ATP-dependent sugar transport complexity in human erythrocytes

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ATP-dependent sugar transport complexity in human erythrocytes

Jeffry M. Leitch and Anthony CarruthersDepartment of Biochemistry and Molecular Pharmacology, Universityof Massachusetts Medical School, Worcester, Massachusetts

Submitted 19 June 2006; accepted in final form 15 August 2006

Leitch JM, Carruthers A. ATP-dependent sugar transportcomplexity in human erythrocytes. Am J Physiol Cell Physiol292: C974 –C986, 2007. First published August 23, 2006;doi:10.1152/ajpcell.00335.2006.—Human erythrocyte glucose sugartransport was examined in resealed red cell ghosts under equilibriumexchange conditions ([sugar]intracellular � [sugar]extracellular, wherebrackets indicate concentration). Exchange 3-O-methylglucose(3MG) import and export are monophasic in the absence of cytoplas-mic ATP but are biphasic when ATP is present. Biphasic exchange isobserved as the rapid filling of a large compartment (66% cellvolume) followed by the slow filling of the remaining cytoplasmicspace. Biphasic exchange at 20 mM 3MG eliminates the possibilitythat the rapid exchange phase represents ATP-dependent 3MG bind-ing to the glucose transport protein (GLUT1; cellular [GLUT1] of�20 �M). Immunofluorescence-activated cell sorting analysis showsthat biphasic exchange does not result from heterogeneity in cell sizeor GLUT1 content. Nucleoside transporter-mediated uridine exchangeproceeds as rapidly as 3MG exchange but is monoexponential regard-less of cytoplasmic [ATP]. This eliminates cellular heterogeneity oran ATP-dependent, nonspecific intracellular diffusion barrier ascauses of biphasic exchange. Red cell ghost 3MG and uridine equi-librium volumes (130 fl) are unaffected by ATP. GLUT1 intrinsicactivity is unchanged during rapid and slow phases of 3MG exchange.Two models for biphasic sugar transport are presented in which 3MGmust overcome a sugar-specific, physical (diffusional), or chemical(isomerization) barrier to equilibrate with cell water. Partial transportinhibition with the use of cytochalasin B or maltose depresses bothrapid and slow phases of transport, thereby eliminating the physicalbarrier hypothesis. We propose that biphasic 3MG transport resultsfrom ATP-dependent, differential transport of 3MG anomers in whichVmax/apparent Km for �-3MG exchange transport is 19-fold greaterthan Vmax/apparent Km for �-3MG transport.

carrier-mediated transport; transport kinetics; transport regulation

A FAMILY OF INTEGRAL MEMBRANE proteins called glucose trans-porters (GLUTs) (40) mediates equilibrative sugar transport inmammalian cells. The glucose transport protein GLUT1 cata-lyzes sugar transport in cells of the reticuloendothelial system(7, 61) and presents an interesting experimental puzzle. Thesteady-state kinetics of GLUT1-mediated sugar transport inrabbit (70), rat (38, 62), and avian (7, 8) erythrocytes and inbasal (insulin-starved) rat adipocytes (76) are consistent withclassical models for carrier-mediated solute transport (5, 47).GLUT1-mediated sugar transport in human red cells, however,displays a kinetic complexity that has proven difficult toreconcile with models for carrier-mediated transport (4, 21, 32,49, 56, 79).

Transport complexity is especially obvious in zero-trans exitand infinite-cis entry conditions (13). In the zero-trans exit

condition, cells are loaded with various starting sugar concen-trations and the initial rate of exit is measured (49, 56), or thecomplete time course of exit is analyzed by using an integratedMichaelis-Menten equation (4, 16, 42, 56). Initial rate mea-surements (49, 56) routinely provide estimates of apparent Km

[Km(app)] for sugar exit that are two to three times lower thanthose obtained by analysis of the complete time course of sugarexit (4, 16, 42, 56). In the infinite-cis sugar uptake experiment,the external sugar level is saturating and the concentration ofintracellular sugar that reduces net sugar uptake by one-half ismeasured. Km(app) for infinite-cis entry is routinely 5- to 10-fold lower than values predicted by classical carrier models forsugar transport (13, 14, 21, 22).

Why is it that human red cell sugar transport displays kineticcomplexity, whereas GLUT1-mediated sugar transport in rab-bit, rat, and avian erythrocytes and in rat adipocytes is consis-tent with sugar transport models? Human, rat, and rabbitGLUT1 share 98.4% identity (3, 7, 61). Six common residuesin rabbit and rat GLUT1 diverge from human GLUT1 se-quence, but all six substitutions show positive scores on thepoint-accepted mutation similarity scale (1), suggesting thateach substitution is unlikely to affect structure or function. Ifsequence divergence is not the cause of transport complexity,GLUT1 phenotypic variation must result from differences incellular environment, GLUT1 expression levels, or artifacts ofmeasurement.

The sugar transport capacity of human red cells is 220- to10,000-fold greater than that of rat basal adipocytes (76), ratred blood cells (38), and avian erythrocytes (24). Naftalin andHolman (63) discussed several ways by which this could giverise to transport complexity. 1) Transport measurements inhuman red blood cells are technically challenging even at lowtemperatures owing to the very high GLUT1 density of humanblood red cells and the high catalytic turnover of GLUT1 (49).Significant back flux of imported sugar during the course of atransport determination would lead to underestimation of netimport. 2) If net cellular sugar import were composed of twosteps (transport followed by intracellular diffusion/distribu-tion), the diffusional step could become rate limiting if thetransport step were sufficiently rapid. Evidence for nonuniformintracellular distribution of sugars has been obtained in bothhuman and rat erythrocytes (4, 36, 38, 62, 64).

According to the diffusional barrier hypothesis, human redcell net sugar import is composed of rapid transport (owing tohigh cellular GLUT1 content) and slow intracellular diffusionand/or distribution. The overall result is one where net sugarimport is rate limited by intracellular diffusion/distribution andnot by transport. Measurements of sugar uptake in rat or rabbit

Address for reprint requests and other correspondence: A. Carruthers,Dept. of Biochemistry and Molecular Pharmacology, Univ. of Massachu-setts Medical School, 364 Plantation St., Worcester, MA 01605 (e-mail:[email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Cell Physiol 292: C974–C986, 2007.First published August 23, 2006; doi:10.1152/ajpcell.00335.2006.

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erythrocytes, however, largely reflect low-capacity GLUT1-mediated transport and thus provide a more accurate descrip-tion of the intrinsic properties of GLUT1.

This hypothesis infers that GLUT1-mediated sugar transportis inherently simple but that operational complexity is causedby factors extrinsic to the transport system. Transport com-plexity is lost in cytosol-depleted human red cell ghosts (16,20, 37, 41) but is preserved if ATP is included in artificialcytosol during red cell ghost resealing (12, 37). Does this meanthat human GLUT1 is uniquely ATP sensitive? HumanGLUT1 is an ATP-binding protein (15, 22) and contains threesequence motifs that form the ATP-binding pocket of humanadenylate kinase (27). One of these domains (GLUT1 residues332–338) has been identified by peptide mapping, by micro-sequencing of proteolyzed, azidoATP-photolabeled humanGLUT1 (43), and by scanning alanine mutagenesis (44) as adomain critically involved in nucleotide-GLUT1 interaction.This domain is unchanged in rabbit and rat GLUT1 sequenceswhere there is no a priori reason to assume that it does notserve a similar function. It is not known, however, whetherrabbit or rat red cell sugar transport is modulated by alteredintracellular ATP. Parental (hamster) and exogenous (human)GLUT1-mediated sugar transport are insensitive to cellularATP depletion in Chinese hamster ovary cells (22, 44),whereas ATP-sensitive, human GLUT1-mediated sugar trans-port is observed in transfected HEK cells (22, 44) and in clone9 cells (73). This suggests that cellular environment influencesGLUT1 phenotype.

These observations stimulate two questions. 1) Is human redcell sugar transport complexity an artifact of measurement? 2)If complexity is real, do human erythrocytes and clone 9 andHEK cells uniquely express cellular factors that partner in ATPmodulation of sugar transport? We address the former questionin the present study. Human red cell membrane GLUT1 con-tent approaches 10% total protein by mass (36), resulting inextremely rapid sugar transport rates at all temperatures studied(8). Measurements of transport are, therefore, complicated bythe necessity for rapid sampling procedures (8, 49) but may besimplified by use of the equilibrium exchange condition (77).In equilibrium exchange, intracellular sugar concentration �extracellular sugar concentration, no net transport occurs, andunidirectional sugar fluxes are measured by addition of radio-tracer sugar either to the external or to the internal medium. Inprinciple, radiotracer fluxes under these conditions are firstorder and thus monoexponential in a uniform population ofcells. We have used this condition to investigate whether sugartransport in red blood cells reflects the properties of transmem-brane sugar flux alone or whether steps subsequent to trans-membrane flux also contribute to overall transport behavior.

MATERIALS AND METHODS

Materials. 3H-labeled 3-O-methylglucose (3MG), 14C-labeled3MG, [3H]uridine (Urd), and [14C]Urd were purchased from Sigma.Rabbit antisera raised against a synthetic carboxy-terminal peptide ofGLUT1 (C-Ab) were obtained from East Acres Biologicals. Sheepantisera raised against tetrameric GLUT1 (�-Ab) were prepared asdescribed previously (34). Fluorescein-conjugated goat anti-rabbitantibody was purchased from Molecular Probes. Fluorescein-conju-gated goat anti-sheep antibody was purchased from Calbiochem.Human blood was purchased from Biological Specialties. Other re-agents were purchased from Sigma.

Solutions. Kaline consisted of (in mM) 150 KCl, 5 MgCl2, 5EGTA, and 5 HEPES (pH 7.4). Lysis buffer contained 10 mMTris �HCl and 2 mM EDTA (pH 8.0). Stripping solution contained 2mM EDTA and 15.4 mM NaOH (pH 12). Sugar-stop solution con-sisted of ice-cold kaline containing 20 �M CCB and 200 �Mphloretin. Urd stop solution consisted of ice-cold kaline containing 50�M S-nitrothioinosine.

Red blood cells. Red blood cells were isolated by washing wholehuman blood in four or more volumes of ice-cold kaline and centri-fuging at 10,000 g for 15 min at 4°C. Serum and buffy coat wereremoved by aspiration, and the wash, centrifugation, aspiration cycleswere repeated until the buffy coat was no longer visible. Cells wereresuspended in four volumes of sugar-free or sugar-containing kalineand incubated for 1 h at 37°C to deplete or load intracellular sugar.

Red cell ghosts. Ghosts were hypotonically lysed by resuspendingwashed red blood cells in 10 volumes of ice-cold lysis buffer for 10min. Membranes were harvested by centrifugation at 27,000 g for 20min. Ghosts were repeatedly washed with lysis buffer and centrifugeduntil the membranes appeared light pink (�3 cycles). Ghosts werethen washed with 10 volumes of ice-cold kaline and collected bycentrifugation at 27,000 g. Harvested membranes were resealed byincubation in four volumes of kaline � 4 mM ATP (37°C ) for 1 h andcollected by centrifugation at 27,000 g for 15 min at 4°C. Resealedghosts were stored on ice until use.

Net 3MG uptake. Sugar-depleted cells or ghosts were incubated in20 volumes of ice-cold kaline containing 100 �M unlabeled 3MG and0.5 �Ci/ml labeled 3MG. Uptake was allowed to proceed for intervalsas short at 6 s to intervals as long as 3 h. Uptake was arrested byaddition of ice-cold stop buffer, and ghosts were centrifuged at 14,000g for 1 min. The supernatant was removed by aspiration, and ghostswere washed with 20 volumes of sugar stop buffer and recentrifuged,and supernatant was aspirated. The ghost pellet was extracted with500 �l of 3% perchloric acid and centrifuged, and samples of the clearsupernatant were counted in duplicate. Zero time points were col-lected by the addition of sugar stop solution to ghosts followed byuptake medium. Samples were then immediately processed. Radioac-tivity associated with cells at zero time was subtracted from allnonzero time points. Equilibrium time points were collected with anovernight incubation. All time points were normalized to the equilib-rium time point. All solutions and tubes used in the assay werepreincubated on ice for 30 min before the start of the experiment.Triplicate samples were processed for each time point.

Initial rate, zero-trans 3MG uptake: time dependence. Sugar-depleted cells or ghosts were allowed to rest in 20 volumes of ice-coldkaline for intervals as short as 30 s or as long as 3 h. Thirty secondsbefore the end of the rest interval, a small volume of 3MG and 0.5�Ci/ml tracer 3MG were added to the suspension to a final concen-tration of 3MG of 100 �M. Uptake was stopped by addition of sugarstop solution, and cells or ghosts were processed as above. A zerotime point was obtained as described above. An equilibrium timepoint was obtained by allowing ghosts to uptake unlabeled and labeled3MG overnight at 4°C. Uptake rates were expressed as micromolesper minute per liter cell water by subtracting the zero time point andnormalizing to the equilibrium time point.

3MG equilibrium exchange, uptake, and exit. Ghosts were resealedin the presence of 0.1, 2.5, 10, or 20 mM unlabeled 3MG and traceamounts (0.5 �Ci/ml) of [14C]3MG and centrifuged, and the super-natant was aspirated. Ghosts were then incubated in 20 volumes ofice-cold kaline containing the same concentration of 3MG and 0.5�Ci/ml [3H]3MG. Exchange was allowed to proceed for time inter-vals as short as 6 s to intervals as long as 10 h. Exchange was thenstopped by the addition of ice-cold sugar stop, and ghosts were treatedas above with zero-trans uptake.

Urd equilibrium exchange, uptake, and exit. Experiments wereperformed as with 3MG equilibrium exchange except that the trans-port substrate was Urd, and Urd stop was used to stop the reaction andto wash the ghosts.

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GLUT1 immunofluorescence microscopy and fluorescence-acti-vated cell sorting analysis. For C-Ab immunofluorescence staining,50 �l of nonfixed unsealed ghosts were washed once in ice-coldkaline and centrifuged at 4°C, and the supernatant was aspirated. A1:500 dilution of C-Ab was added to the pellet, the suspension wasincubated at 4°C for 2 h and centrifuged, and the supernatant wasaspirated. Ghosts were washed with ice-cold kaline. Secondary anti-body (1:500 dilution) was added to the pellet, and the suspension wasincubated at 4°C for 1 h and centrifuged, and the supernatant wasremoved by aspiration. Ghosts were washed five times with ice-coldkaline and brought to a final volume of 500 �l. For �-Ab staining,resealed ghosts were incubated with a 1:500 dilution of �-Ab at 37°Cfor 1 h and then processed as above. For microscopy, 10 �l of thesuspension were added to a polylysine-coated coverslip and adheredby low-speed centrifugation. Coverslips were washed five times with10 ml of ice-cold kaline. Fluorescence microscopy was performed onan Olympus BX-51 fluorescence microscope. For fluorescence-acti-vated cell sorting (FACS) analysis, 50 �l of the immunofluorescence-stained ghosts were mixed with �1 ml of FACS buffer in a FACSsample tube. FACS analysis was performed with a Becton DickinsonFACS scan sampled for 10,000 counts and analyzed with CellQuest 3.3.

Ghost-accessible volumes. Ghosts were prepared and resealed withand without intracellular ATP. After ghosts were resealed, 2.5 mM3MG or Urd and tracer [3H]3MG or [3H]Urd and [14C]sucrose wereadded to the ghosts, and the radiotracers were allowed to equilibratewith cell water. The samples were centrifuged at 14,000 g for 1 min,and known volumes of supernatant and pellet were sampled andcounted for both isotopes. Ghosts were counted on a hemocytometer.Tracer accessible volume (Vacc) was calculated as

Vacc �

�1 �14Cp

14Cs� 3HpVs

3Hs

Cell number(1)

where 14C and 3H refer to the respective tracers (dpm), V is thesample volume, and subscripts S and P refer to supernatant and pellet,respectively. In this manner, the extracellular volume of the pellet issubtracted by measuring the sucrose space. Alternatively, ghosts wereresealed with 2.5 mM 3MG and 2.5 mM Urd plus [3H]3MG and[14C]Urd or [14C]3MG and [3H]Urd radiotracers. At 0 and 15 h afterresealing was completed, ghosts were sedimented by centrifugationand the supernatant was sampled. The ghost pellets were washedtwice with 20 �M CCB, 200 �M phloretin, and 50 �M S-nitrothioi-nosine in ice-cold kaline. Known volumes of supernatant and pelletwere counted for 3MG and Urd, and pellet-to-supernatant ratios werecomputed.

Red blood cell and ghost electron microscopy. Scanning electronmicroscopy (SEM) of red blood cells and ghosts was carried out aspreviously described (35). Red blood cells and ghost cells were fixedby immersion in 2.5% (vol/vol) glutaraldehyde in 0.5 M sodiumphosphate buffer (pH 7.2) for 1 h at room temperature. Fixed sampleswere then washed three times in the same buffer. After the third wash,the cells were postfixed for 1 h in 1% osmium tetroxide (wt/vol) in thephosphate buffer, washed three times in buffer, and left overnight at4°C. Samples were dehydrated through a graded series of ethanol to100%, and then the bottoms of the tubes were excised, place in poroussample holders, and critical point dried in liquid CO2 . After thedrying step, tubes were emptied onto aluminum SEM stubs coatedwith adhesive carbon tape. The edges were then painted with silverconductive paste, and the SEM stubs were sputter coated with Au/Pd(80/20). The specimens were then examined by an ETEC autoscanscanning electron microscope at 20-kV accelerating voltage.

Circular dichroism. Freshly dissolved �-3MG or equilibrium so-lutions of 3MG (each 20 mM) were prepared in kaline, and thecircular dichroism of the sugar was monitored. Data were collected at

4°C by a Jasco J-810 spectropolarimeter and a 0.1-cm pathlengthcuvette.

Proton NMR. Data were collected at 24°C on a 400-MHz Ox-ford NMR.

Curve-fitting procedures. Where appropriate, data sets were ana-lyzed by nonlinear regression using the software packages proFit(version 6.06; Quantum Soft, Uetikon am See, Switzerland) or Igor-Pro (version 5; Wavemetrics, Lake Oswego, OR). Numerical integra-tion was performed by using Berkeley Madonna X (version 8.3.14).

RESULTS

Effect of ATP on 3MG equilibrium exchange transport. Theeffect of intracellular ATP on equilibrium exchange 3MGtransport at 4°C was monitored over the course of 5 h bymeasuring the simultaneous uptake of tracer [3H]3MG and exitof [14C]3MG (Fig. 1). Resealed ghosts lacking intracellularATP show an exchange time course that follows a single,simple exponential rise or decay for uptake and exit, respec-tively. This is expected for a passive transport process charac-terized by a single rate-limiting step and occurring in a uniformpopulation of cells. When ghosts are resealed with 4 mMintracellular ATP, however, the time course changes, becom-ing biphasic for both unidirectional uptake and exit. The earlyphase of transport is accelerated fourfold in the presence ofATP. After 10 min when one-half to two-thirds of unidirec-tional exchange is complete, the rate of transport declines bymore than 20-fold to a rate approaching 10-fold slower thantransport in the absence of ATP. Table 1 summarizes fitparameters for ghosts lacking and containing ATP. Equilib-rium exchange time courses have been studied previously bythis laboratory, but biphasic transport was observed only at thelowest 3MG concentration employed (0.1 mM) (23), wherebiphasic exchange may be related to GLUT1 sugar binding.That study did not monitor transport beyond 5 min, whichexplains why biphasic equilibrium exchange transport at higher3MG concentrations was not observed.

One explanation for biphasic transport in the presence ofATP is that a significant subpopulation of ghosts spontaneouslyreseal before ATP is introduced so that the measurementssample a mixed population of ATP-containing (fast) and ATP-lacking (slow) ghosts. This simple hypothesis is refuted by theobservation that the slow phase of 3MG transport in ATP-containing ghosts is nearly 10 times slower than transport inATP-free ghosts.

Red cell ghosts present a relatively uniform population ofcell sizes and GLUT1 content. A mixed population of ghostcell sizes or GLUT1 content could account for biphasic 3MGexchange transport. As cell size decreases, the surface area-to-volume ratio increases, thereby increasing the rate of GLUT1-mediated equilibration of radiotracer sugar. Altered GLUT1content (or activity) at the membrane surface also determinesthe rate constant for radiotracer equilibration.

GLUT1 content was assayed by immunofluorescence mi-croscopy of fixed or living cells and by FACS. Figure 2, A–C,shows fluorescence micrographs of ghosts stained with anti-bodies directed to cytoplasmic (Fig. 2A) or extracellular (Fig.2, B and C) GLUT1 epitopes. The use of cytoplasmic epitope-directed antibodies (C-Ab) necessitates cell fixation/permeabi-lization. FACS analysis of GLUT1 staining by exofacial anti-bodies (�-Ab) suggests a uniform population of cells � ATP(Fig. 2E). C-Ab staining of unsealed ghosts � ATP produces

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similar results (data not shown). Cell size was quantified byFACS analysis of single cell light scattering. Ghosts show asingle population of scattering intensities as detected at eitherlow (data not shown) or high angles (Fig. 2D) and scattering isindependent of cytoplasmic ATP concentration.

Effect of ATP on human equilibrative nucleoside transporter1-mediated Urd exchange transport. Previous models haverationalized sugar transport complexity by hypothesizing anunstirred layer beneath the red cell membrane (4, 62, 63).Although predicted deviations from simple Michaelis-Mentenkinetics at low 3MG concentrations are observed for sugar exitin human and rat red blood cells (36, 38), direct evidence forthis unstirred layer is not available.

Human equilibrative nucleoside transporter-1 (ENT1)-medi-ated Urd transport was examined to determine whether theputative unstirred layer is sugar transport specific. Figure 3shows the time course of 2.5 mM Urd exchange in ghosts �ATP. Although there are 100-fold fewer nucleoside transport-ers per red blood cell than there are glucose transporters, Km

for Urd transport is �100 times lower than Km(app) for GLUT1-mediated 3MG transport (44). The apparent rate constant fortransport of any species (k) is proportional to the ratio of Vmax

to Km(app) (75); hence, sugar transport and Urd transportproceed at similar rates. Urd uptake and exit are monoexpo-nential in ghosts regardless of cellular ATP content (Fig. 3). Ifan intracellular unstirred layer or diffusional barrier does exist,it must be selective for GLUT1 substrates. Monophasic Urdexchange transport in ghosts lacking and containing ATP alsoargues against multiple cell sizes in resealed ghosts.

Effect of intracellular ATP on accessible sugar and Urdvolumes. Table 2 summarizes two experiments in which theintracellular 3MG and Urd spaces of red cell ghosts weremeasured in the absence and presence of 4 mM intracellularATP. 3MG and Urd spaces are identical and are unaffected byinclusion of 4 mM ATP during resealing. Differences betweenexperiments may result from cell counting errors caused bylow contrast of red cell ghosts in the hemocytometer. Becausetransport experiments span several hours, the time dependenceof cell volumes was also investigated. The normalized internal-to-external ratios of 3MG and Urd concentrations � ATP at 0and 15 h are shown in Fig. 4. There is no change in theaccessible volume over that time.

Effect of preincubation time on initial rates and time coursesof transport. We have found that ATP levels are reduced by�50% during the 5 h of an exchange time course experiment(data not shown). Although AMP and ADP do not directlyaffect transport, they serve as competitive inhibitors of ATPbinding to GLUT1 (15). It is possible, therefore, that nucleo-tide-dependent changes in GLUT1 intrinsic activity occur dur-ing the time course of our measurements. To examine thispossibility, both the time course of net 3MG uptake and theinstantaneous rate of 3MG uptake were measured in ATP-freeand ATP-containing ghosts before and after 5 h of mock(sugar-free) transport. Figure 5A shows that ATP modulationof zero-trans uptake persists after 5 h. No change in theinstantaneous (initial) rate of zero-trans 3MG uptake is ob-served after 5 h (Fig. 5B). Biphasic exchange transport persistsin ghosts that have previously exchanged 3MG for 15 h beforethey are assayed for radiotracer exchange (Fig. 5C). These dataindicate that time-dependent changes in the intrinsic activity ofGLUT1 do not account for biphasic sugar transport.

Fig. 1. Time course of 3-O-methylglucose (3MG) equilibrium exchangeuptake and exit in red cell ghosts containing or lacking intracellular ATP.Ordinate, fractional equilibration; abscissa, time in minutes (note log scale). A:exchange of 2.5 mM 3MG in red cell ghosts resealed without intracellularATP. Uptake (F) follows a single-exponential rise, and exit (E) follows asingle-exponential decay. Solid curves drawn through the points are computedby nonlinear regression and take the following forms: (1 � e�kt) for uptake ande�kt for exit, where k is the observed rate constant and t is time. Dashed linesshow the least-squares computed fits for uptake and exit (Eq. 3) where Km(app)

for uptake and exit are 38.8 and 38.2 mM, respectively, and Vmax for uptakeand exit are 3.65 and 4.07 mmol � l�1 �min�1, respectively. B: exchange of 2.5mM 3MG in red cell ghosts resealed with 4 mM intracellular ATP. Uptake (F)and exit (E) follow biexponential kinetics. Solid curves drawn through thepoints are computed by nonlinear regression of the following forms: A(1 �e�k1t) (1 � A)(1 � e�k2t) for uptake and A(e�k1t) (1 � A)(e�k2t) for exit,where k1 is the observed rate constant for the fast phase, k2 is the slow phaserate constant, and A is the fractional component of total uptake or exitdescribed by the fast phase. Values of k, A, k1, and k2 are shown in Table 1.Dashed lines show the least-squares computed fits for uptake and exit (Eq. 2)where Km(app) for uptake and exit of �-3MG are 37.1 and 24.8 mM, respec-tively, and Vmax for uptake and exit of �-3MG are 0.79 and 1.42mmol � l�1 �min�1, respectively. Km(app) for uptake and exit of �-3MG are 4.7and 3.0 mM, respectively, and Vmax for uptake and exit of �-3MG are 2.41 and2.88 mmol � l�1 �min�1, respectively.

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ATP modulation of red cell ghost morphology. Red bloodcells and ghosts containing or lacking ATP were examined byscanning electron microscopy. Figure 6 shows the morpholog-ical changes induced by ATP depletion. Red cell membranesbecome spherical and highly crenated after ghosting in theabsence of ATP. Ghost resealing in the presence of MgATPreverses this effect.

DISCUSSION

Human erythrocyte sugar transport displays a kinetic com-plexity that is not explained by available models for carrier-mediated facilitated diffusion (4, 21, 32, 49, 57, 79). Rat,rabbit, and pigeon erythrocyte sugar transport are, by contrast,compatible with carrier models (38, 62, 70, 74). Several factorssuggest that human red blood cell sugar transport complexity isdirectly attributable to the very high GLUT1 content of humanerythrocytes, which, because the resulting sugar transport isextremely rapid, contributes to inaccuracies in sugar flux de-terminations (4, 14, 63). Studies with resealed erythrocyteghosts further suggest that cytosolic factors contribute to trans-port complexity (11, 12, 16, 37).

Naftalin and Holman (63) proposed that an unstirred layer atthe cytosolic surface of the membrane would increase Km(app)

for rapid sugar exit because, during the course of an experi-ment, the concentration of sugar beneath the membrane fallsmore precipitously than total intracellular sugar. Evidence foran unstirred layer comes from three sources. Net 3MG uptakein human red blood cells displays biphasic kinetics, suggestingrapid equilibration with an unstirred layer and slower equili-bration with bulk cytosol (23). Steady-state sugar exit inhuman red blood cells deviates from Michaelis-Menten kinet-ics at low intracellular sugar concentration in a way that ischaracteristic of an intracellular unstirred layer (10, 28). Coun-terflow experiments in rat red blood cells suggest a nonuniformdistribution of unlabeled and labeled sugars within the cell(38, 62).

The unstirred layer hypothesis was modified after the dis-covery of an ATP-dependent, high-affinity GLUT1 sugar bind-ing site (36), which, it was hypothesized, might also explainbiphasic sugar uptake. Sugar translocation and subsequentbinding at an endofacial GLUT1 domain account for the fastphase of transport. The slower phase of transport was ascribedto slow release into cytosol, a process limited by a diffusionalbarrier or cage formed by endofacial GLUT1 domains (COOHterminus and cytoplasmic loop-6). Although delayed within the

cage, the probability of sugar reassociation with the exit siteand of translocation back to interstitium is high. The net effectis reduced net sugar import. This model predicts that the size ofthe fast transport component in any cell is proportional toGLUT1 concentration. Transport complexity, however, shouldbe independent of cellular GLUT1 concentration because dif-fusionally restricted sugar release results from properties in-trinsic to each GLUT1 molecule. The nominal “absence” oftransport complexity in rat, rabbit, and avian red blood cellsrefutes this hypothesis.

To eliminate complexities inherent to zero-trans experi-ments, we measured transport under equilibrium exchangeconditions where no net change in sugar concentration occurs.Several studies have examined GLUT1-mediated sugar equi-librium exchange in detail. Weiser et al. (77) measured ex-change uptake and exit in both cold-stored and fresh human redblood cells. They concluded that exchange kinetics weremonophasic but used early time points (30 s) in their analysisand thus missed the second slower phase. Previous studies bythis laboratory (23), which used times of �5 min, demon-strated biphasic sugar exchange transport in intact red bloodcells but only at the lowest 3MG concentration tested (1mM). This led to the conclusion that biphasic transport was dueto sugar binding in the cell. However, we now know thatdetection and analysis of the slower phase of transport at 4°Crequire measurements beyond 5 min; this prompted us toextend our analysis of equilibrium exchange transport to latertime points.

When red cell ghosts are nominally ATP free, radiotracer3MG equilibrium exchange follows simple monophasic kinet-ics. Exchange in ATP-loaded red cell ghosts shows biphasicexchange kinetics at all 3MG concentrations tested. Neitherfast nor slow phases of exchange transport can be attributed tosugar complexation by GLUT1 because GLUT1 is at 20 �Mwhereas 3MG ranges from 0.1 to 20 mM. ATP-dependentdeviation from monophasic exchange kinetics is not due toheterogeneity in ghost size, cellular GLUT1 content, orGLUT1 activity. Monophasic Urd exchange kinetics in thepresence of intracellular ATP also argue against ATP-depen-dent ghost heterogeneity.

Urd exchange kinetics imply that biphasic 3MG exchangekinetics are either GLUT1 or sugar specific. If a sugar-specificcompartment exists in ATP-loaded cells, then the 3MG spaceof ATP-loaded cells should be greater than the Urd space. ATPremoval should be without effect on the 3MG space but will

Table 1. Effect of substrate concentration and ATP on rate constants and components of 3MG equilibrium exchange

Without ATP With ATP

kobs Fast kobs Slow kobs Fast Component Size

Uptake Exit Uptake Exit Uptake Exit Uptake Exit

3MG100 �M 0.168�0.010 0.132�0.010 0.357�0.043 0.671�0.070 0.012�0.004 0.040�0.005 0.722�0.039 0.575�0.0322.5 mM 0.090�0.004 0.100�0.004 0.372�0.019 0.392�0.007 0.015�0.002 0.018�0.005 0.651�0.018 0.819�0.00510 mM 0.104�0.004 0.109�0.004 0.320�0.013 0.287�0.007 0.012�0.001 0.026�0.001 0.681�0.014 0.768�0.00520 mM 0.098�0.008 0.122�0.006 0.386�0.050 0.167�0.012 0.025�0.003 0.009�0.001 0.468�0.038 0.708�0.026

Means�SE 0.115�0.018 0.116�0.007 0.359�0.014 0.379�0.108 0.016�0.003 0.023�0.007 0.631�0.056 0.718�0.053

All rate constants are first order and have units of min�1 and are shown as means � SE. kobs, Observed rate constant. Fast component size represents the size[fraction of total 3-O-methylglucose (3MG) accessible space] of the exchange transport component described by the fast rate constant and is shown asmeans � SE. The size of the component described by the slow rate constant is 1 minus fast fractional size.

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increase the Urd space. This was not observed. If a specificsugar-binding compartment explains the ATP-dependent slowphase of exchange transport, then the 3MG space of ATP-loaded cells should be greater than the Urd space, whereas

ATP depletion will reduce the 3MG space of the cell to that ofthe Urd space, which remains unchanged. The observation thatATP has no affect on 3MG or Urd space refutes both hypotheses.

Fig. 2. Effect of intracellular ATP on ghost size and GLUT1 content. Immu-nofluorescence labeling and microscopy of fixed and permeabilized red bloodcells stained with C-Ab (A); living, nonfixed ghosts resealed without ATP andstained with �-Ab (B); and living nonfixed ghosts resealed with 4 mM ATPstained with �-Ab (C). D: fluorescence-activated cell sorting (FACS) analysis(large-angle light scattering) was used to assay ghost size in cells lacking (lightgray) or containing (dark gray) intracellular ATP. E: GLUT1 content wasmeasured by FACS analysis of �-Ab staining. For D and E, ordinate showsnumber of cells and abscissa shows intensity of signal (note log scale).

Fig. 3. Time course of uridine (Urd) equilibrium exchange uptake and exit withand without intracellular ATP. Ordinate shows fractional equilibration, and ab-scissa shows time in minutes (note log scale). A: exchange of 2.5 mM Urd in redcell ghosts resealed without intracellular ATP. B: exchange of 2.5 mM 3MG in redcell ghosts resealed with 4 mM intracellular ATP. Uptake (F) follows a single-exponential rise, and exit (E) follows a single-exponential decay. Curves drawnthrough the points are computed by nonlinear regression and take the followingforms: (1 � e�kt) for uptake and e�kt for exit. Measured means � SE are asfollows: for A (0 ATP), uptake k � 0.079 � 0.004 and exit k � 0.071 � .004; forB (4 mM ATP), uptake k � 0.076 � 0.005 and exit k � 0.065 � 0.005.

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The possibility of a time-dependent change in the intrinsickinetics of transport over the 5 h required for measurements isrefuted by two observations: 1) instantaneous 3MG transportrates do not change with time and 2) biphasic exchange ofradiolabeled 3MG is recapitulated after 15 h of exchange ofunlabeled sugar. In summary, these results suggest two serialbarriers to 3MG transport. The first barrier is translocationthrough GLUT1, whereas the nature of the second barrier isunknown. Two possibilities exist: the barrier is a physicalbarrier (e.g., permeability barrier or unstirred layer) or thebarrier is a chemical barrier (e.g., sugar is reversibly convertedto a second transported species).

The physical barrier hypothesis. A physical barrier modelfor ATP-dependent, GLUT1-mediated biphasic 3MG equilib-rium exchange is illustrated in Fig. 7A. In normal red bloodcells, GLUT1 is anisotropically distributed in the plasma mem-brane. The cytosol, however, contains two diffusionally iso-lated compartments (e.g., see Ref. 28). Cytosol adjacent toGLUT1-enriched membrane rafts (compartment 1) is accessi-ble to newly imported sugars. Cytosol adjacent to GLUT1-depleted membrane (compartment 2) is inaccessible to newlyimported sugars. Thus sugar transport rapidly fills compart-ment 1, whereas equilibration of compartment 2 requires in-tracellular diffusion across the barrier separating the compart-ments. The Urd transporter ENT1, by contrast, is uniformlydistributed in the membrane such that both 3MG compartmentsare accessible to imported Urd. With Mg-ATP depletion-induced echinocytosis (48), GLUT1 becomes uniformly dis-tributed in the plasma membrane and/or the diffusion barrierbetween cytoplasmic compartments is lost, making the entirecellular space directly accessible to transported 3MG.

This model predicts 1) agents that promote spheroechinocyteformation will induce monophasic 3MG exchange kinetics, 2)GLUT1 membrane localization is different from ENT1 local-ization, 3) diffusion-mediated 3MG transport in discocytesshould be monophasic, and 4) slowing transport by use ofinhibitors will inhibit the fast component of transport but notthe slow phase.

The chemical barrier hypothesis. A chemical barrier de-scribes sugar conversion to a second molecular species withinthe cell (Fig. 7B). A sugar could, for example, be metabolized,undergo anomerization, or become complexed to a bindingpartner. If metabolized, the transformed species must remain asubstrate for bidirectional transport (by either GLUT1 or othertransporters); otherwise, the cytoplasmic distribution spaceavailable to sugar plus its metabolite would exceed cell watervolume. 3MG is, however, a nonmetabolizable sugar that is nottransformed after transport either into or out of the cell (51,52). We therefore reject the metabolism hypothesis. The bind-ing partner hypothesis is refuted by the observation of equalUrd and 3MG equilibration spaces.

Differential transport of sugar anomers could explain bipha-sic sugar transport. D-Glucose and 3MG anomerize slowly inaqueous solution between �- and �-anomers (half-time � 10min at 37°C and 250 min at 4°C; equilibrium ratio of �- to�-anomer of �33:66) (45). The anomeric hydroxyl group(located at C-1) is oriented below the pyranose ring in �-D-glucose and 3MG but is equatorial for the �-anomers. Couldthe orientation of this hydroxyl group affect transporter affinityand/or capacity for substrate? Epimers of D-glucose (glucosestereoisomers with different hydroxyl group orientations atspecific ring carbons) show strikingly different affinities for thered cell sugar transporter. Studies with D-galactose (a C-4epimer of D-glucose), D-allose (C-3), or D-mannose (C-2)indicate that opposite hydroxy configurations at C-4, C-3, orC-2 cause a 12-, infinite, or 3-fold, respectively, increase inapparent inhibition constant (relative to D-glucose) for inhibi-tion of sorbose uptake by red blood cells (6). Where it has beenstudied (e.g., D-galactose), there is no accompanying change inVmax for sugar transport (31).

The equilibrium ratio of �-3MG to �-3MG at 4–20°C is65:35, which is very close to the relative sizes of fast and slow3MG compartments observed in ATP-loaded red cell ghosts(see Table 1). If biphasic sugar transport is explained bydifferential transport of �- and �-anomers, several predictionsfollow: 1) �-3MG is the preferred GLUT1 substrate in thepresence of ATP (ratio of fast to slow transport rates of �19:1;see Table 1). In the absence of ATP, 3MG anomers aretransported equally; therefore, 2) equilibrium compartmentsizes in ATP-containing cells are proportional to equilibriumanomer distributions, 3) acceleration of mutarotation to a rateseveralfold faster than the slow rate of transport eliminates theslow phase of transport, and 4) slowing transport by use ofinhibitors will inhibit both the fast and slow phases of transportbecause both phases are carrier mediated.

Testing the barrier hypotheses. The physical and chemicalbarrier hypotheses predict mutually exclusive outcomes for thepartial inhibition test. The physical barrier hypothesis predictsthat only the rapid phase of transport is inhibited by transportinhibitors because the slow phase is a GLUT1-independent

Fig. 4. Effect of time on accessible 3MG (gray bars) and Urd (dark bars)volumes of ghosts lacking or containing 4 mM ATP. Ordinate, accessiblevolume normalized to Urd; abscissa, key indicating the time of measurementand cellular ATP content. Results are means � SE of triplicate measurements.

Table 2. Effect of ATP on substrate-accessibleinternal volume

Substrate

0 ATP ATP

1 2 1 2

3MG, fl 134�8 100�8 129�5 81�6Uridine, fl 133�13 98�9 133�7 77�4

Values are means � SE. 1 and 2 refer to the experiments in the study.

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event (diffusion). The chemical barrier hypothesis predictsinhibition of both phases because each is GLUT1 mediated.Our results show that both fast and slow phases are inhibited bycytochalasin B (Fig. 8) and by maltose (39). We thereforeconclude the physical barrier hypothesis is untenable.

Refining the anomer (chemical barrier) hypothesis. Figure9A illustrates a simulated exchange uptake of �- and �-3MG.Figure 9 models experimental data to a mechanism in which �-and �-3MG compete for GLUT1-mediated uptake and where�-3MG is transported with greater capacity and affinity than�-3MG. Exchange transport is assumed to follow simpleMichaelis-Menten kinetics (14, 38, 47), and uptake of radio-tracer 3MG (Q) is given by

uptake � v�oi � v�

oi � v�io � v�

io (2)

where

v�oi �

V��Qo

K��1 ��

K�

So ��

K�

�Qo � S0�� Qo

,

v�oi �

V��Qo

K��1 ��

K�

So ��

K�

�Qo � So�� Qo

v�io �

V��Qi

K��1 ��

K�

Si ��

K�

�Qi � Si�� Qi

,

v�io �

V��Qi

K��1 ��

K�

Si ��

K�

�Qi � Si�� Qi

where v�oi and v�

oi are unidirectional uptake of radiolabeled �-and �-3MG, v�

io and v�io are unidirectional exit of radiolabeled

�- and �-3MG, V� is Vmax for exchange transport of �-3MG,K� is Km for �-3MG transport, V� is Vmax for exchangetransport of �-3MG, K� is Km for �-3MG transport, Si and So

are the total unlabeled intra- and extracellular 3MG concentra-tions, Qi and Qo are the total radiolabeled intra- and extracel-lular 3MG concentrations, � is the fraction of S or Q that existsas �-3MG (0.35), and � is the fraction of S or Q that exists as�-3MG (0.65).

Total (radiolabeled �- plus �-3MG) exchange uptake data at2.5 mM 3MG and 10 �M radiolabeled 3MG (4°C) were

Fig. 5. Effect of time on initial rates of transport and equilibrium exchangetime course. A: zero-trans uptake of 100 �M 3MG in red cell ghosts resealedwith (F) and without (E) intracellular ATP and permitted to rest in 20 volumesof ice-cold kaline for 5 h. Ordinate, fractional equilibration; abscissa, time inminutes. Curves drawn through the points were computed by nonlinearregression to assume the following biexponential form: A(1 � e�k1t) (1 �A)(1 � e�k2t). In the absence of ATP, A � 0.12 � 0.03, k1 � 2.6 � 1.5 min�1,and k2 � 0.06 � 0.01 min�1. In the presence of ATP, A � 0.43 � 0.03, k1 �2.2 � 0.3 min�1, and k2 � 0.10 � 0.01 min�1. B: time dependence of initialrates of zero-trans uptake plus (open bars) and minus (gray bars) ATP. Ghostswere allowed to rest in kaline for a specific time after which a small volumeof 3MG to make a final concentration of 100 �M was added, and the initial rateof transport was measured over a 30-s interval. y-axis, uptake rate in mmol � lcell water�1 �min�1; x-axis, time of preincubation before measurement ofuptake rate. C: time course of 2.5 mM 3MG equilibrium exchange in thepresence of 4 mM intracellular ATP measured immediately after resealing (E)or after 12 h of unlabeled 3MG equilibrium exchange (F). Curves were drawnby nonlinear regression assuming exchange is described by A(1 � e�k1t) (1 � A)(1 � e�k2t). Results are as follows: for control, A � 0.72 � 0.03, k1

� 0 .372 � 0.054 min�1, k2 � 0.017 � 0.007 min�1, and 2 � 0.011; for 12 hpostcontrol, A � 0.59 � 0.07, k1 � 0 .411 � 0.116 min�1, k2 � 0.032 � 0.013min�1, and 2 � 0.023.

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simulated by fourth-order Runge Kutta numerical integration(Fig. 9A). Vmax and Km(app) parameters for �- and �-3MGtransport were varied by an iterative least-squares procedureuntil the deviation between observed and predicted uptakereached a minimum. The result suggests that �-3MG [Vmax �2.4 mmol � l�1 �min�1 and Km(app) � 4.7 mM] is a more effi-cient exchange substrate than is �-3MG [Vmax � 0.8mmol � l�1 �min�1 and Km(app) � 37.1 mM]. Figure 9 alsoshows the observed kinetics of �-3MG mutarotation at 4°C asmeasured by circular dichroism. 3MG mutarotation is signifi-cantly slower than exchange uptake of �-3MG, a prerequisiteof the anomer (chemical-barrier) model for sugar transport.The complete 3MG exchange data set of Table 1 has beenreanalyzed by this procedure (e.g., see Fig. 1B, dashed curvefits), and the results are summarized in Table 3. V�, K�, V�, andK� are independent of 3MG concentration and direction ofradiotracer 3MG movement (uptake or exit). V� is 2.5-foldgreater than V�, whereas K� is 7.4-fold greater than K�. Thusthe major impact of reorientation of the anomeric hydroxylgroup to the �-configuration is a sevenfold loss in affinity ofsugar binding. In the absence of intracellular ATP, radiolabeled3MG exchange transport is consistent with a single transportprocess and is described by

uptake � voi � vio (3)

where

voi �VQo

K�1 �So

K� � Qo

, vio �VQi

K�1 �Si

K� � Qi

where V and K are Vmax and Km(app), respectively, for 3MGexchange transport. Analysis of exchange transport in theabsence of ATP (dashed curve fits of Fig. 1A, Table 3)indicates Km(app) and Vmax are 35 mM and 5.2mmol � l�1 �min�1, respectively, indicating that ATP depletionresults in the loss of high-affinity �-3MG binding. ATP-GLUT1 interaction is thus a primary determinant of high-affinity �-3MG exchange transport.

Few previous studies have examined the transport of �- and�-D-glucose in red blood cells (17, 26, 58). Unlike liver, whichcontains significant mutarotase activity (10), the human redblood cell and red cell ghosts lack mutarotase (17). Preliminaryin vitro measurements indicate that 3MG is not a substrate forporcine mutarotase and that mutarotase-acceleration of D-glu-cose anomerization is unaffected by 4 mM ATP (Leitch andCarruthers, unpublished observations). Our observations are,therefore, not explained by ATP-dependent inhibition of eryth-rocyte mutarotase. When freshly dissolved as pure anomers, �-and �-D-glucose are relatively equipotent in their inhibition ofthe initial rate of radiolabeled D-glucose uptake from an equi-librium mixture of D-[�-14C]glucose and D-[�-14C]glucose(17). This suggests that differences in rate constants for trans-port of �- and �-anomers reflect differential capacity fortransport rather than differences in binding affinity, a conclu-sion that stands in contrast to our analysis of 3MG exchange

Fig. 6. Effect of intracellular ATP on ghost size and shape. Scanning electronmicroscopy of red blood cells (A), resealed ghosts lacking ATP (B), andresealed ghosts containing 4 mM intracellular ATP (C). Bars � 5 �m.

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transport. We therefore reexamined zero-trans [14C]3MG up-take in red cell ghosts containing 4 mM ATP. Proton NMR offreshly dissolved, unlabeled �-3MG and of an equilibriumsolution of 3MG indicates that “�-3MG” is in fact 80%�-anomer and 20% �-anomer. The equilibrium solution con-tains a 35-to-65 ratio of �- to �-3MG. These sugar solutionswere used as inhibitors of [3H]3MG uptake at 4°C. [3H]3MGis an equilibrium mixture of [�-3H]3MG and [�-3H]3MG.Uptake was measured at 8 nM [3H]3MG with increasingamounts of unlabeled 3MG (0–10 mM). Figure 9B shows that�-3MG and equilibrium 3MG inhibit unidirectional [3H]3MGuptake half-maximally at 0.6–1.2 mM (see x-intercepts ofcurve fits). This is consistent with our earlier study (17)comparing �- and �-D-glucose affinities and suggests onlysmall differences in the affinity of GLUT1 for �- and �-3MG.However, the slope of the inhibition data employing equilib-rium 3MG is significantly greater than the slope of the �-3MGinhibition data. At radiolabeled 3MG concentrations unla-beled 3MG concentrations, the ratio of control radiolabeled3MG uptake to inhibited 3MG uptake (vc/vi) increases linearlywith total concentration of unlabeled 3MG and is characterizedby the slope

�

K�

��

K�

(4)

where � and � are the fractional amounts of �- and �-3MGpresent in the uptake medium and K� and K� are Km(app) foruptake of �- and �-3MG, respectively. Using the computedslopes of Figure 9B and the known ratios of �- to �-anomerspresent in solution, we calculate that K� and K� for zero-transnet �- and �-3MG uptake are 5.29 � 0.47 and 0.77 � 0.13mM, respectively (Table 3). These affinity constants are sig-nificantly lower than those for exchange transport. However,Km(app) for zero-trans D-glucose and galactose zero-trans up-take are 14- and 11-fold lower, respectively, than Km(app) for

Fig. 7. Models for ATP-induced red blood cell ghost morphological change andGLUT1 nonisotropic distribution. A: physical barrier. With sufficiently highintracellular Mg2 and ATP concentrations, red blood cells and ghosts arebiconcave and GLUT1 is restricted to specific areas of the membrane. Cytoplasmicspace that is adjacent to GLUT1-depleted plasma membrane is diffusionallyisolated from cytoplasmic space adjacent to GLUT1-enriched membrane. ENT1 isisotropically distributed in the membrane so that Urd is transported into bothcytoplasmic compartments simultaneously. ATP and/or Mg2 depletion results inphosphatidylserine redistribution and echinocyte formation. GLUT1 surface dis-tribution in spheroechinocytes is isotropic and/or diffusional isolation of cytoplas-mic spaces is lost resulting in monophasic 3MG exchange. B: chemical barrier.Sugars undergo spontaneous but slow anomerization between �- and �-forms inaqueous solution [k is �0.002–0.003 min�1 at 4°C (17); see also Fig. 9A]. Twosubunits of tetrameric GLUT1 are shown in cartoon form in the lipid bilayer.Extracellular �- and �-D-glucose are imported equally in the absence of ATP but�-D-glucose is preferred when GLUT1 is complexed with intracellular ATP. The�- and �-sugars compete for uptake and exit. Although �-sugars equilibrate mostrapidly across the membrane, mutarotation to �-sugar ensures that both �- and�-sugars are available in the cytoplasm. The consequences for transport includeunderestimation of Km(app) and Vmax for net sugar uptake, overestimation of Km(app)

for sugar exit, and underestimation of Km(app) exit into saturating sugar solutions.

Fig. 8. Time course of 2.5 mM 3MG equilibrium exchange uptake and exit inred cell ghosts containing intracellular 4 mM ATP and exposed to 5 �M CCB.Ordinate, fractional equilibration; abscissa, time in minutes (note log scale).Uptake (E) and exit (F) follow biexponential kinetics. Curves drawn throughthe points are computed by nonlinear regression of the following forms: A(1 �e�k1t) (1 � A)(1 � e�k2t) for uptake and A(e�k1t) (1 � A)(e�k2t).Computed values of k1, k2, and A are as follows: uptake k1 � 0.050 � 0.022min�1, k2 � 0.005 � 0.001 min�1, A � 0.234 � 0.064, and 2 � 0.026; exitk1 � 0.069 � 0.016 min�1, k2 � 0.004 � 0.001 min�1, A � 0.307 � 0.034, 2 � 0.0178.

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exchange uptake of the same sugars (18, 29, 30). This moredetailed analysis strongly supports the hypothesis that GLUT1shows greater affinity for �-3MG than �-3MG.

Physiological significance. A preference for �-D-glucose intransport and/or metabolism has been reported for liver (10, 54,59), pancreatic beta cells (53, 55, 60, 71), muscle (80), adipose(70), and yeast cells (25). Cardiac muscle hexokinase displays

high affinity but low capacity for �-D-glucose and low affinitybut high capacity for �-D-glucose (50). Brain, retina erythro-cytes, and lens cells appear to prefer �-D-glucose or show nopreference (17, 26, 52, 59, 65, 67, 68). �-D-Glucose is morepotent than �-D-glucose in inhibiting vagally mediated secre-tion of gastric acid (77). These observations emphasize thetissue-specific physiology of anomer transport and utilizationand further suggest that cells expressing GLUT1 or GLUT3may prefer the �-anomer, whereas cells expressing GLUT2 orGLUT4 may prefer the �-anomer.

Significance to previous transport studies. These findingsmay necessitate reevaluation of red cell glucose transportdeterminations. Transport measurements are normally obtainedby initial rate analysis over intervals of 1 min or less. If initialrates of transport are derived largely from uptake of �-sugar,they are related to (and must be corrected for) the amount of�-sugar present in solution during the measurement. ThusKm(app) and Vmax parameters may be overestimated by 1.5-fold.Furthermore, computed rates and Km(app) must be corrected forcompetitive inhibition by the omnipresent, lower affinity�-sugar. This result could also explain (see Refs. 2 and 78 forlucid expositions) how integrated rate equation analysis of timecourse data results in overestimation of Km(app) for transportbecause all time courses are artificially extended owing toslower transport of �-sugar. This laboratory has previouslyinterpreted the slow phase of sugar import as diffusionallylimited release of translocated sugar into cytoplasm (8, 9). Thisinterpretation is no longer tenable.

Conclusion. ATP-containing human red blood cell ghostsexhibit biphasic equilibrium exchange 3MG transport at sugarconcentrations ranging from 1 to 20 mM. This observation isinconsistent with previous models for ATP modulation ofGLUT1 activity. We propose that ATP-GLUT1 interactions

Fig. 9. A: simulated time course of �- and �-3MG equilibrium exchangeuptake in red cell ghosts containing intracellular ATP. Ordinate, fractionalequilibration; abscissa, time in minutes (note log scale). E, Measured exchangeuptake of radiolabeled 3MG in cells equilibrated with 2.5 mM unlabeled 3MG(see Fig. 1B). Three curves were simulated using Eq. 2 and have the followingconstants: for �-3MG, Km(app) � 37.1 mM and Vmax � 0.79 mmol � l�1 �min�1;for �-3MG, Km(app) � 4.7 mM and Vmax � 2.41 mmol � l�1 �min�1. ��represents the sum of �-3MG and �-3MG uptake. The time course of �-3MGmutarotation at 4°C (�ª �; F) was measured by CD at 193 nm using a freshlyprepared 20 mM �-3MG solution. Ordinate, CD at 193 nm in mdeg; abscissa,time in minutes. The curve drawn through the points is a single exponentialcomputed by nonlinear regression and is characterized by a first-order rateconstant of 0.0029 � 0.0002 min�1. Dashed lines indicate the half-times (min)for �-3MG uptake (1.65 min), �-3MG uptake (41 min), and 3MG mutarotation(239 min). B: analysis of inhibition of radiolabeled 3MG uptake in ATP-containing ghosts (8 nM [�-3H]MG plus [�-3H]3MG) by freshly prepared,unlabeled �-3MG (80:20 �- to �-3MG; E) or by an equilibrium unlabeled3MG solution (35:65 �- to �-3MG; F). Ordinate: ratio of 3MG uptake in theabsence of unlabeled 3MG to uptake in the presence of unlabeled 3MG.Abscissa, 3MG concentration in mM. Straight lines drawn through the pointswere computed by nonlinear regression and are characterized by the slope ��/K� �/K�. Results are as follows: for �-3MG (dashed line), K� � 4.59 mM,K� � 0.58 mM; for equilibrium 3MG (continuous line), K� � 6 mM, K� �0.97 mM.

Table 3. Kinetics of 3MG transport

Parameter

n 0 mm ATPi 4 mM ATPiExchange Zero-trans uptake

K� 8 27.5�2.44K� 8 3.74�0.21V� 8 1.28�0.11V� 8 3.20�0.25k� 8 0.05�0.00k� 8 0.86�0.06K 8 34.9�1.62V 8 5.21�0.31k 8 0.15�0.01

K� 4 5.29�0.49K� 4 0.77�0.13

Values are means � SE of n (no. of experiments in triplicate). Equilibriumexchange and zero-trans 3MG transport were measured in red cell ghostscontaining or lacking 4 mM intracellular ATP (ATPi). The time course ofexchange uptake and exit at 0.1, 2.5 (see Fig. 1), 10, and 20 �M 3MG wasanalyzed according to Eq. 2 (exchange transport plus ATP) or Eq. 3 (exchangetransport, 0 ATP) by an iterative least-squares procedure to obtain K� and K�

(apparent Km for equilibrium exchange transport of �- and �-3MG), V� and V�

(Vmax for equilibrium exchange transport of �- and �-3MG), k� and k�

(Vmax/apparent Km for equilibrium exchange transport of �- and �-3MG), andK and V (apparent Km and Vmax for equilibrium exchange 3MG transport in theabsence of ATPi). 3MG inhibition of the initial rate of radiolabeled 3MGuptake by ATP-containing ghosts was measured by equilibrium (35:65) or80:20 �- to �-3MG solutions (see Fig. 9B) to obtain K� and K� [apparentinhibition constant (Ki) for �- and �-3MG inhibition of zero-trans 3MGuptake].

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promote preferential transport of �-3MG and that extendedtime courses of sugar transport in human red blood cells revealdeviations from simple kinetics owing to low-affinity, low-capacity �-3MG transport and continuous �-3MG and �-3MGmutarotation. In the absence of ATP, both anomers may betransported equally or differences in the kinetics of �-3MG and�-3MG become too small to measure with existing methodol-ogies. Differential transport of 3MG anomers (and thus trans-port complexity) is only observable in cells where rate con-stants for 3MG transport are greater than those for 3MGmutarotation.

GRANTS

This work was supported by National Institute of Diabetes and Digestiveand Kidney Diseases Grants DK-36081 and DK-44888.

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