to three H ions replace the three K ions, and(out) refers to the outside of the cell for plasma membraneCa2ATPase or to the lumen of the sarco(endo)plasmicreticulum for the Ca2ATPase of that membrane.

a. X-Ray Structures of the Ca2ATPase Suggest Its MechanismThe X-ray structures of the 994-residue Ca2ATPase

from rabbit muscle sarcoplasmic reticulum [also known asSERCA for sarco(endo)plasmic reticulum Ca2-ATPase]in its complexes with a variety of analogs of ATP and itscomponents have been determined, the first of which were re-ported by Toyoshima. SERCA, which constitutes 90% of SRmembrane protein, is a 140--long monomeric protein thatclosely resembles the subunit of the (NaK)ATPase(Fig. 20-20). It contains a 10-helix transmembrane domain(M), an actuator domain (A), a nucleotide-binding do-main (N) to which the ATP binds, and a phosphorylationdomain (P) that contains the phosphorylatable Asp 351.The two Ca2 ions are bound in a cavity, similar to that inthe (NaK)ATPase, which is formed, in large part, bythe disruption of helices M4 and M6 in this region.

Four of these structures, all determined by Poul Nissen,collectively suggest a mechanism of action of P-type ATPasesresulting from a vectorial series of conformational changesdriven by ATP binding, hydrolysis, and release (Figs. 20-22):

1. Let us start with the X-ray structure of SERCA inits E2 conformation in complex with the inhibitor thapsi-gargin (TG; Fig. 22-23a), which stabilizes SERCAs E2state, and the nonhydrolyzable ATP analog adenosine-5-(,-methylene)triphosphate (AMPPCP; Fig. 22-23b).This structure provides a model for the E2 ATP state(Fig. 20-22, upper left). The exchange of 2 to 3 protons for2 cytosolic Ca2 ions causes a rotation of the A domainthat occludes the binding cavity. This activates the ATPasecatalytic site to phosphorylate Asp 351 yielding ADP,which releases the N domain from the P domain dueto the cleavage of the ATPs ,-phosphodiester bondthat had previously linked these two domains. A modelfor the resulting Ca2E1P ADP state is provided by theX-ray structure of SERCA in its E1 state with Asp 351


Figure 20-23 Molecular formulas of several SERCA inhibitors. (a) Thapsigargin (TG), a product of the plant Thapsiagarganica, (b) AMPPCP, and (c) AMPPN.

Thapsigargin (TG)(a)

OH

OH

H

O

O

O

OO O

OO

O

O

P

O

O

A

CH2CH2

H HH H

OH OH

OP

O

O

Adenosine-5-(,-methylene)triphosphate(AMPPCP)

O

O

O

P

O

O

(b)

Adenosine-5-(-amino)diphosphate(AMPPN)

P O

A

CH2

H HH H

OH OH

ONH2 O

O

O

P

O

O

(c)

JWCL281_c20_744-788.qxd 7/1/10 7:18 AM Page 763

C. (HK)ATPase of Gastric Mucosa

Parietal cells of the mammalian gastric mucosa secrete HClat a concentration of 0.15M (pH 0.8). Since the cytosolicpH of these cells is 7.4, this represents a pH difference of6.6 units, the largest known in eukaryotic cells.The secretedprotons are derived from the intracellular hydration ofCO2 by carbonic anhydrase:

The secretion of H involves the participation of an(HK)ATPase, an electroneutral antiport with struc-ture and properties similar to that of Ca2ATPase. Likeother P-type ATPases, it is phosphorylated during thetransport process. In this case, however, the K, which en-ters the cell as H is pumped out, is subsequently external-ized by its electroneutral cotransport with Cl. HCl istherefore the overall transported product.

a. Cimetidine and Omeprazole Prevent GastricUlcers and HeartburnFor many years, effective treatment of peptic ulcers,

which was a frequently fatal condition caused by the attackof stomach acid on the gastric mucosa, often required sur-gical removal of the affected portions of the stomach orsometimes the entire stomach. The discovery, by JamesBlack, of cimetidine,

which inhibits stomach acid secretion, all but eliminatedthe need for this dangerous and debilitating surgery. The(HK)ATPase of the gastric mucosa is activated by his-tamine stimulation of the H2-receptor in a process medi-ated by cAMP. Cimetidine (trade name Tagamet), whichbecame available in 1976, was the first of several widelyused drugs that competitively inhibit the binding of hista-mine to this receptor. These drugs likewise alleviate thesymptoms of gastroesophageal reflux disease (GERD,commonly known as heartburn), which is caused by the re-guritation of stomach acid into the esophagus, a widespreadand painful condition that, when chronic, can damage theesophagus and cause esophageal cancer. Heartburn canalso be relieved by antacids but the effects of cimetidineand its analogs are longer lasting (610 hours vs 12 hoursfor antacids, although the former require 30 minutes totake effect) and can be taken before meals to preventheartburn from occurring. A drawback of most of thesesubstances is that they inhibit several cytochrome P450sand thereby interfere with the metabolism of numerouswidely used drugs (Section 15-4Bc).

CH2S

N

CH2

Cimetidine

CH2

N

NH NHC CH3

C

NHN

H3C

CH2CH2 NH3

NHN

+

Histamine

CO2 H2O HCO3 H


complex with AMPPCP and AlF4, which is covalently

linked to Asp 351 to form a trigonal bipyramidal mimic ofthe hydrolytic transition state (Fig. 16-6b).

4. The dephosphorylation of Asp 351, as stimulated bythe previous binding of ATP, motivates conformationalchanges that opens a channel to the cytosol that permitsthe exchange of protons with Ca2 ions, thereby complet-ing the catalytic cycle.

This mechanism explains how P-type ATPases transportcations against their steep electrochemical gradients.

b. Calmodulin Regulates the Plasma Membrane Ca2 PumpFor a cell to maintain its proper physiological state, it

must regulate the activities of its ion pumps precisely. Theregulation of the Ca2 pump in the plasma membrane iscontrolled by the level of Ca2 through the mediation ofcalmodulin (CaM).This ubiquitous eukaryotic Ca2-bindingprotein participates in numerous cellular regulatoryprocesses including, as we have seen, the control of glyco-gen metabolism (Section 18-3Ce).

Ca2Calmodulin activates the Ca2ATPase of plasmamembranes. The activation, as deduced from the study of theisolated ATPase, results in a decrease in its KM for Ca

2 from20 to 0.5 M. Ca2CaM activates the Ca2 pump by bindingto an inhibitory polypeptide segment of the pump in a man-ner similar to the way in which Ca2CaM activates its tar-get protein kinases (Section 18-3Cf). Evidence supportingthis mechanism comes from proteolytically excising theCa2 pumps CaM-binding polypeptide, yielding a truncatedpump that is active even in the absence of CaM. Syntheticpeptides corresponding to this CaM-binding segment notonly bind Ca2CaM but inhibit the truncated pump by in-creasing its KM for Ca

2 and decreasing its Vmax. This sug-gests that, in the absence of Ca2CaM, the CaM-bindingsegment of the pump interacts with the rest of the protein soas to inhibit its activity. When the Ca2 concentration in-creases, Ca2CaM forms and binds to the CaM-bindingsegment of the pump in a way that causes it to dissociatefrom the rest of the pump, thereby relieving the inhibition.

Now we can see how Ca2 regulates its own cytoplasmicconcentration: At Ca2 levels below calmodulins 1 Mdissociation constant for Ca2, the Ca2ATPase is relativelyinactive due to autoinhibition by its CaM-binding segment. If,however, the [Ca2] rises to this level, Ca2 binds to calmod-ulin, which, in turn, binds to the CaM-binding segment so asto relieve the inhibition, thereby activating the Ca2 pump:

(CaM* indicates activated calmodulin). This interaction de-creases the pumps KM for Ca

2 to below the ambient [Ca2],thereby causing Ca2 to be pumped out of the cytosol.Whenthe [Ca2] decreases sufficiently, Ca2 dissociates fromcalmodulin and this series of events reverses itself, thereby in-activating the pump.The entire system is therefore analogousto a basement sump pump that is automatically activated bya float when the water reaches a preset level.

Ca2CaM* pump(active)Ca2 CaM Ca2CaM* pump(inactive)

JWCL281_c20_744-788.qxd 7/1/10 7:19 AM Page 764

energy phosphoryl donor for ATP synthesis in the pyruvatekinase reaction of glycolysis; Section 17-2J). The PTS simul-taneously transports and phosphorylates sugars. Since the cellmembrane is impermeable to sugar phosphates, once theyenter the cell, they remain there. Some of the PTS-transportedsugars are listed in Table 20-2.

The PTS system involves two soluble cytoplasmic pro-teins, Enzyme I (EI) and HPr (for histidine-containingphosphocarrier protein), which participate in the transportof all sugars (Fig. 20-24). In addition, for each sugar the sys-tem transports, there is a specific transmembrane transportprotein EII, which consists of at least three functional com-ponents: two that are cytoplasmic, EIIA and EIIB, and atransmembrane channel, EIIC. These three components as-sociate differently in different EIIs. In E. coli, for example,EIIA, EIIB, and EIIC are separate subunits in cellobiose-specific EII; EIIB and EIIC are covalently linked and EIIAis separate in glucose-specific EII; and all three componentsare present on a single peptide in mannitol-specific EII.

Glucose transport, which resembles that of other sugars,involves the transfer of a phosphoryl group from PEP toglucose with net inversion of configuration about the phos-phorus atom. Since each phosphoryl transfer involves inver-sion (Section 16-2A), an odd number of transfers must be

Omeprazole (trade names Prilosec and Nexium),

directly inhibits the (HK)ATPase by forming anadduct with the side chain of its Cys 831. Since its introduc-tion in 1989, omeprazole has greatly supplanted the use ofcimetidine and its analogs due to the irreversible nature ofits inhibition (which reduces acid secretion by up to 99%)and its lack of drugdrug interactions. Hence, omeprazole ispresently one of the most frequently used drugs worldwide.

D. Group Translocation

Group translocation is a variation of ATP-driven activetransport that most bacteria use to import certain sugars. Itis required for many bacterial processes, both useful andharmful (to humans), such as those that produce cheese, soysauce, and dental cavities. It differs from active transport inthat the molecules transported are simultaneously modifiedchemically. The most extensively studied example of grouptranslocation is the phosphoenolpyruvate-dependent phos-photransferase system (PTS) of E. coli discovered by SaulRoseman in 1964. Phosphoenolpyruvate

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