pH dependence and compartmentalization of zinc transported

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pH dependence and compartmentalization of zinc transportedacross plasma membrane of rat cortical neurons

ROBERT A. COLVINProgram in Neuroscience, Department of Biological Sciences, Ohio University, Athens, Ohio 45701

Received 19 March 2001; accepted in final form 17 October 2001

Colvin, Robert A. pH dependence and compartmental-ization of zinc transported across plasma membrane of ratcortical neurons. Am J Physiol Cell Physiol 282: C317C329,2002. First published October 17, 2001; 10.1152/ajpcell.00143.2001.In this study, Zn2 transport in rat corticalneurons was characterized by successfully combining radio-active tracer experiments with spectrofluorometry and fluo-rescence microscopy. Cortical neurons showed a time-depen-dent and saturable transport of 65Zn2 with an apparentaffinity of 1520 M. 65Zn2 transport was pH dependentand was decreased by extracellular acidification and in-

creased by intracellular acidification. Compartmentalizationof newly transported Zn2 was assessed with the Zn2-selective fluorescent dye zinquin. Resting cortical neuronsshowed uniform punctate labeling that was found in cellprocesses and the soma, suggesting extrasynaptic compart-mentalization of Zn2. Depletion of intracellular Zn2 withthe membrane-permeant chelator N,N,N,N-tetrakis(2-pyri-dylmethyl)-ethylenediamine (TPEN) resulted in the com-plete loss of punctate zinquin labeling. After Zn2 depletion,punctate zinquin labeling was rapidly restored when cellswere placed in 30 M Zn2, pH 7.4. However, rapid restora-tion of punctate zinquin labeling was not observed when cellswere placed in 30 M Zn2, pH 6.0. These data were con-firmed in parallel 65Zn2 transport experiments.

pH; zinquin; carboxyseminaphthorhodofluor-1 fluorescence;ion transport; transition elements; primary culture

IT IS KNOWN THAT Zn2 can enter neurons by two distinctpathways. The first pathway is transporter mediatedand slow, occurs under resting conditions, and is ob-served at concentrations of Zn2 well below 100 M;relatively small amounts of Zn2 enter the cell by thispathway (5, 8, 9). The second pathway is channelmediated and rapid, requires depolarization or recep-tor activation, and is seen at concentrations of Zn2

100 M; greater amounts of Zn2 enter the cell bythis pathway (29, 30, 32). The transporter-mediated

pathway can be easily distinguished from channelpathways by its insensitivity to various channel block-ers (5, 9) (e.g., MK801, GYKI 53466, nifedipine, FTX-3.3, -conotoxin GVIA, and CNQX). Previously, thetransporter-mediated pathway was studied in synap-tosomes (34) and hippocampal slices (19), but themechanisms of Zn2 transport were never elucidated.More recently, this laboratory has studied (6, 8, 9) the

mechanisms of 65Zn2 transport in rat brain plasmamembrane vesicles. 65Zn2 transport showed satura-tion with increasing concentrations of 65Zn2, and65Zn2 influx was inhibited when extravesicular pHwas lowered. The transport mechanism appeared re-versible in that both pH-dependent Zn2 influx andefflux were observed. Together, these studies led to aworking hypothesis: plasma membrane transport ofZn2 is pH dependent because it depends on a Zn2/nH antiport mechanism.

The recent cloning of cDNA coding for several pro-teins associated with plasma membrane Zn2 trans-port function, i.e., DMT1 (17), hZIP1 (15), hZIP2 (14),and ZnT-1 (28), has provided new molecular and ge-netic tools with which to address the mechanism ofplasma membrane Zn2 transport. All the cloned Zn2

transporters are apparently independent of cellularenergy stores, show no dependence on Na, K, or Cl

concentrations, and show an apparent affinity for Zn2

in the micromolar range (14, 15, 17, 28). Expressionstudies have revealed interesting pH effects on Zn2

transport mediated by either DMT1 (17, 35) or hZIP1and hZIP2 (14, 15). DMT1 shows nonspecific divalent

metal transport (most notably iron transport in theintestine) coupled with protons (i.e., low extracellularpH stimulates Fe2 uptake when the protein is ex-pressed in Xenopus oocytes; Ref. 17). When DMT1 wastransiently expressed in COS-7 cells, Fe2 uptake wasgreatly increased and showed a dependence on extra-cellular pH (35). In contrast to expression studies inXenopus oocytes, Fe2 uptake was inhibited in trans-fected COS-7 cells by extracellular acidification, andtransport had an optimal pH of 6.8. Thus it is still notclear what effect changes in extracellular or intracel-lular pH should have on DMT1 function. hZIP is thehuman homolog of the ZIP family of Zn2 transporterscloned from yeast and plants (16). Three human ZIP

gene products have been identified (hZIP13); bothhZIP1 and hZIP2 have been functionally characterized(14, 15). Zn2 transport observed in K562 erythroleu-kemia cells expressing either hZIP1 or hZIP2 wasinhibited by lowering extracellular pH (14). The inhi-bition at low pH was reversed in cells expressing hZIP2by addition of HCO

3

, leading the authors of that study

Address for reprint requests and other correspondence: R. A.Colvin, Dept. of Biological Sciences, Ohio Univ., Athens, OH 45701(E-mail: [email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Cell Physiol 282: C317C329, 2002.First published October 17, 2001; 10.1152/ajpcell.00143.2001.

0363-6143/02 $5.00 Copyright 2002 the American Physiological Societyhttp://www.ajpcell.org C317


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to suggest a Zn2-HCO3 cotransport mechanism.

ZnT-1 is thought to function primarily as a Zn2 effluxprotein (21), and its transport mechanism and pHdependence have yet to be clearly established. It is notknown which of the above proteins might be responsi-ble for Zn2 transport in cortical neurons.

Although Zn2 is an abundant element in the brain,its cellular homeostasis and compartmentalization are

poorly understood. Once inside the neuron, Zn2 isthought to exist in at least four distinct cellular pools(13). The first pool is composed of metalloproteins (e.g.,metalloenzymes and transcription factors) that usetightly bound Zn2 as a required cofactor to carry outtheir cellular functions. The second pool constitutesZn2 bound to cytoplasmic metallothionein-III (MT-III), which is thought to be a reservoir and buffer ofcytosolic Zn2 (12, 22, 27). The third pool, cytosolic freeZn2, is maintained at very low levels probably wellbelow 100 nM (1, 5, 23, 29), presumably by the actionsof cytosolic MT-III. The fourth pool is compartmental-ized Zn2. Experimental evidence shows that manyeukaryotic cell types contain compartmentalized Zn2

and that cytoplasmic organelles can sequester Zn2

(10, 25). A well-characterized compartment containingZn2 in neurons is synaptic vesicles of glutamatergicneurons of the cerebral cortex and in particular thehippocampal formation (13).

The present studies were designed to address someof the gaps in our knowledge of Zn2 transport andhomeostasis in cortical neurons. The results provideconvincing evidence of a pH-dependent transportmechanism for Zn2 in the plasma membrane of corti-cal neurons in primary culture. Zn2 influx was fa-vored when 1) the flow of ions was down a concentra-tion gradient and 2) the cell interior was acidic with

respect to the extracellular medium. In addition, evi-dence was obtained of the extrasynaptic compartmen-talization of newly transported Zn2.

MATERIALS AND METHODS

Primary culture of cortical neurons. Primary culture ofembryonic (embryonic day 18) cortical neurons was per-formed as described previously (7, 24). Brains were removedfrom the skulls and kept moist in Hanks balanced saltsolution (HBSS; without Mg2 and Ca2) for further dissec-tion. With a dissecting microscope, the cerebral cortex wascarefully separated by blunt dissection from the brain stemdiencephalon, olfactory bulbs, and cerebellum, which wasdiscarded. Next the meninges and choroid plexus werestripped away. The cerebral hemispheres were cut into small

pieces (about 4 pieces for each hemisphere) and trypsinizedin HBSS at room temperature. After trypsinization, nervecells were dissociated by gentle trituration through the nar-row opening of a fire-polished Pasteur pipette. The dissoci-ated neurons suspended in HBSS were plated on 24-wellculture plates (Falcon) or culture plates containing sterilizedcoverslips coated with polyethylenimine (50% solution;Sigma, St. Louis, MO), which was diluted 1:1,000 in boratebuffer. The cortical neurons were allowed to attach to theplates or coverslips at 37C and 5% CO2 in 1 ml of MEMsolution (GIBCO BRL) supplemented with 10 mM sodiumbicarbonate, 2 mM L-glutamine, 1 mM pyruvate, 20 mM KCl,10% glucose, and 10% (vol/vol) heat-inactivated fetal bovine

serum. The desired cell density was obtained by adjustingthe volume of cell suspension added to each plate (finalplating density was 5 105 cells/ml). The medium wasreplaced with fresh supplemented MEM after 36 h and 24 hlater switched to 1 ml of Neurobasal medium (GIBCO BRL)supplemented with 0.5 mM glutamine and 2% B27 (GIBCOBRL). Use of serum-free culture medium, which does notfavor glial proliferation, allows the culture of cortical neuronsin the near absence of glial cells.

Measurement of 65Zn2 transport. Cortical neurons (47days in vitro) attached to 24-well plates were assayed for65Zn2 transport as follows (all buffers were at 37C). Eachwell of a 24-well plate was first washed with Lockes buffer(in mM: 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1.0 MgCl2, 5 HEPES,and 10 glucose, pH 7.4). Various pretreatments (e.g., NH3/NH4 prepulse) were performed at this point to prepare thecells for subsequent 65Zn2 transport assay. The transportexperiment was initiated by switching to Lockes buffer (pH7.4 or 6.0 or various other conditions) containing 65Zn2

(NEN, Boston, MA). Depending on the experiment, 65Zn2

was mixed with nonradioactive Zn2 to obtain a final concen-tration in the range of 0.0010.005 Ci/l. The specific ac-tivity of 65Zn2 (expressed as cpm/nmol Zn2) was deter-

mined for each experiment by assaying an aliquot of eachsolution containing 65Zn2 for radioactivity. The cells wereincubated for various times at 37C. Normally, 5% of thetotal Zn2 in the reaction buffer was taken up by the cells. Toterminate a transport reaction, the buffer containing 65Zn2

was rapidly removed by aspiration and replaced with ice-coldLockes buffer (pH 7.4) without Zn2 added. Next, the wellswere washed three times with 0.5 ml of ice-cold wash buffercontaining Lockes buffer (without Ca2 and Mg2 added)and 1 mM EGTA (pH 7.4). The cells were lysed by freezethawing (70C). The lysed cells in each well were resus-pended in 250 l of buffer, of which 200 l were used to assayradioactivity and 40 l were used to determine protein.Protein concentration was determined by a Bio-Rad methodwith bovine serum albumin as a standard, and 65Zn2 was

determined in a gamma counter. With the specific activityand protein concentration, counts per minute were convertedto nanomoles per milligram for each well. In experimentsusing65Zn2, parallel experiments were done in pH 6 Locke sbuffer containing 1 mM Cd2. The results from these exper-iments (nmol/mg) were subtracted from total 65Zn2 up-take to obtain 65Zn2 transport and nonspecific 65Zn2 bind-ing. Cortical cells are firmly attached to the culture platesand can easily withstand several buffer washes. This was

verified by viewing the cells in each well before and after thewashing procedure was complete.

MTT assay. Many manipulations described in these ex-periments have the potential of causing cell death, and there-fore assessments of acute cell viability were performed.3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-

mide (MTT

) conversion was determined colorometrically asdescribed elsewhere (24). Parallel cultures to be used forMTT measurements received the same experimental ma-nipulations but were not exposed to 65Zn2 used for trans-port. Instead, these cultures were allowed to remain in freshLockes buffer for an additional 24 h to allow time for acuteeffects on cell survival to occur. After 24 h the cells wereassayed for MTT cleavage and conversion. Activities ob-tained after experimental manipulations were comparedwith cells that were switched only to fresh Locke s buffer for24 h. Negligible cell death was observed in high-densitycortical cultures exposed only to fresh Lockes buffer for 24 h,as judged by MTT assay.

C318 PH-DEPENDENT ZN2 TRANSPORT IN RAT CORTICAL NEURONS

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Microscopy and zinquin labeling. Cells were cultured asdescribed in Primary culture of cortical neurons in cultureplates containing sterilized glass coverslips. For visualiza-tion of intracellular Zn2 with zinquin, the medium wasdiscarded and replaced with fresh Lockes buffer before anexperiment was started. After various experimental settingsat 37C, the medium was discarded and the glass coverslipswere washed twice with 1 ml of Lockes buffer. One milliliterof Lockes buffer was left in the culture well to prevent thecells from drying out. A 5 mM concentrate of zinquin ester(freshly dissolved in DMSO) was diluted directly into thewells to produce a final concentration of 25 M. Althoughzinquin is highly permeable to lipid bilayers, the ester formwas used so that only intracellular zinquin would contributeto the observed signal. Zinquin experiments were also per-formed at 12.5 M with similar results. The culture plateswere reincubated at 37C for 30 min to allow zinquin to bindspecifically to intracellular Zn2. The cells were washedthree times with fresh Lockes buffer (pH 7.4) and thenimmediately mounted on glass slides with Aquatex (for im-mediate viewing) to prevent leakage of zinquin. Coverslipsinverted on microscope slides were examined using a NikonEclipse 600 with epifluorescence and differential interferencecontrast (DIC) optics, equipped with a SPOT RT digital

camera for image capture. Zinquin fluorescence was observedusing an ultraviolet filter block (EF-4 UV 2E/C DAPI filterblock, EX 330380). Zinquin labeling experiments were re-peated in at least three different cultures. The digital imagespresented are qualitatively similar and representative ofwhat was observed in all three experiments. Zinquin esterwas obtained from Luminus (Adelaide, SA, Australia).

Carboxyseminaphthorhodofluor-1 fluorescence. Corticalneurons attached to glass coverslips were loaded with car-boxyseminaphthorhodofluor (SNARF)-1 by incubation for 30min in Lockes buffer (pH 7.4) at 37C containing 5 M 5-(and 6)-carboxy SNARF-1 acetoxymethyl ester, acetate (Mo-lecular Probes, Eugene, OR). The cells were then washedwith 1 ml of Lockes buffer before pH measurement. Cellsattached to coverslips were held in a cuvette at an 45 angle

to the incident light beam by a coverslip holder (HitachiInstruments, San Jose, CA). To switch buffer solutions, thecoverslip and holder were lifted out of the cuvette and quicklyplaced into a waiting cuvette containing the next desiredbuffer. SNARF-1 fluorescence was measured in a fluores-cence spectrophotometer (Hitachi F-2000) with excitation at514 nm and emission wavelengths of 585 and 630 nm. Thefluorescence ratio F585/F630 was calibrated in separate exper-iments with cells treated with 10 M nigericin in Lockesbuffers of various pH containing 120 mM KCl (2). With thecalibration data, intracellular pH was calculated directlyfrom F585/F630. F585/F630 was a linear function of pH over pH

values between 6.4 and 8.0. In each experiment, to correct forscattered light/autofluorescence, data obtained from cellstreated as above but without incubation with SNARF-1 were

subtracted from the data obtained with SNARF-1.Buffer preparation. All buffers were pH adjusted on the

day of the experiment. HEPES buffers without HCO3 added

were first adjusted to the desired pH with the addition ofNaOH or HCl and then bubbled with 100% O2 for 5 min toremove dissolved CO2. The buffers were then checked againand usually required a small additional adjustment withNaOH or HCl to bring them to the desired pH. These bufferswere considered to be nominally free of HCO3

during anexperiment. HEPES buffers containing 5 mM HCO3

wereprepared as above with the addition of 5 mM NaHCO3 andthen were bubbled with 95% O2-5% CO2 for 5 min. Subse-quently, these buffers were checked and usually required a

small additional adjustment with NaOH or HCl as above.Control experiments had 5 mM NaCl added instead ofNaHCO3 and were bubbled with O2 as above. Experimentswere performed in room air, but all solutions were covered tominimize the amount of CO2 that could dissolve in or escapefrom the buffer during an experiment.

Statistical analysis. Each preparation of cortical neurons(1 pregnant rat) normally yields three to four 24-well platesat the density used for these experiments. The data pre-sented (except data in Fig. 6, which include up to 4 differentpreparations) are the means of multiple determinations withwells from the same 24-well plate. All the data presentedwere repeated in at least two and normally at least threedifferent preparations of cortical neurons with similar re-sults, although variation did exist when different prepara-tions were compared. Potential sources of variation includedifferences in cell density and animal variation. Statisticalanalysis and nonlinear curve fitting were accomplished withGraphPad Prism software (San Diego, CA). Data were ana-lyzed by t-test, one-way ANOVA with Tukeys post test, or,where appropriate, two-way ANOVA.

RESULTS

Previous studies from this laboratory (6, 8) charac-terized a robust, saturable, pH-dependent 65Zn2

transport in plasma membrane vesicles isolated fromadult rat brain. The present study used cortical neu-rons in primary culture derived from fetal rat cortex tocharacterize 65Zn2 transport. Because it is wellknown that high concentrations of heavy metals aretoxic to neurons, determining the toxicity of the vari-ous conditions to be used in this study was necessary.To accomplish this, cells were exposed to various ex-perimental conditions for a brief exposure (5 min) or along exposure (60 min), and then cell death was deter-mined 24 h later with the MTT conversion assay. It

was found that exposure of cells to concentrations ofZn2 as high as 300 M in Lockes buffer for as long as60 min did not result in significant cell death whenassayed 24 h later, similar to results recently reportedby Sheline et al. (31). Likewise, changing buffer pH toeither 6 or 8 from 7.4 did not result in evidence ofincreased cell death. La3 (1 mM) was used in plasmamembrane vesicle experiments to inhibit 65Zn2 trans-port; however, La3 was quite toxic to neurons evenafter a brief exposure. Cd2 (1 mM) was much less toxicto the neurons than La3. No cell death was observedafter a brief exposure, although after the 60-min expo-sure, some cell death was evident 24 h later. Variousother transition elements (Co2, Mn2, Cu2, and

Fe2

) showed intermediate toxicity less than La3

butmore than Cd2. Nickel was found to be similar to Cd2

in toxicity.In this study, 65Zn2 uptake was measured under

conditions in which transport-mediated pathways pre-dominate over channel-mediated pathways for 65Zn2

influx (i.e., Zn2was 100 M, extracellular Ca2 andMg2 were at physiological concentrations, and nonde-polarizing conditions were used). Under these condi-tions, 65Zn2 transport is insensitive to calcium andglutamate channel blockers (9). The effect of varioustransition elements on 65Zn2 uptake is shown in Fig.

C319PH-DEPENDENT ZN2 TRANSPORT IN RAT CORTICAL NEURONS



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1. Neurons in culture were exposed to various transi-tion elements (Cd2, Co2, Mn2, Cu2, Fe2, Ni2) inLockes buffer for 5 min with 30 M 65Zn2. The tran-sition elements were included at a concentration of 300M (conditions that for the most part do not produceacute toxicity). All the transition elements producedsignificant inhibition of 65Zn2 accumulation. How-ever, inhibition was incomplete. Increasing the concen-

tration of the transition elements to 1 mM produced agreater level of inhibition, albeit still incomplete. Thegreatest inhibition was seen with Cd2. The reductionof extracellular pH to 6.0 reduced the total 65Zn2

uptake by50%. Inhibition by transition elements wasstill evident (except Mn2), even at pH 6.0. In thisexperiment, the inhibitory effects of transition ele-ments and extracellular acidification appeared to beadditive.

The 65Zn2 uptake measured under the above condi-tions probably included 65Zn2 binding to the surfaceof neurons and culture plates, which was resistant toremoval by EGTA. To characterize 65Zn2 binding bet-ter, the time course of 65Zn2 uptake was determined(see Fig. 2, which illustrates the effects of pH and Cd2

inhibition). Incubation of neurons with 30 M 65Zn2

pH 7.4 resulted in a slow accumulation of 65Zn2,which began to approach a plateau after 3060 min.Changing extracellular pH to 8 enhanced 65Zn2 up-take (most noticeably after 30 min of exposure),whereas reduction of extracellular pH to 6 inhibited65Zn2 uptake (Fig. 2A). The addition of 1 mM Cd2 atpH 7.4 reduced 65Zn2 uptake to a level similar to thatseen at pH 6. The addition of 1 mM Cd2 at pH 6further reduced 65Zn2uptake to nearly that seen afterisotope dilution by addition of 1 mM nonradioactiveZn2 (Fig. 2B). Again, the effects of Cd2 and extracel-

lular acidification appeared to be additive. In the pres-ence of 1 mM Cd2 and pH 6.0, the 65Zn2 signalclosely approximates nonspecific binding, as defined by

isotope dilution, and suggests near-complete inhibitionof transport without acute toxicity, particularly whenexposures are kept brief.

Figure 3 shows 65Zn2 uptake as a function of in-creasing 65Zn2 concentration. 65Zn2 uptake showedsaturation with respect to increasing65Zn2 concentra-tion, and the Michaelis-Menten constant (Km) and themaximum rate of65Zn2 transport (Vmax) were estimated

by computer-assisted nonlinear curve fitting to a rectan-gular hyperbola (Fig. 3). The Km obtained (1520 M)was similar to that obtained with plasma membranevesicles when La3 was used as an inhibitor (6). In thepresence of 1 mM Cd2 and pH 6.0, a small but measur-able component of nonspecific 65Zn2 binding remainedthat was resistant to removal by EGTA (see also Fig. 2).Decreasing extracellular pH (in the absence of Cd2)decreased the estimated Vmax without changing the esti-mated Km (consistent with this finding, vesicle studieshave shown that lowering pH produces noncompetitiveinhibition of 65Zn2 influx). The dashed line in Fig. 3shows the curve fit for the corresponding data with 1 mMCd2-pH 6.0 subtracted from pH 7.4 (no effect was ob-served by subtracting the 1 mM Cd2-pH 6.0 data on theestimate of Km, confirming that at low concentrations ofZn2 the contribution of nonspecific binding to measuredZn2 transport was small and insignificant). A Hill plot ofthese data yielded a slope for the Hill coefficient 1,consistent with the transport of a single zinc ion perturnover of the transport mechanism(s), similar to thatobtained in plasma membrane vesicles (6). The inhibitionseen by lowering extracellular pH was completely revers-ible. After a brief exposure (5 min) to pH 6.0 Lockesbuffer, 65Zn2 transport measured in pH 7.4 Lockesbuffer was similar to that seen in cells preincubated inpH 7.4 buffer (data not shown).

The effect of changing extracellular pH on Zn2

transport in cortical neurons is shown in Fig. 4. Zn2

transport in cortical neurons showed a clear monopha-

Fig. 1. 65Zn2 uptake was measured asdescribed in MATERIALS AND METHODS inLockes buffer at 37C. The total concen-tration of ZnCl2 in the uptake buffer was30 M; the various transition elementsshown were added at a concentration of300 M (as chloride salts). The iron solu-tion contained equimolar ascorbic acid. Af-

ter 5 min the reaction was stopped andassayed for 65Zn2 uptake. Each pointrepresents the mean SE of 3 replicatewells. Each transition element caused asignificant decrease in 65Zn2 uptake com-pared with control, as did lowering extra-cellular pH to 6.0 (P 0.001). Althoughthe mean was noticeably larger for 65Zn2

uptake in pH 6.0 than for 65Zn2 uptake inpH 6.0 with the addition of Cd2, the dif-ference was not significant (P 0.05).




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sic dependence on extracellular pH (between 6 and 8),increasing with increasing pH and approaching a max-imum at pH 8. The data were fit to a sigmoidal dose-response curve with an estimated EC50 of pH 6.77.

Next, cortical neurons were preincubated for 20 min inLockes buffer containing 1 mM iodoacetate and KCN(without glucose) and 65Zn2 accumulation was as-sayed for an additional 5 min under these same condi-tions. Metabolic inhibition failed to inhibit 65Zn2

transport; on the contrary, a small increase in 65Zn2

was observed (control with glucose: 2.25 0.17nmol mg1 5 min1; without glucose, 1 mM iodoacce-tate, and KCN: 2.81 0.19 nmol mg1 5 min1;means SE; n 6; P 0.05). The small increase in65Zn2 transport during metabolic inhibition may bethe result of intracellular acidification (see below). Fi-nally, it was determined whether Na, Cl, Ca2, orMg2 was required for 65Zn2 transport. Removal of

Ca2

and Mg2

from the Lockes buffer did not affect65Zn2 transport (providing additional support for thelack of influence of channel-mediated pathways of Zn2

influx), and substitution of either thiocyanate for Cl

or choline or Li for Na caused a small but statisti-cally insignificant increase in 65Zn2 transport (datanot shown). Thus cortical neurons in primary cultureexhibit 65Zn2 transport with properties that closelymirror results obtained with plasma membrane vesiclepreparations including 1) saturation with respect toincreasing Zn2 concentrations in the micromolarrange, 2) extracellular pH dependence, 3) inhibition by

Fig. 2. Time course of65Zn2 uptake by rat cortical neurons. Corticalneurons were exposed to various Locke s buffers containing 30 MZn2 for the time periods indicated and then assayed for 65Zn2

uptake. Buffer conditions were as follows. A: s, pH 7.4; , pH 8; , pH6. B: s, pH 7.4; }, 1 mM Cd2-pH 7.4; F,1 mM Cd2-pH 6; , 1 mMZn2-pH 7.4. Each point represents the mean SE of 2 replicatewells. Two-way ANOVA showed a significant effect of time, 1 mMCd2, pH 8.0, pH 6.0, 1 mM Cd2-pH 6.0, and 1 mM Zn2 on 65Zn2

uptake (P 0.01).

Fig. 3. 65Zn2 uptake was measured as described in Fig. 1 butdetermined after only 3 min. Cortical neurons were exposed tovarious concentrations of 65Zn2 (3, 10, 20, 40, 60, and 75 M) inLockes buffer as follows: E, pH 7.4 (Km 16.8 M, Vmax 1.39nmol mg1 min1); dashed line, curve fit to the subtracted data, pH7.4 pH 61 mM Cd2 (Km 17.2 M, Vmax 1.07nmol mg1 min1); F, p H 61 mM Cd2 (linear regression, r2 0.72). The curves drawn represent the nonlinear fit of the data to arectangular hyperbola, from which was obtained the above kineticconstants. Each point represents the mean of 2 replicate wells.

Fig. 4. Cortical neurons were exposed to various buffers adjusted tothe pH indicated, and 65Zn2 transport was measured. The reactionwas allowed to proceed for 5 min and contained 30 M 65Zn2. Thecurve drawn represents the nonlinear fit of the data to a sigmoidaldose-response relationship from which was obtained EC50 pH6.77.. A parallel experiment was run in which the transport reactionbuffer was pH 6-1 mM Cd2. The value obtained (1.06 nmol/mg) wassubtracted from the value from reactions without Cd2. Each barrepresents the mean SE of 4 replicate wells.




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other transition elements, and 4) transport indepen-dent of cellular energy stores.

On the basis of our published data (6, 8), we hypoth-esized that pH effects are fundamental to the transportmechanism, perhaps mediated by an antiport process.Therefore, it was important to show in cortical neu-rons, consistent with the antiporter hypothesis, thatintracellular acidification would enhance plasma mem-

brane 65Zn2 transport. To test this hypothesis, intra-cellular acidification was induced in cortical neuronsby the NH3/NH4

prepulse method (2). To confirm thatintracellular acidification occurred, intracellular pHwas monitored by SNARF-1 fluorescence. The intracel-lular pH of resting cells in Lockes buffer (nominallyHCO3

free) was estimated to be between 7.4 and 7.5and was largely unaffected by several buffer changesover a 20-min period (Fig. 5A). Next, cells were exposedto 20 mM NH4Cl in Lockes buffer with or without 30M ethylisopropylamiloride (EIPA) for 5 min (Fig. 5B).Thirty micromolar EIPA was included to inhibitNa/H exchange, in hopes of prolonging intracellularacidification. In HEPES buffer, cortical neurons arethought to rely heavily on Na/H exchange for intra-cellular pH homeostasis (26), which is partially inhib-ited by amiloride derivatives. During the exposure toNH4Cl the neurons took up NH3, as evidenced by a risein intracellular pH (pHi 7.8; Fig. 5). The addition of30 M EIPA had no effect on pHi, which graduallyreturned to 7.4 by the end of the 5-min incubation.These data are very similar to those obtained in corti-cal cell cultures by Siesjo and coworkers (26). Finally,the cells were returned to Lockes buffer (pH 7.4, with-out NH4Cl) with or without 30 M EIPA. During thistime, the neurons rapidly lost NH3, as evidenced by animmediate drop in pHi (pHi 6.8 with EIPA added).

Again, despite the addition of 30 M EIPA, pHi subse-quently rose to reach pH 7.2 after 10 min. No effect onpHi was observed when neurons were exposed to EIPAin pH 7.4 Lockes buffer (data not shown). On the otherhand, intracellular acidification without EIPA addedwas blunted and pHi was at least 0.2 pH units higher(Fig. 5B). As shown in Fig. 6A, conditions that resultedin the greatest intracellular acidification (NH4-EIPA)during exposure to 30 M 65Zn2 enhanced 65Zn2

transport. The enhancement of 65Zn2 transport wasnot the result of the addition of either NH4Cl or EIPA,because neither compound when added alone had aneffect on 65Zn2 transport (Fig. 6A). To be certain thatthe increase in 65Zn2 transport was the result of

intracellular acidification rather than a response to theaddition of either NH4Cl or EIPA, intracellular acidi-fication was induced by the addition of the weak acidbutyrate. Cortical neurons were preincubated with 20mM butyrate in Lockes buffer (pH 7.4) for 20 min andthen assayed for 65Zn2 transport. It can be seen inFig. 6B that intracellular acidification induced by ad-dition of the weak acid butyrate resulted in an increasein 65Zn2 transport. Finally, a recent report (20)showed nimodipine-sensitive Zn2 currents in corticalneurons that are enhanced by extracellular acidity. Torule out the possibility that the increase in 65Zn2

transport seen with intracellular acidification was aresult of this pathway, cortical cells were acidified bythe NH3/NH4

prepulse method and 65Zn2 transport

was measured in the presence of 1 M nimodipine. Noeffect of nimodipine on the enhancement of 65Zn2

transport after intracellular acidification was seen(control 1.35 0.053, control 1 M nimodipine1.28 0.031, NH4 2.17 0.053, NH4 1 M nimo-dipine 2.48 0.104 nmol mg1 3 min1; means SE;n 4). Thus convincing evidence has been presentedfor the enhancement of65Zn2 transport by intracellu-lar acidification.

A possible candidate that could mediate the Zn2

transport activity seen in the present studies is hZIP2(14, 15), which shows pH dependence similar to that

Fig. 5. The effect of changes in NH3/NH4 concentration on intra-cellular pH (pHi) measured by carboxyseminaphthorhodofluor(SNARF)-1 fluorescence. High-density cultures attached to cover-slips were exposed to 5 M SNARF-1 acetoxymethyl ester for 30 minat 37C. The cells were washed with Locke s buffer (pH 7.4), and thecoverslips were then mounted in a cuvette equipped with a coverslipholder. Control cells (A) were exposed to 2 changes of Locke s bufferpH 7.4 without the addition of NH4Cl or ethylisopropylamiloride(EIPA), demonstrating no significant effect on pHi of buffer changesalone. Cells were acid loaded (B) by a 5-min exposure to 20 mMNH3/NH4 in pH 7.4 Lockes buffer with or without the addition of 30M EIPA followed by switching to Lockes buffer pH 7.4 with orwithout 30 M EIPA. The data plotted represent the means of 3coverslips from the same preparation of cells.




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observed in the present study and appears to be aZn2-HCO3

cotransporter. To test for the presence ofhZIP2 activity, the effect of added HCO3

on 65Zn2

transport was determined. As can be seen in Fig. 7,addition of 5 mM HCO3

failed to reverse the inhibitionof65Zn2 transport produced by lowering extracellularpH. Addition of 5 mM HCO3

had no measurable effecton 65Zn2 transport at pH 7.4 compared with 65Zn2

transport in the same buffer nominally free of HCO3,

suggesting that hZIP2 activity was not present underthese conditions. This finding is not surprising because

hZIP2 expression has been observed in only prostateand uterine epithelial cells to date (14, 15). On theother hand, hZIP1 activity, which does not show HCO3

dependence but is inhibited by extracellular acidifica-tion (15), has been shown to be expressed in mosthuman tissues and may therefore contribute to 65Zn2

transport in rat cortical neurons.To provide even more convincing evidence of pH

dependent Zn2 transport in cortical neurons, proof ofchanges in intracellular Zn2 after Zn2 transport wasobtained with zinquin and fluorescence microscopy.Digital fluorescent images of cortical cultures labeledwith zinquin paired with the corresponding DIC imageof the same field are shown in Fig. 8. Fig. 8A2 showsresting cortical neurons labeled for 30 min at 37C with25 M zinquin after a 25-min preincubation in Locke sbuffer pH 7.4. Cultures were plated at high densitysimilar to conditions used for 65Zn2 transport assays.Zinquin labeling was punctate rather than diffuse.This suggests that zinquin was unable to report thepresence of free cytosolic Zn2. This finding is notsurprising, because zinquin is known to partition intobilayers (33), and is consistent with the notion that inresting cortical neurons, free cytosolic Zn2 is main-tained at submicromolar levels. A uniform punctatelabeling pattern was observed in nearly all corticalneurons, which included the soma and cell processes.Zinquin labeling was not observed in the nucleus.Punctate zinquin labeling was observed within regionsof neurons that were devoid of any visible appositionbetween cells (putative synaptic contacts).

It was next determined whether zinquin labelingwould be altered when the neurons were briefly incu-bated with Zn2 under conditions used for measuring

Fig. 7. Cortical cells were washed, and then reaction buffer contain-ing 30 M 65Zn2 was added: Lockes buffer pH 7.4 bubbled with O2(pH 7.4); Lockes buffer pH 7.4-5 mM NaHCO3 bubbled with 95%O2-5% CO2 (pH 7.4 NaHCO3); Lockes buffer pH 6.0 bubbled withO2 (pH 6.0); Lockes buffer pH 6-5 mM NaHCO3 bubbled with 95%O2-5% CO2 (pH 6 NaHCO3). The reaction was allowed to proceedfor 5 min. In each case, parallel experiments were run in which theuptake reaction buffer was pH 6-1 mM Cd2 with or without theaddition of 5 mM NaHCO3 (no effect of the addition of 5 mM NaHCO3was observed) . The value obtained was subtracted from the reac-tions without Cd2. Each bar represents the mean SE of triplicatewells. Both pH 7.4 and pH 7.4NaHCO3 were significantly differentcompared with pH 6.0 (P 0.001).

Fig. 6. A: cells were washed and then incubated in 3 successivebuffer changes designed to induce intracellular acidification. Eachbuffer was incubated with the cells for 5 min. Control, 3 changes ofLockes buffer pH 7.4; NH4/EIPA, 20 mM NH4Cl-30 M EIPA-pH 7.4followed by 2 more changes of Lockes buffer pH 7.4-30 M EIPA;NH4, 20 mM NH4Cl-pH 7.4 followed by 2 more changes of Lockesbuffer pH 7.4; EIPA, 3 changes of Locke s buffer 30 M EIPA-pH 7.4.The final buffer (uptake reaction buffer) in each experiment con-

tained 30 M

65

Zn

2

. All buffers were nominally HCO3

free andbubbled with O2. Each bar represents the mean SE of replicatewells from 4 different preparations; n 4. NH4/EIPA was signifi-cantly different from all other means (P 0.05).B: cells were washedas in A and then were preincubated in either Lockes buffer pH 7.4(control) or Lockes buffer pH 7.4 with the addition of 20 mMbutyrate (butyrate) for 20 min at 37C. Next, an uptake reactionbuffer (containing 20 mM butyrate) was added that contained 30 M65Zn2; reaction time was 5 min. Each bar represents the mean SEof replicate wells from 2 different preparations; n 4. Butyrate wassignificantly different from control (unpaired t-test, P 0.05). Forboth A and B, parallel experiments were run in which the uptakereaction buffer was pH 6.0 Lockes buffer containing 1 mM Cd2. Thedata plotted are the result of subtracting 65Zn2 transport obtainedfor each condition from those obtained in pH 6.0 Lockes buffercontaining 1 mM Cd2.




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65Zn2 transport. Figure 8B2 shows zinquin labeling incortical neurons after a 20-min preincubation inLockes buffer pH 7.4 followed by incubation with 30M Zn2 for 5 min (conditions identical to those usedfor 65Zn2 transport assays). No discernable differencecould be seen in zinquin labeling when comparingresting neurons with or without prior Zn2 exposure.Apparently, free cytosolic Zn2 rose slightly, if at all,

under these conditions, well below the detection limitof zinquin. Similarly, the pattern of punctate labelingwas unaltered by incubation with Zn2.

Although attempts at observing changes in intracel-lular Zn2 in resting cells were unsuccessful, it wasthought likely that such changes should be easier todetect after cellular Zn2 depletion. In Fig. 8C2, neu-rons were exposed to 25 M N,N,N,N-tetrakis(2-pyri-dylmethyl)-ethylenediamine (TPEN), a cell-permeantZn2 chelator, for 20 min. This buffer was removed,and then fresh Lockes buffer pH 7.4 without Zn2wasadded for a 5-min incubation. Finally, this buffer was

removed, and fresh Lockes buffer pH 7.4 containing 25M zinquin was added. Punctate zinquin labeling wasmetal dependent, as evidenced by its TPEN sensitivity.Neurons labeled with zinquin after exposure to TPENfollowed by a 5-min exposure to 30 M Zn2 in Lockesbuffer pH 7.4 are shown in Fig. 8D2. The uniformpunctate labeling returned (compare Fig. 8D2 with C2)within neuronal processes and cell bodies of neurons

(although it might appear that the intensity of zinquinlabeling was slightly greater in Fig. 8D2 than in eitherA2 or B2, such quantitative assessments are not pos-sible with zinquin). This recovery of punctate labelingafter TPEN-induced depletion was clearly pH dependentas shown in Fig. 8E2, which shows neurons exposed toTPEN followed by a 5-min exposure to 30 M Zn2 inLockes buffer pH 6.0. Zinquin labeling was nearly ab-sent, similar to TPEN treatment alone. These data pro-vide convincing evidence that pH-dependent transport ofextracellular Zn2 results in the loading of the samecytoplasmic organelles that are labeled with zinquin.

Fig. 8. Digital images of cortical neu-rons by fluorescence in the presence of25 M zinquin in Lockes buffer pH 7.4(A2); fluorescence in the presence of 25M zinquin after exposure to 30 MZn2 in Lockes buffer pH 7.4 for 5 min(B2); fluorescence in the presence of 25M zinquin after 20-min pretreatmentwith 25 M TPEN in Lockes buffer pH7.4 (C2); fluorescence in the presence of25 M zinquin after 20-min pretreat-ment with 25 M TPEN followed bythe addition of 30 M Zn2 in Lockesbuffer pH 7.4 for 5 min (D2); fluores-cence in the presence of 25 M zinquinafter 20-min pretreatment with 25 MTPEN followed by the addition of 30M Zn2 in pH 6 Lockes buffer for 5min (E2); fluorescence in the presenceof 25 M zinquin after 20-min pre-treatment with 25 M TPEN followedby the addition of 30 M Zn2 in pH7.4 Lockes buffer with 20 M pyri-thione added for 5 min (F2). A1F1,differential interference contrast im-ages corresponding with A2F2.




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Finally, as a positive control, zinquin labeling wasobserved after Zn2 depletion with TPEN followed byexposure to 20 M pyrithione (a well-characterizedZn2 ionophore) and 30 M Zn2 (in Lockes buffer, pH7.4) for 5 min. Pyrithione treatment should result inlarge increases in free cytosolic Zn2 that should bedetected by zinquin. Indeed, a diffuse labeling wasobservable in the cell bodies, which was not seen pre-

viously. This suggested that in the presence of pyri-thione and Zn2, free cytosolic Zn2 increased to mi-cromolar levels that were detectable by zinquin. Thesedata indicate that, although zinquin partitions intobilayers, significant amounts remain in the cytosol.Additional punctate labeling was clearly seen in neu-ronal processes and cell bodies (compare Fig. 8F2 withB2). However, additional punctate labeling may resultfrom the ionophoric activity of zinquin (see DISCUSSION).These data provide additional evidence that zinquinlabeling was Zn2 dependent and can detect new Zn2

that enters cortical neurons.

To strengthen this argument, parallel experimentswere performed that show the effects of TPEN andpyrithione on 65Zn2 transport in cortical neurons (Fig.9). Each bar represents subtracted data, i.e., 65Zn2

transport under the conditions indicated after subtrac-tion of65Zn2 binding in Lockes buffer pH 6 with 1 mMCd2 added. A negative value indicates that the 65Zn2

binding measured under those conditions was less

than that obtained in Lockes buffer pH 6 with 1 mMCd2 added. The first bar shows control cells exposed toLockes buffer pH 7.4 for 20 min and then exposed tothe same buffer containing 30 M 65Zn2 for an addi-tional 5 min. If 20 M pyrithione was included with the65Zn2, a significant increase in uptake was observed.Data obtained with fluorescent indicators for Zn2

(Ref. 23; Fig. 8) suggest that cytosolic concentrations ofZn2 reach micromolar levels after pyrithione treat-ment. Therefore, it was expected that the increase in65Zn2 uptake would be much greater than what wasobserved. It is likely that during the washes with

Fig. 8 (Continued)




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EGTA, pyrithione remaining in the membrane pro-vided an efficient efflux channel releasing significantamounts of the 65Zn2 that was accumulated duringthe previous pyrithione incubation when 65Zn2 waspresent. Brief Zn2 depletion induced by TPEN treat-ment (20 min) was not toxic to the neurons, becausethey were able to take up 65Zn2 in Lockes buffer pH7.4 to nearly the same level as neurons without priorexposure to TPEN and pyrithione had its expectedlarge increase in 65Zn2 uptake as well. These resultsare consistent with zinquin labeling experiments (Fig.8F2). In contrast, cells exposed to 30 M 65Zn2 in pH6.0 buffer were unable to take up significant 65Zn2

after TPEN-induced Zn2 depletion. Again, these re-sults are consistent with zinquin labeling (Fig. 8E2).When TPEN was present in the Lockes buffer used for65Zn2 transport, 65Zn2 binding levels were near zero,lower than that obtained in Lockes buffer pH 6 with 1mM Cd2 added. It should be noted that although 1

mM Cd2

-pH 6 was used to define nonspecific65

Zn2

binding, Cd2 was not used with zinquin. This is be-cause zinquin fluorescence is increased by Cd2 (albeitto a much lesser degree than Zn2).

DISCUSSION

pH-dependent Zn2 binding or Zn2 transport? Ra-dionuclide accumulation, when used to directly mea-sure Zn2 fluxes in cultured cells, offers the advan-tages of 1) absolute certainty that only Zn2 is beingmeasured and 2) unequaled sensitivity and precision.Unfortunately, it is often difficult to distinguish true

influx/efflux from changes in cell surface binding.Therefore, when radionuclide accumulation is used,the data must satisfy several criteria associated withtransport phenomena. 1) The process must occur underexperimental conditions that mimic the physiologicalconcentrations of major ions found in vivo and showsaturation with respect to increasing Zn2 concentra-tions. 65Zn2uptake occurred under physiological ionic

conditions and showed saturation kinetics with respectto increasing concentrations of 65Zn2. The calculatedaffinity for the putative transport process was similarto that reported for known mammalian Zn2 trans-porters (14, 15, 17, 28). 2) Transported 65Zn2 must beresistant to repeated extracellular chelation. Trans-ported 65Zn2 was resistant to repeated extracellularchelation by the high-affinity membrane-impermeantchelator EGTA. 3) The transport process must inde-pendently depend on both intracellular and extracellu-lar conditions. The transport of 65Zn2 independentlydepended on both intracellular and extracellular pH.That is, 65Zn2 transport was inhibited by extracellu-lar acidification and stimulated by intracellular acidi-fication. Each effect was observed when the corre-sponding intracellular or extracellular pH was normal.4) Experiments must provide evidence that intracellu-lar Zn2 levels change as a result of Zn2 transport.Under resting conditions, incubation with Zn2 re-sulted in little if any change in intracellular Zn2 asevidenced by zinquin fluorescence. This was not sur-prising because Marin and coworkers (23) showed onlysmall increases in N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) fluorescence in cortical neurons af-ter exposure to 10 M Zn2. In contrast, 65Zn2uptakecan readily be observed under these same conditionsthat roughly equates to 1 fmol Zn2 taken up per cell.

Studies in plasma membrane vesicles (8) suggest thatZn2/Zn2 exchange does occur under these conditions.Thus the net uptake of Zn2 under these conditions isprobably quite small. On the other hand, changes inzinquin labeling after TPEN depletion showed goodagreement with 65Zn2 transport studies, including pHdependence. After cellular Zn2 depletion, newlytransported Zn2 appeared to be directed to preexist-ing cytoplasmic organelles. Because all four of thesecriteria have been met, these data provide convincingevidence of a pH-dependent Zn2 transport process incortical neurons. Even so, the possibility exists that aportion of the 65Zn2 signal could result from covalentbonds with protein functional groups on the membrane

surface. However, neither Zn2

nor Cd2

is particu-larly redox active (e.g., Ca2 is a much more powerfulreducing agent than either Zn2 or Cd2), making itunlikely that covalent bonds between Zn2 and aminoacid functional groups would readily form at neutralpH.

Zn2/nH antiport mechanism and identity of thetransporter. An appealing mechanistic explanation forthese data is that the pH gradient across the plasmamembrane, in particular its direction being opposite tothe direction of Zn2 flux, is a principal determinant ofthe extent of 65Zn2 transport. These data are clearly

Fig. 9. Cortical neurons were washed and then incubated in Lockesbuffer for 20 min at 37C followed by 5 min in Lockes buffercontaining 30 M 65Zn2 with the following conditions: 7.4/7.4, 1stand 2nd buffer were Lockes pH 7.4; 7.4/Pyr: 1st buffer Lockes pH7.4followed by Lockes pH 7.4-20 M pyrithione; TPEN/7.4: 1st bufferLockes pH 7.4-TPEN 25 M followed by Lockes pH 7.4; TPEN/Pyr:1st buffer Lockes pH 7.4-TPEN 25 M followed by Lockes pH 7.4-20M pyrithione; TPEN/pH 6: 1st buffer Locke s pH 7.4-TPEN 25 Mfollowed by Lockes pH 6.0; TPEN/TPEN: 1st buffer Lockes pH7.4-TPEN 25 M followed by the same. In each case, parallel exper-

iments were run in which the uptake reaction buffer was pH 6.0Lockes buffer containing 1 mM Cd2 and subtracted from the cor-responding reaction without Cd2. Each bar represents the mean SE of triplicate wells. 7.4/Pyr and TPEN/Pyr were significantlydifferent compared with 7.4/7.4 (P 0.001). There was no significantdifference between 7.4/7.4 and TPEN/7.4, 7.4/Pyr and TPEN/Pyr, orTPEN/pH 6 and TPEN/TPEN (P 0.05).




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consistent with a Zn2/nH antiport mechanism (8)but do not provide direct evidence that such a mecha-nism exists. A direct test of this hypothesis will be todetermine whether Zn2 transport is associated withH flux as evidenced, for example, by changes in pH i.The addition of extracellular micromolar Zn2 (at pH7.4) has little or no effect on pHi of cortical neurons(data not shown). The H flux associated with Zn2

transport under these conditions may be small anddifficult to measure because cortical neurons have alarge intrinsic buffer capacity (20 mmol/pH unit; Ref.26). Another approach would be to attempt to measureZn2-dependent H fluxes after an acid load. Unfortu-nately, cortical neurons contain an active Na/H ex-changer (net H flux 3 mmol l1 min1; Ref. 26)that is resistant to inhibition by EIPA (see Fig. 5B),thus making it difficult to measure a small change inH flux. Our laboratory is currently working on exper-iments in which pHi is measured in the absence of Na

(to inhibit Na/H exchange) in hopes of uncovering asmall Zn2-dependent H flux. Finally, an approachthat has been attempted is to load cortical neuronswith Zn2 using the ionophore pyrithione and deter-mine changes in pHi. In fact, it has been reported thatsuch treatments resulted in a prolonged intracellularacidification of cultured neurons (10a), which can bereversed on application of TPEN. Unfortunately, Reyn-olds and coworkers (10a) were unable to find any directevidence of Zn2/H antiport in their studies. Thus it islikely that large elevations in intracellular or extracel-lular Zn2 affect pHi by several different mechanisms.For example, micromolar Zn2 has been shown to in-hibit Na/H exchanger activity (36).

The pH-dependent pathway for Zn2 transport de-scribed in these studies may mediate transport by

other transition elements. However, eukaryotes haveevolved specific mechanisms for the transport of ironand copper, which are quite distinct from the Zn2

transport mechanism characterized in the presentstudies. Unlike Zn2, iron and copper are redox activemetals and are sequestered in nonreductive forms asthey are transported into cells and moved throughsubcellular compartments (11). In addition, the effluxof copper appears to be mediated by a Cu-ATPaseinvolved in Menkes and Wilson diseases (18). No ex-perimental evidence suggests the existence of a similarZn2-ATPase involved in Zn2 efflux. The biochemicalbasis of the pH-dependent Zn2 transport may residein histidine residues in the putative Zn2 transporter.

In previous studies of 65Zn2

transport in purifiedplasma membrane vesicles, we showed (8) that theapparent Km for proton effects on

65Zn2 transport was0.3 M (i.e., pH 6.5). In the present study, the EC50estimated from the effect of changing extracellular pHon 65Zn2 transport was pH 6.8 (see Fig. 4). Histidineis the only amino acid with a pK(6.0) close to this rangeand is known to be a component of metal bindingmotifs of many proteins. A histidine residue could beinvolved in the Zn2 binding/translocation site of thetransporter. High pH would dissociate a proton fromhistidine that could participate in a translocation of

protons coupled to movement of Zn2 or induce a con-formational change in the protein that would be asso-ciated with the translocation step. It should be notedthat the predicted amino acid sequences of ZnT-1 (28),hZIP1 (15), and hZIP2 (14) all show a histidine-con-taining metal binding motif.

Ideally, one could take advantage of the well-charac-terized pH dependence of plasma membrane Zn2

transport in cortical neurons to examine the pH depen-dence of cloned Zn2 transporters and, by comparison,develop reasonable hypotheses about the identity ofthe transporter in cortical neurons. As detailed in theintroduction, examination of the literature reveals anincomplete picture of the pH dependence of DMT1 (17,35) and ZnT-1, although both genes are expressed incortical cultures. On the other hand, 65Zn2 uptake byK562 erythroleukemia cells expressing either hZIP1 orhZIP2 was clearly inhibited by lowering extracellularpH (14, 15). The pH dependence of Zn2 transport incells expressing hZIP1 is similar to that found in thepresent study for cortical neurons (see Fig. 7). Unfor-tunately, the rat homolog of hZIP has not been identi-fied and functionally characterized. Although we areunable to determine with certainty which Zn2 trans-porter(s) in cortical neurons is responsible for pH-dependent Zn2 transport, it appears that the ZIPfamily proteins are potentially interesting candidatesworthy of additional study.

Evidence for extrasynaptic compartmentalization ofZn2. In the present study, evidence is presented forthe extrasynaptic compartmentalization of Zn2 inpreexisting cytoplasmic organelles. These conclusionsare based on the pattern of zinquin labeling observedin cortical neurons (see Fig. 8). The uniform, albeitpunctate, zinquin labeling pattern and its occurrence

in cellular regions apparently devoid of cell-to-cell ap-position (putative synaptic contacts) suggested thatzinquin labeling was not restricted to presynaptic ves-icles.

In light of the physical properties of zinquin (33),several concerns can be raised as to whether the pres-ence of zinquin will itself perturb Zn2 compartmen-talization. 1) This could occur first by cytosolic zinquinacting as a sink for intracellular Zn2. This phenom-enon is very unlikely because of zinquin s low affinityfor Zn2. Zinquin will not perturb the status of MT-III-bound Zn2 in the cytosol (binding affinity of MT-III forZn2 is much too high to be affected by zinquin), andfree cytosolic Zn2 concentrations are well below the

binding affinity of the zinquin-Zn2

complex. 2) It wasreported that high concentrations of zinquin can act asan ionophore for Zn2 at acidic pH (33). Could zinquinload cytoplasmic compartments with Zn2 by virtue ofits ionophoric properties? Probably not, because theionophoric effects of zinquin are concentration depen-dent and are negligible at the concentrations used inthe proposed studies (25 M). In addition, zinquinwould only be able to affect cytoplasmic compartmentswhen Zn2 concentrations are micromolar and pH is 5.In this study, only when pyrithione was added wouldcytosolic Zn2 concentrations be expected to reach mi-




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cromolar levels. Thus only the pyrithione experimentsare subject to this potential artifact. In addition, it isnot possible that the ionophore properties of zinquincould assist in loading cytoplasmic compartments withZn2 after TPEN depletion. By design, these compart-ments were loaded with Zn2 before zinquin incuba-tion. Could zinquin deplete acidic organelles contain-ing high concentrations of Zn2? This certainly is

possible but would still be expected to be a small effectat the concentrations of zinquin used in the presentstudy. 3) One can question whether zinquin will beselective in its interaction with intracellular mem-branes, such that some Zn2 containing compartmentswould be missed. Because the Zn2-zinquin complexcan be charged, the membrane partitioning can beaffected by membrane charge. However, this is a qual-itative effect, because zinquin partitions into all typesof lipid bilayers and shows good stability in zwitteri-onic bilayers. Thus it is unlikely that preferentialmembrane partitioning of zinquin occurred in thisstudy.

The identification of the subcellular organelles thatcorrespond to zinquin-labeled structures in neurons isnot known for certain. Glutamate-containing synapticvesicles are known to contain chelatable Zn2 (13) andmay account for a fraction of the compartmentalizedZn2 seen in resting neurons. A possible candidate forthe extrasynaptic compartmentalization of Zn2wouldbe mitochondria, because it has been known for manyyears that mitochondria transport Zn2 (3). Mitochon-dria have a subcellular distribution in neurons that issimilar to the distribution of zinquin labeling, albeiteven more numerous. It seems very likely that duringpyrithione-induced loading, large quantities of Zn2

enter the cell and would be shuttled into mitochondria.

This has been suggested to occur when toxic levels ofZn2 are induced in neurons by depolarization andexposure to high extracellular Zn2 (4, 30). Therefore itis plausible that the increased quantity of punctatezinquin labeling seen after pyrithione treatment rep-resents, at least in part, an increase in mitochondrialZn2 sequestration.

Biological significance of pH-dependent Zn2 trans-port. The results with zinquin demonstrate that, afterZn2 depletion with TPEN, pH-dependent plasmamembrane Zn2 transport can supply Zn2 directly tosubcellular compartments and effect a rapid refilling ofthese stores in the presence of an inwardly directedZn2 gradient and physiological pH values. This sug-

gests that the plasma membrane pH-dependent path-way may provide the means for released Zn2 to reen-ter neurons. Such recycling of histochemically reactiveZn2 has been observed after electrical stimulation ofthe mossy fibers innervating the hippocampus (19).The pH-dependent pathway may also provide the pri-mary means for Zn2 entry into neurons under restingconditions as well. Because the cortical cultures usedin the present studies may contain as much as 10%glial cells, a similar pH-dependent Zn2 transport mayoccur in glial cells. Eventually, a better understandingof plasma membrane transport mechanisms for Zn2

and the fate of transported Zn2 will aid in the clari-fication of the role that Zn2plays in selective neuronaldeath after transient brain ischemia. Although ourknowledge of the mechanisms of plasma membraneZn2 transport and homeostasis is growing, there isstill much more to be learned.

This work would not have been possible without the technical

assistance of Philip A. Carter, Jason Zaros, and Lynn Bowman.Special thanks go to Mark Berryman, Department of BiomedicalSciences, Ohio University, who supplied the microscope and digitalcamera setup. Also contributing to this work was TatsuhikoKawaguchi, a summer undergraduate research fellow in cellular andmolecular biology.

This work was supported by National Institute on Aging GrantAG-17741.

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pH dependence and compartmentalization of zinc transported

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