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

pH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical neurons
ROBERT A. COLVIN Program in Neuroscience, Department of Biological Sciences, Ohio University, Athens, Ohio 45701
Received 19 March 2001; accepted in nal form 17 October 2001

Colvin, Robert A. pH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical 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 cortical neurons was characterized by successfully combining radioactive tracer experiments with spectrouorometry and uorescence microscopy. Cortical neurons showed a time-dependent and saturable transport of 65Zn2 with an apparent afnity of 1520 M. 65Zn2 transport was pH dependent and was decreased by extracellular acidication and increased by intracellular acidication. Compartmentalization of newly transported Zn2 was assessed with the Zn2 selective uorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn2 . Depletion of intracellular Zn2 with the membrane-permeant chelator N,N,N ,N -tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) resulted in the complete loss of punctate zinquin labeling. After Zn2 depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 M Zn2 , pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 M Zn2 , pH 6.0. These data were conrmed in parallel 65Zn2 transport experiments. pH; zinquin; carboxyseminaphthorhodouor-1 uorescence; ion transport; transition elements; primary culture

Zn2 can enter neurons by two distinct pathways. The rst pathway is transporter mediated and slow, occurs under resting conditions, and is observed at concentrations of Zn2 well below 100 M; relatively small amounts of Zn2 enter the cell by this pathway (5, 8, 9). The second pathway is channel mediated and rapid, requires depolarization or receptor activation, and is seen at concentrations of Zn2 100 M; greater amounts of Zn2 enter the cell by this pathway (29, 30, 32). The transporter-mediated pathway can be easily distinguished from channel pathways by its insensitivity to various channel blockers (5, 9) (e.g., MK801, GYKI 53466, nifedipine, FTX3.3, -conotoxin GVIA, and CNQX). Previously, the transporter-mediated pathway was studied in synaptosomes (34) and hippocampal slices (19), but the mechanisms of Zn2 transport were never elucidated. More recently, this laboratory has studied (6, 8, 9) the
IT IS KNOWN THAT

mechanisms of 65Zn2 transport in rat brain plasma membrane vesicles. 65Zn2 transport showed saturation with increasing concentrations of 65Zn2 , and 65 Zn2 inux was inhibited when extravesicular pH was lowered. The transport mechanism appeared reversible in that both pH-dependent Zn2 inux and efux were observed. Together, these studies led to a working hypothesis: plasma membrane transport of Zn2 is pH dependent because it depends on a Zn2 / nH antiport mechanism. The recent cloning of cDNA coding for several proteins associated with plasma membrane Zn2 transport function, i.e., DMT1 (17), hZIP1 (15), hZIP2 (14), and ZnT-1 (28), has provided new molecular and genetic tools with which to address the mechanism of plasma membrane Zn2 transport. All the cloned Zn2 transporters are apparently independent of cellular energy stores, show no dependence on Na , K , or Cl concentrations, and show an apparent afnity for Zn2 in the micromolar range (14, 15, 17, 28). Expression studies have revealed interesting pH effects on Zn2 transport mediated by either DMT1 (17, 35) or hZIP1 and hZIP2 (14, 15). DMT1 shows nonspecic divalent metal transport (most notably iron transport in the intestine) coupled with protons (i.e., low extracellular pH stimulates Fe2 uptake when the protein is expressed in Xenopus oocytes; Ref. 17). When DMT1 was transiently expressed in COS-7 cells, Fe2 uptake was greatly increased and showed a dependence on extracellular pH (35). In contrast to expression studies in Xenopus oocytes, Fe2 uptake was inhibited in transfected COS-7 cells by extracellular acidication, and transport had an optimal pH of 6.8. Thus it is still not clear what effect changes in extracellular or intracellular pH should have on DMT1 function. hZIP is the human homolog of the ZIP family of Zn2 transporters cloned from yeast and plants (16). Three human ZIP gene products have been identied (hZIP13); both hZIP1 and hZIP2 have been functionally characterized (14, 15). Zn2 transport observed in K562 erythroleukemia cells expressing either hZIP1 or hZIP2 was inhibited by lowering extracellular pH (14). The inhibition at low pH was reversed in cells expressing hZIP2 by addition of HCO3 , leading the authors of that study
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Address for reprint requests and other correspondence: R. A. Colvin, Dept. of Biological Sciences, Ohio Univ., Athens, OH 45701 (E-mail: colvin@ohio.edu). http://www.ajpcell.org

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to suggest a Zn2 -HCO3 cotransport mechanism. ZnT-1 is thought to function primarily as a Zn2 efux protein (21), and its transport mechanism and pH dependence have yet to be clearly established. It is not known which of the above proteins might be responsible 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 is thought to exist in at least four distinct cellular pools (13). The rst pool is composed of metalloproteins (e.g., metalloenzymes and transcription factors) that use tightly bound Zn2 as a required cofactor to carry out their cellular functions. The second pool constitutes Zn2 bound to cytoplasmic metallothionein-III (MTIII), which is thought to be a reservoir and buffer of cytosolic Zn2 (12, 22, 27). The third pool, cytosolic free Zn2 , is maintained at very low levels probably well below 100 nM (1, 5, 23, 29), presumably by the actions of cytosolic MT-III. The fourth pool is compartmentalized Zn2 . Experimental evidence shows that many eukaryotic cell types contain compartmentalized Zn2 and that cytoplasmic organelles can sequester Zn2 (10, 25). A well-characterized compartment containing Zn2 in neurons is synaptic vesicles of glutamatergic neurons of the cerebral cortex and in particular the hippocampal formation (13). The present studies were designed to address some of the gaps in our knowledge of Zn2 transport and homeostasis in cortical neurons. The results provide convincing evidence of a pH-dependent transport mechanism for Zn2 in the plasma membrane of cortical neurons in primary culture. Zn2 inux was favored when 1) the ow of ions was down a concentration gradient and 2) the cell interior was acidic with respect to the extracellular medium. In addition, evidence was obtained of the extrasynaptic compartmentalization of newly transported Zn2 .
MATERIALS AND METHODS

Primary culture of cortical neurons. Primary culture of embryonic (embryonic day 18) cortical neurons was performed as described previously (7, 24). Brains were removed from the skulls and kept moist in Hanks balanced salt solution (HBSS; without Mg2 and Ca2 ) for further dissection. With a dissecting microscope, the cerebral cortex was carefully separated by blunt dissection from the brain stem diencephalon, olfactory bulbs, and cerebellum, which was discarded. Next the meninges and choroid plexus were stripped away. The cerebral hemispheres were cut into small pieces (about 4 pieces for each hemisphere) and trypsinized in HBSS at room temperature. After trypsinization, nerve cells were dissociated by gentle trituration through the narrow opening of a re-polished Pasteur pipette. The dissociated neurons suspended in HBSS were plated on 24-well culture plates (Falcon) or culture plates containing sterilized coverslips coated with polyethylenimine (50% solution; Sigma, St. Louis, MO), which was diluted 1:1,000 in borate buffer. The cortical neurons were allowed to attach to the plates or coverslips at 37C and 5% CO2 in 1 ml of MEM solution (GIBCO BRL) supplemented with 10 mM sodium bicarbonate, 2 mM L-glutamine, 1 mM pyruvate, 20 mM KCl, 10% glucose, and 10% (vol/vol) heat-inactivated fetal bovine
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serum. The desired cell density was obtained by adjusting the volume of cell suspension added to each plate (nal plating density was 5 105 cells/ml). The medium was replaced with fresh supplemented MEM after 36 h and 24 h later switched to 1 ml of Neurobasal medium (GIBCO BRL) supplemented with 0.5 mM glutamine and 2% B27 (GIBCO BRL). Use of serum-free culture medium, which does not favor glial proliferation, allows the culture of cortical neurons in the near absence of glial cells. Measurement of 65Zn2 transport. Cortical neurons (47 days in vitro) attached to 24-well plates were assayed for 65 Zn2 transport as follows (all buffers were at 37C). Each well of a 24-well plate was rst 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 the cells for subsequent 65Zn2 transport assay. The transport experiment was initiated by switching to Lockes buffer (pH 7.4 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 nal concentration in the range of 0.0010.005 Ci/ l. The specic activity of 65Zn2 (expressed as cpm/nmol Zn2 ) was determined for each experiment by assaying an aliquot of each solution containing 65Zn2 for radioactivity. The cells were incubated for various times at 37C. Normally, 5% of the total Zn2 in the reaction buffer was taken up by the cells. To terminate a transport reaction, the buffer containing 65Zn2 was rapidly removed by aspiration and replaced with ice-cold Lockes buffer (pH 7.4) without Zn2 added. Next, the wells were washed three times with 0.5 ml of ice-cold wash buffer containing Lockes buffer (without Ca2 and Mg2 added) and 1 mM EGTA (pH 7.4). The cells were lysed by freeze thawing ( 70C). The lysed cells in each well were resuspended in 250 l of buffer, of which 200 l were used to assay radioactivity and 40 l were used to determine protein. Protein concentration was determined by a Bio-Rad method with bovine serum albumin as a standard, and 65Zn2 was determined in a gamma counter. With the specic activity and protein concentration, counts per minute were converted to nanomoles per milligram for each well. In experiments using 65Zn2 , parallel experiments were done in pH 6 Lockes buffer containing 1 mM Cd2 . The results from these experiments (nmol/mg) were subtracted from total 65Zn2 uptake to obtain 65Zn2 transport and nonspecic 65Zn2 binding. Cortical cells are rmly attached to the culture plates and can easily withstand several buffer washes. This was veried by viewing the cells in each well before and after the washing procedure was complete. MTT assay. Many manipulations described in these experiments have the potential of causing cell death, and therefore assessments of acute cell viability were performed. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT ) conversion was determined colorometrically as described elsewhere (24). Parallel cultures to be used for MTT measurements received the same experimental manipulations but were not exposed to 65Zn2 used for transport. Instead, these cultures were allowed to remain in fresh Lockes buffer for an additional 24 h to allow time for acute effects on cell survival to occur. After 24 h the cells were assayed for MTT cleavage and conversion. Activities obtained after experimental manipulations were compared with cells that were switched only to fresh Lockes buffer for 24 h. Negligible cell death was observed in high-density cortical cultures exposed only to fresh Lockes buffer for 24 h, as judged by MTT assay.
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Microscopy and zinquin labeling. Cells were cultured as described in Primary culture of cortical neurons in culture plates containing sterilized glass coverslips. For visualization of intracellular Zn2 with zinquin, the medium was discarded and replaced with fresh Lockes buffer before an experiment was started. After various experimental settings at 37C, the medium was discarded and the glass coverslips were washed twice with 1 ml of Lockes buffer. One milliliter of Lockes buffer was left in the culture well to prevent the cells from drying out. A 5 mM concentrate of zinquin ester (freshly dissolved in DMSO) was diluted directly into the wells to produce a nal concentration of 25 M. Although zinquin is highly permeable to lipid bilayers, the ester form was used so that only intracellular zinquin would contribute to the observed signal. Zinquin experiments were also performed at 12.5 M with similar results. The culture plates were reincubated at 37C for 30 min to allow zinquin to bind specically to intracellular Zn2 . The cells were washed three times with fresh Lockes buffer (pH 7.4) and then immediately mounted on glass slides with Aquatex (for immediate viewing) to prevent leakage of zinquin. Coverslips inverted on microscope slides were examined using a Nikon Eclipse 600 with epiuorescence and differential interference contrast (DIC) optics, equipped with a SPOT RT digital camera for image capture. Zinquin uorescence was observed using an ultraviolet lter block (EF-4 UV 2E/C DAPI lter block, EX 330380). Zinquin labeling experiments were repeated in at least three different cultures. The digital images presented are qualitatively similar and representative of what was observed in all three experiments. Zinquin ester was obtained from Luminus (Adelaide, SA, Australia). Carboxyseminaphthorhodouor-1 uorescence. Cortical neurons attached to glass coverslips were loaded with carboxyseminaphthorhodouor (SNARF)-1 by incubation for 30 min in Lockes buffer (pH 7.4) at 37C containing 5 M 5(and 6)-carboxy SNARF-1 acetoxymethyl ester, acetate (Molecular Probes, Eugene, OR). The cells were then washed with 1 ml of Lockes buffer before pH measurement. Cells attached to coverslips were held in a cuvette at an 45 angle to the incident light beam by a coverslip holder (Hitachi Instruments, San Jose, CA). To switch buffer solutions, the coverslip and holder were lifted out of the cuvette and quickly placed into a waiting cuvette containing the next desired buffer. SNARF-1 uorescence was measured in a uorescence spectrophotometer (Hitachi F-2000) with excitation at 514 nm and emission wavelengths of 585 and 630 nm. The uorescence ratio F585 /F630 was calibrated in separate experiments with cells treated with 10 M nigericin in Lockes buffers of various pH containing 120 mM KCl (2). With the calibration data, intracellular pH was calculated directly from F585 /F630. F585 /F630 was a linear function of pH over pH values between 6.4 and 8.0. In each experiment, to correct for scattered light/autouorescence, data obtained from cells treated 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 rst adjusted to the desired pH with the addition of NaOH or HCl and then bubbled with 100% O2 for 5 min to remove dissolved CO2. The buffers were then checked again and usually required a small additional adjustment with NaOH or HCl to bring them to the desired pH. These buffers were considered to be nominally free of HCO3 during an experiment. HEPES buffers containing 5 mM HCO3 were prepared as above with the addition of 5 mM NaHCO3 and then were bubbled with 95% O2-5% CO2 for 5 min. Subsequently, these buffers were checked and usually required a
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small additional adjustment with NaOH or HCl as above. Control experiments had 5 mM NaCl added instead of NaHCO3 and were bubbled with O2 as above. Experiments were performed in room air, but all solutions were covered to minimize the amount of CO2 that could dissolve in or escape from the buffer during an experiment. Statistical analysis. Each preparation of cortical neurons (1 pregnant rat) normally yields three to four 24-well plates at the density used for these experiments. The data presented (except data in Fig. 6, which include up to 4 different preparations) are the means of multiple determinations with wells from the same 24-well plate. All the data presented were repeated in at least two and normally at least three different preparations of cortical neurons with similar results, although variation did exist when different preparations were compared. Potential sources of variation include differences in cell density and animal variation. Statistical analysis and nonlinear curve tting were accomplished with GraphPad Prism software (San Diego, CA). Data were analyzed by t-test, one-way ANOVA with Tukeys post test, or, where appropriate, two-way ANOVA.
RESULTS

Previous studies from this laboratory (6, 8) characterized a robust, saturable, pH-dependent 65Zn2 transport in plasma membrane vesicles isolated from adult rat brain. The present study used cortical neurons in primary culture derived from fetal rat cortex to characterize 65Zn2 transport. Because it is well known that high concentrations of heavy metals are toxic to neurons, determining the toxicity of the various conditions to be used in this study was necessary. To accomplish this, cells were exposed to various experimental conditions for a brief exposure (5 min) or a long exposure (60 min), and then cell death was determined 24 h later with the MTT conversion assay. It was found that exposure of cells to concentrations of Zn2 as high as 300 M in Lockes buffer for as long as 60 min did not result in signicant cell death when assayed 24 h later, similar to results recently reported by Sheline et al. (31). Likewise, changing buffer pH to either 6 or 8 from 7.4 did not result in evidence of increased cell death. La3 (1 mM) was used in plasma membrane vesicle experiments to inhibit 65Zn2 transport; however, La3 was quite toxic to neurons even after a brief exposure. Cd2 (1 mM) was much less toxic to the neurons than La3 . No cell death was observed after a brief exposure, although after the 60-min exposure, some cell death was evident 24 h later. Various other transition elements (Co2 , Mn2 , Cu2 , and Fe2 ) showed intermediate toxicity less than La3 but more 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 predominate over channel-mediated pathways for 65Zn2 inux (i.e., Zn2 was 100 M, extracellular Ca2 and Mg2 were at physiological concentrations, and nondepolarizing conditions were used). Under these conditions, 65Zn2 transport is insensitive to calcium and glutamate channel blockers (9). The effect of various transition elements on 65Zn2 uptake is shown in Fig.
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1. Neurons in culture were exposed to various transition elements (Cd2 , Co2 , Mn2 , Cu2 , Fe2 , Ni2 ) in Lockes buffer for 5 min with 30 M 65Zn2 . The transition elements were included at a concentration of 300 M (conditions that for the most part do not produce acute toxicity). All the transition elements produced signicant inhibition of 65Zn2 accumulation. However, inhibition was incomplete. Increasing the concentration of the transition elements to 1 mM produced a greater level of inhibition, albeit still incomplete. The greatest inhibition was seen with Cd2 . The reduction of extracellular pH to 6.0 reduced the total 65Zn2 uptake by 50%. Inhibition by transition elements was still evident (except Mn2 ), even at pH 6.0. In this experiment, the inhibitory effects of transition elements and extracellular acidication appeared to be additive. The 65Zn2 uptake measured under the above conditions probably included 65Zn2 binding to the surface of neurons and culture plates, which was resistant to removal by EGTA. To characterize 65Zn2 binding better, 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 uptake (most noticeably after 30 min of exposure), whereas reduction of extracellular pH to 6 inhibited 65 Zn2 uptake (Fig. 2A). The addition of 1 mM Cd2 at pH 7.4 reduced 65Zn2 uptake to a level similar to that seen at pH 6. The addition of 1 mM Cd2 at pH 6 further reduced 65Zn2 uptake to nearly that seen after isotope dilution by addition of 1 mM nonradioactive Zn2 (Fig. 2B). Again, the effects of Cd2 and extracellular acidication appeared to be additive. In the presence of 1 mM Cd2 and pH 6.0, the 65Zn2 signal closely approximates nonspecic binding, as dened by

isotope dilution, and suggests near-complete inhibition of transport without acute toxicity, particularly when exposures are kept brief. Figure 3 shows 65Zn2 uptake as a function of increasing 65Zn2 concentration. 65Zn2 uptake showed saturation with respect to increasing 65Zn2 concentration, and the Michaelis-Menten constant (Km) and the maximum rate of 65Zn2 transport (Vmax) were estimated by computer-assisted nonlinear curve tting to a rectangular hyperbola (Fig. 3). The Km obtained (1520 M) was similar to that obtained with plasma membrane vesicles when La3 was used as an inhibitor (6). In the presence of 1 mM Cd2 and pH 6.0, a small but measurable component of nonspecic 65Zn2 binding remained that 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 estimated Km (consistent with this nding, vesicle studies have shown that lowering pH produces noncompetitive inhibition of 65Zn2 inux). The dashed line in Fig. 3 shows the curve t for the corresponding data with 1 mM Cd2 -pH 6.0 subtracted from pH 7.4 (no effect was observed by subtracting the 1 mM Cd2 -pH 6.0 data on the estimate of Km, conrming that at low concentrations of Zn2 the contribution of nonspecic binding to measured Zn2 transport was small and insignicant). A Hill plot of these data yielded a slope for the Hill coefcient 1, consistent with the transport of a single zinc ion per turnover of the transport mechanism(s), similar to that obtained in plasma membrane vesicles (6). The inhibition seen by lowering extracellular pH was completely reversible. After a brief exposure (5 min) to pH 6.0 Lockes buffer, 65Zn2 transport measured in pH 7.4 Lockes buffer was similar to that seen in cells preincubated in pH 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 as described in MATERIALS AND METHODS in Lockes buffer at 37C. The total concentration of ZnCl2 in the uptake buffer was 30 M; the various transition elements shown were added at a concentration of 300 M (as chloride salts). The iron solution contained equimolar ascorbic acid. After 5 min the reaction was stopped and assayed for 65Zn2 uptake. Each point represents the mean SE of 3 replicate wells. Each transition element caused a 65 signicant decrease in Zn2 uptake compared with control, as did lowering extracellular pH to 6.0 (P 0.001). Although the mean was noticeably larger for 65Zn2 uptake in pH 6.0 than for 65Zn2 uptake in pH 6.0 with the addition of Cd2 , the difference was not signicant (P 0.05).

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Fig. 2. Time course of 65Zn2 uptake by rat cortical neurons. Cortical neurons were exposed to various Lockes buffers containing 30 M Zn2 for the time periods indicated and then assayed for 65Zn2 uptake. Buffer conditions were as follows. A: s, pH 7.4; , pH 8; , pH 6. B: s, pH 7.4; }, 1 mM Cd2 -pH 7.4; F,1 mM Cd2 -pH 6; , 1 mM Zn2 -pH 7.4. Each point represents the mean SE of 2 replicate wells. Two-way ANOVA showed a signicant effect of time, 1 mM Cd2 , pH 8.0, pH 6.0, 1 mM Cd2 -pH 6.0, and 1 mM Zn2 on 65Zn2 uptake (P 0.01).

Fig. 4. Cortical neurons were exposed to various buffers adjusted to the pH indicated, and 65Zn2 transport was measured. The reaction was allowed to proceed for 5 min and contained 30 M 65Zn2 . The curve drawn represents the nonlinear t of the data to a sigmoidal dose-response relationship from which was obtained EC50 pH 6.77.. A parallel experiment was run in which the transport reaction buffer was pH 6-1 mM Cd2 . The value obtained (1.06 nmol/mg) was subtracted from the value from reactions without Cd2 . Each bar represents the mean SE of 4 replicate wells.

sic dependence on extracellular pH (between 6 and 8), increasing with increasing pH and approaching a maximum at pH 8. The data were t to a sigmoidal doseresponse curve with an estimated EC50 of pH 6.77.

Fig. 3. 65Zn2 uptake was measured as described in Fig. 1 but determined after only 3 min. Cortical neurons were exposed to various concentrations of 65Zn2 (3, 10, 20, 40, 60, and 75 M) in Lockes buffer as follows: E, pH 7.4 (Km 16.8 M, Vmax 1.39 nmol mg 1 min 1); dashed line, curve t to the subtracted data, pH 7.4 pH 61 mM Cd2 (Km 17.2 M, Vmax 1.07 nmol mg 1 min 1); F, pH 61 mM Cd2 (linear regression, r2 0.72). The curves drawn represent the nonlinear t of the data to a rectangular hyperbola, from which was obtained the above kinetic constants. Each point represents the mean of 2 replicate wells. AJP-Cell Physiol VOL

Next, cortical neurons were preincubated for 20 min in Lockes buffer containing 1 mM iodoacetate and KCN (without glucose) and 65Zn2 accumulation was assayed for an additional 5 min under these same conditions. Metabolic inhibition failed to inhibit 65Zn2 transport; on the contrary, a small increase in 65Zn2 was observed (control with glucose: 2.25 0.17 nmol mg 1 5 min 1; without glucose, 1 mM iodoaccetate, and KCN: 2.81 0.19 nmol mg 1 5 min 1; means SE; n 6; P 0.05). The small increase in 65 Zn2 transport during metabolic inhibition may be the result of intracellular acidication (see below). Finally, it was determined whether Na , Cl , Ca2 , or Mg2 was required for 65Zn2 transport. Removal of Ca2 and Mg2 from the Lockes buffer did not affect 65 Zn2 transport (providing additional support for the lack of inuence of channel-mediated pathways of Zn2 inux), and substitution of either thiocyanate for Cl or choline or Li for Na caused a small but statistically insignicant increase in 65Zn2 transport (data not shown). Thus cortical neurons in primary culture exhibit 65Zn2 transport with properties that closely mirror results obtained with plasma membrane vesicle preparations including 1) saturation with respect to increasing Zn2 concentrations in the micromolar range, 2) extracellular pH dependence, 3) inhibition by
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other transition elements, and 4) transport independent of cellular energy stores. On the basis of our published data (6, 8), we hypothesized that pH effects are fundamental to the transport mechanism, perhaps mediated by an antiport process. Therefore, it was important to show in cortical neurons, consistent with the antiporter hypothesis, that intracellular acidication would enhance plasma membrane 65Zn2 transport. To test this hypothesis, intracellular acidication was induced in cortical neurons by the NH3/NH4 prepulse method (2). To conrm that intracellular acidication occurred, intracellular pH was monitored by SNARF-1 uorescence. The intracellular pH of resting cells in Lockes buffer (nominally HCO3 free) was estimated to be between 7.4 and 7.5 and was largely unaffected by several buffer changes over a 20-min period (Fig. 5A). Next, cells were exposed to 20 mM NH4Cl in Lockes buffer with or without 30 M ethylisopropylamiloride (EIPA) for 5 min (Fig. 5B). Thirty micromolar EIPA was included to inhibit Na /H exchange, in hopes of prolonging intracellular acidication. In HEPES buffer, cortical neurons are thought to rely heavily on Na /H exchange for intracellular pH homeostasis (26), which is partially inhibited by amiloride derivatives. During the exposure to NH4Cl the neurons took up NH3, as evidenced by a rise in intracellular pH (pHi 7.8; Fig. 5). The addition of 30 M EIPA had no effect on pHi, which gradually returned to 7.4 by the end of the 5-min incubation. These data are very similar to those obtained in cortical cell cultures by Siesjo and coworkers (26). Finally, the cells were returned to Lockes buffer (pH 7.4, without NH4Cl) with or without 30 M EIPA. During this time, the neurons rapidly lost NH3, as evidenced by an immediate drop in pHi (pHi 6.8 with EIPA added). Again, despite the addition of 30 M EIPA, pHi subsequently rose to reach pH 7.2 after 10 min. No effect on pHi was observed when neurons were exposed to EIPA in pH 7.4 Lockes buffer (data not shown). On the other hand, intracellular acidication without EIPA added was blunted and pHi was at least 0.2 pH units higher (Fig. 5B). As shown in Fig. 6A, conditions that resulted in the greatest intracellular acidication (NH4-EIPA) during exposure to 30 M 65Zn2 enhanced 65Zn2 transport. The enhancement of 65Zn2 transport was not the result of the addition of either NH4Cl or EIPA, because neither compound when added alone had an effect on 65Zn2 transport (Fig. 6A). To be certain that the increase in 65Zn2 transport was the result of intracellular acidication rather than a response to the addition of either NH4Cl or EIPA, intracellular acidication was induced by the addition of the weak acid butyrate. Cortical neurons were preincubated with 20 mM butyrate in Lockes buffer (pH 7.4) for 20 min and then assayed for 65Zn2 transport. It can be seen in Fig. 6B that intracellular acidication induced by addition of the weak acid butyrate resulted in an increase in 65Zn2 transport. Finally, a recent report (20) showed nimodipine-sensitive Zn2 currents in cortical neurons that are enhanced by extracellular acidity. To rule out the possibility that the increase in 65Zn2
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Fig. 5. The effect of changes in NH3/NH4 concentration on intracellular pH (pHi) measured by carboxyseminaphthorhodouor (SNARF)-1 uorescence. High-density cultures attached to coverslips were exposed to 5 M SNARF-1 acetoxymethyl ester for 30 min at 37C. The cells were washed with Lockes buffer (pH 7.4), and the coverslips were then mounted in a cuvette equipped with a coverslip holder. Control cells (A) were exposed to 2 changes of Lockes buffer pH 7.4 without the addition of NH4Cl or ethylisopropylamiloride (EIPA), demonstrating no signicant effect on pHi of buffer changes alone. Cells were acid loaded (B) by a 5-min exposure to 20 mM NH3/NH4 in pH 7.4 Lockes buffer with or without the addition of 30 M EIPA followed by switching to Lockes buffer pH 7.4 with or without 30 M EIPA. The data plotted represent the means of 3 coverslips from the same preparation of cells.

transport seen with intracellular acidication was a result of this pathway, cortical cells were acidied by the NH3/NH4 prepulse method and 65Zn2 transport was measured in the presence of 1 M nimodipine. No effect of nimodipine on the enhancement of 65Zn2 transport after intracellular acidication was seen (control 1.35 0.053, control 1 M nimodipine 1.28 0.031, NH4 2.17 0.053, NH4 1 M nimodipine 2.48 0.104 nmol mg 1 3 min 1; means SE; n 4). Thus convincing evidence has been presented for the enhancement of 65Zn2 transport by intracellular acidication. 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
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Fig. 6. A: cells were washed and then incubated in 3 successive buffer changes designed to induce intracellular acidication. Each buffer was incubated with the cells for 5 min. Control, 3 changes of Lockes buffer pH 7.4; NH4/EIPA, 20 mM NH4Cl-30 M EIPA-pH 7.4 followed 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 Lockes buffer pH 7.4; EIPA, 3 changes of Lockes buffer 30 M EIPA-pH 7.4. The nal buffer (uptake reaction buffer) in each experiment contained 30 M 65Zn2 . All buffers were nominally HCO3 free and bubbled with O2. Each bar represents the mean SE of replicate wells from 4 different preparations; n 4. NH4 /EIPA was signicantly different from all other means (P 0.05). B: cells were washed as 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 mM butyrate (butyrate) for 20 min at 37C. Next, an uptake reaction buffer (containing 20 mM butyrate) was added that contained 30 M 65 Zn2 ; reaction time was 5 min. Each bar represents the mean SE of replicate wells from 2 different preparations; n 4. Butyrate was signicantly different from control (unpaired t-test, P 0.05). For both A and B, parallel experiments were run in which the uptake reaction buffer was pH 6.0 Lockes buffer containing 1 mM Cd2 . The data plotted are the result of subtracting 65Zn2 transport obtained for each condition from those obtained in pH 6.0 Lockes buffer containing 1 mM Cd2 .

hZIP2 expression has been observed in only prostate and uterine epithelial cells to date (14, 15). On the other hand, hZIP1 activity, which does not show HCO3 dependence but is inhibited by extracellular acidication (15), has been shown to be expressed in most human 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 of changes in intracellular Zn2 after Zn2 transport was obtained with zinquin and uorescence microscopy. Digital uorescent images of cortical cultures labeled with zinquin paired with the corresponding DIC image of the same eld are shown in Fig. 8. Fig. 8A2 shows resting cortical neurons labeled for 30 min at 37C with 25 M zinquin after a 25-min preincubation in Lockes buffer pH 7.4. Cultures were plated at high density similar to conditions used for 65Zn2 transport assays. Zinquin labeling was punctate rather than diffuse. This suggests that zinquin was unable to report the presence of free cytosolic Zn2 . This nding is not surprising, because zinquin is known to partition into bilayers (33), and is consistent with the notion that in resting cortical neurons, free cytosolic Zn2 is maintained at submicromolar levels. A uniform punctate labeling pattern was observed in nearly all cortical neurons, which included the soma and cell processes. Zinquin labeling was not observed in the nucleus. Punctate zinquin labeling was observed within regions of neurons that were devoid of any visible apposition between cells (putative synaptic contacts). It was next determined whether zinquin labeling would be altered when the neurons were briey incubated with Zn2 under conditions used for measuring

observed in the present study and appears to be a Zn2 -HCO3 cotransporter. To test for the presence of hZIP2 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 inhibition of 65Zn2 transport produced by lowering extracellular pH. Addition of 5 mM HCO3 had no measurable effect on 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 under these conditions. This nding is not surprising because
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Fig. 7. Cortical cells were washed, and then reaction buffer containing 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 with O2 (pH 6.0); Lockes buffer pH 6-5 mM NaHCO3 bubbled with 95% O2-5% CO2 (pH 6 NaHCO3). The reaction was allowed to proceed for 5 min. In each case, parallel experiments were run in which the uptake reaction buffer was pH 6-1 mM Cd2 with or without the addition of 5 mM NaHCO3 (no effect of the addition of 5 mM NaHCO3 was observed) . The value obtained was subtracted from the reactions without Cd2 . Each bar represents the mean SE of triplicate wells. Both pH 7.4 and pH 7.4 NaHCO3 were signicantly different compared with pH 6.0 (P 0.001). www.ajpcell.org

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Fig. 8. Digital images of cortical neurons by uorescence in the presence of 25 M zinquin in Lockes buffer pH 7.4 (A2); uorescence in the presence of 25 M zinquin after exposure to 30 M Zn2 in Lockes buffer pH 7.4 for 5 min (B2); uorescence in the presence of 25 M zinquin after 20-min pretreatment with 25 M TPEN in Lockes buffer pH 7.4 (C2); uorescence in the presence of 25 M zinquin after 20-min pretreatment with 25 M TPEN followed by the addition of 30 M Zn2 in Lockes buffer pH 7.4 for 5 min (D2); uorescence in the presence of 25 M zinquin after 20-min pretreatment with 25 M TPEN followed by the addition of 30 M Zn2 in pH 6 Lockes buffer for 5 min (E2); uorescence in the presence of 25 M zinquin after 20-min pretreatment with 25 M TPEN followed by the addition of 30 M Zn2 in pH 7.4 Lockes buffer with 20 M pyrithione added for 5 min (F2). A1F1, differential interference contrast images corresponding with A2F2.

Zn2 transport. Figure 8B2 shows zinquin labeling in cortical neurons after a 20-min preincubation in Lockes buffer pH 7.4 followed by incubation with 30 M Zn2 for 5 min (conditions identical to those used for 65Zn2 transport assays). No discernable difference could be seen in zinquin labeling when comparing resting neurons with or without prior Zn2 exposure. Apparently, free cytosolic Zn2 rose slightly, if at all, under these conditions, well below the detection limit of zinquin. Similarly, the pattern of punctate labeling was unaltered by incubation with Zn2 . Although attempts at observing changes in intracellular Zn2 in resting cells were unsuccessful, it was thought likely that such changes should be easier to detect after cellular Zn2 depletion. In Fig. 8C2, neurons were exposed to 25 M N,N,N ,N -tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), a cell-permeant Zn2 chelator, for 20 min. This buffer was removed, and then fresh Lockes buffer pH 7.4 without Zn2 was added for a 5-min incubation. Finally, this buffer was
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removed, and fresh Lockes buffer pH 7.4 containing 25 M zinquin was added. Punctate zinquin labeling was metal dependent, as evidenced by its TPEN sensitivity. Neurons labeled with zinquin after exposure to TPEN followed by a 5-min exposure to 30 M Zn2 in Lockes buffer pH 7.4 are shown in Fig. 8D2. The uniform punctate labeling returned (compare Fig. 8D2 with C2) within neuronal processes and cell bodies of neurons (although it might appear that the intensity of zinquin labeling was slightly greater in Fig. 8D2 than in either A2 or B2, such quantitative assessments are not possible with zinquin). This recovery of punctate labeling after TPEN-induced depletion was clearly pH dependent as shown in Fig. 8E2, which shows neurons exposed to TPEN followed by a 5-min exposure to 30 M Zn2 in Lockes buffer pH 6.0. Zinquin labeling was nearly absent, similar to TPEN treatment alone. These data provide convincing evidence that pH-dependent transport of extracellular Zn2 results in the loading of the same cytoplasmic organelles that are labeled with zinquin.
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Fig. 8 (Continued)

Finally, as a positive control, zinquin labeling was observed after Zn2 depletion with TPEN followed by exposure to 20 M pyrithione (a well-characterized Zn2 ionophore) and 30 M Zn2 (in Lockes buffer, pH 7.4) for 5 min. Pyrithione treatment should result in large increases in free cytosolic Zn2 that should be detected by zinquin. Indeed, a diffuse labeling was observable in the cell bodies, which was not seen previously. This suggested that in the presence of pyrithione and Zn2 , free cytosolic Zn2 increased to micromolar levels that were detectable by zinquin. These data indicate that, although zinquin partitions into bilayers, signicant amounts remain in the cytosol. Additional punctate labeling was clearly seen in neuronal processes and cell bodies (compare Fig. 8F2 with B2). However, additional punctate labeling may result from the ionophoric activity of zinquin (see DISCUSSION). These data provide additional evidence that zinquin labeling was Zn2 dependent and can detect new Zn2 that enters cortical neurons.
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To strengthen this argument, parallel experiments were performed that show the effects of TPEN and pyrithione on 65Zn2 transport in cortical neurons (Fig. 9). Each bar represents subtracted data, i.e., 65Zn2 transport under the conditions indicated after subtraction of 65Zn2 binding in Lockes buffer pH 6 with 1 mM Cd2 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 mM Cd2 added. The rst bar shows control cells exposed to Lockes buffer pH 7.4 for 20 min and then exposed to the same buffer containing 30 M 65Zn2 for an additional 5 min. If 20 M pyrithione was included with the 65 Zn2 , a signicant increase in uptake was observed. Data obtained with uorescent indicators for Zn2 (Ref. 23; Fig. 8) suggest that cytosolic concentrations of Zn2 reach micromolar levels after pyrithione treatment. Therefore, it was expected that the increase in 65 Zn2 uptake would be much greater than what was observed. It is likely that during the washes with
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Fig. 9. Cortical neurons were washed and then incubated in Lockes buffer for 20 min at 37C followed by 5 min in Lockes buffer containing 30 M 65Zn2 with the following conditions: 7.4/7.4, 1st and 2nd buffer were Lockes pH 7.4; 7.4/Pyr: 1st buffer Lockes pH 7.4 followed by Lockes pH 7.4-20 M pyrithione; TPEN/7.4: 1st buffer Lockes 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 7.4-20 M pyrithione; TPEN/pH 6: 1st buffer Lockes pH 7.4-TPEN 25 M followed by Lockes pH 6.0; TPEN/TPEN: 1st buffer Lockes pH 7.4-TPEN 25 M followed by the same. In each case, parallel experiments were run in which the uptake reaction buffer was pH 6.0 Lockes buffer containing 1 mM Cd2 and subtracted from the corresponding reaction without Cd2 . Each bar represents the mean SE of triplicate wells. 7.4/Pyr and TPEN/Pyr were signicantly different compared with 7.4/7.4 (P 0.001). There was no signicant difference between 7.4/7.4 and TPEN/7.4, 7.4/Pyr and TPEN/Pyr, or TPEN/pH 6 and TPEN/TPEN (P 0.05).

EGTA, pyrithione remaining in the membrane provided an efcient efux channel releasing signicant amounts of the 65Zn2 that was accumulated during the previous pyrithione incubation when 65Zn2 was present. Brief Zn2 depletion induced by TPEN treatment (20 min) was not toxic to the neurons, because they were able to take up 65Zn2 in Lockes buffer pH 7.4 to nearly the same level as neurons without prior exposure to TPEN and pyrithione had its expected large increase in 65Zn2 uptake as well. These results are consistent with zinquin labeling experiments (Fig. 8F2). In contrast, cells exposed to 30 M 65Zn2 in pH 6.0 buffer were unable to take up signicant 65Zn2 after TPEN-induced Zn2 depletion. Again, these results are consistent with zinquin labeling (Fig. 8E2). When TPEN was present in the Lockes buffer used for 65 Zn2 transport, 65Zn2 binding levels were near zero, lower than that obtained in Lockes buffer pH 6 with 1 mM Cd2 added. It should be noted that although 1 mM Cd2 -pH 6 was used to dene nonspecic 65Zn2 binding, Cd2 was not used with zinquin. This is because zinquin uorescence is increased by Cd2 (albeit to a much lesser degree than Zn2 ).
DISCUSSION

pH-dependent Zn2 binding or Zn2 transport? Radionuclide accumulation, when used to directly measure Zn2 uxes in cultured cells, offers the advantages of 1) absolute certainty that only Zn2 is being measured and 2) unequaled sensitivity and precision. Unfortunately, it is often difcult to distinguish true
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inux/efux from changes in cell surface binding. Therefore, when radionuclide accumulation is used, the data must satisfy several criteria associated with transport phenomena. 1) The process must occur under experimental conditions that mimic the physiological concentrations of major ions found in vivo and show saturation with respect to increasing Zn2 concentrations. 65Zn2 uptake occurred under physiological ionic conditions and showed saturation kinetics with respect to increasing concentrations of 65Zn2 . The calculated afnity for the putative transport process was similar to that reported for known mammalian Zn2 transporters (14, 15, 17, 28). 2) Transported 65Zn2 must be resistant to repeated extracellular chelation. Transported 65Zn2 was resistant to repeated extracellular chelation by the high-afnity membrane-impermeant chelator EGTA. 3) The transport process must independently depend on both intracellular and extracellular conditions. The transport of 65Zn2 independently depended on both intracellular and extracellular pH. That is, 65Zn2 transport was inhibited by extracellular acidication and stimulated by intracellular acidication. Each effect was observed when the corresponding intracellular or extracellular pH was normal. 4) Experiments must provide evidence that intracellular Zn2 levels change as a result of Zn2 transport. Under resting conditions, incubation with Zn2 resulted in little if any change in intracellular Zn2 as evidenced by zinquin uorescence. This was not surprising because Marin and coworkers (23) showed only small increases in N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide (TSQ) uorescence in cortical neurons after exposure to 10 M Zn2 . In contrast, 65Zn2 uptake can readily be observed under these same conditions that roughly equates to 1 fmol Zn2 taken up per cell. Studies in plasma membrane vesicles (8) suggest that Zn2 /Zn2 exchange does occur under these conditions. Thus the net uptake of Zn2 under these conditions is probably quite small. On the other hand, changes in zinquin labeling after TPEN depletion showed good agreement with 65Zn2 transport studies, including pH dependence. After cellular Zn2 depletion, newly transported Zn2 appeared to be directed to preexisting cytoplasmic organelles. Because all four of these criteria have been met, these data provide convincing evidence of a pH-dependent Zn2 transport process in cortical neurons. Even so, the possibility exists that a portion of the 65Zn2 signal could result from covalent bonds with protein functional groups on the membrane surface. However, neither Zn2 nor Cd2 is particularly redox active (e.g., Ca2 is a much more powerful reducing agent than either Zn2 or Cd2 ), making it unlikely that covalent bonds between Zn2 and amino acid functional groups would readily form at neutral pH. Zn2 /nH antiport mechanism and identity of the transporter. An appealing mechanistic explanation for these data is that the pH gradient across the plasma membrane, in particular its direction being opposite to the direction of Zn2 ux, is a principal determinant of the extent of 65Zn2 transport. These data are clearly
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consistent with a Zn2 /nH antiport mechanism (8) but do not provide direct evidence that such a mechanism exists. A direct test of this hypothesis will be to determine whether Zn2 transport is associated with H ux as evidenced, for example, by changes in pHi. The addition of extracellular micromolar Zn2 (at pH 7.4) has little or no effect on pHi of cortical neurons (data not shown). The H ux associated with Zn2 transport under these conditions may be small and difcult to measure because cortical neurons have a large intrinsic buffer capacity (20 mmol/pH unit; Ref. 26). Another approach would be to attempt to measure Zn2 -dependent H uxes after an acid load. Unfortunately, cortical neurons contain an active Na /H exchanger (net H ux 3 mmol l 1 min 1; Ref. 26) that is resistant to inhibition by EIPA (see Fig. 5B), thus making it difcult to measure a small change in H ux. Our laboratory is currently working on experiments in which pHi is measured in the absence of Na (to inhibit Na /H exchange) in hopes of uncovering a small Zn2 -dependent H ux. Finally, an approach that has been attempted is to load cortical neurons with Zn2 using the ionophore pyrithione and determine changes in pHi. In fact, it has been reported that such treatments resulted in a prolonged intracellular acidication of cultured neurons (10a), which can be reversed on application of TPEN. Unfortunately, Reynolds and coworkers (10a) were unable to nd any direct evidence of Zn2 /H antiport in their studies. Thus it is likely that large elevations in intracellular or extracellular Zn2 affect pHi by several different mechanisms. For example, micromolar Zn2 has been shown to inhibit Na /H exchanger activity (36). The pH-dependent pathway for Zn2 transport described in these studies may mediate transport by other transition elements. However, eukaryotes have evolved specic mechanisms for the transport of iron and copper, which are quite distinct from the Zn2 transport mechanism characterized in the present studies. Unlike Zn2 , iron and copper are redox active metals and are sequestered in nonreductive forms as they are transported into cells and moved through subcellular compartments (11). In addition, the efux of copper appears to be mediated by a Cu-ATPase involved in Menkes and Wilson diseases (18). No experimental evidence suggests the existence of a similar Zn2 -ATPase involved in Zn2 efux. The biochemical basis of the pH-dependent Zn2 transport may reside in histidine residues in the putative Zn2 transporter. In previous studies of 65Zn2 transport in puried plasma membrane vesicles, we showed (8) that the apparent Km for proton effects on 65Zn2 transport was 0.3 M (i.e., pH 6.5). In the present study, the EC50 estimated from the effect of changing extracellular pH on 65Zn2 transport was pH 6.8 (see Fig. 4). Histidine is the only amino acid with a pK (6.0) close to this range and is known to be a component of metal binding motifs of many proteins. A histidine residue could be involved in the Zn2 binding/translocation site of the transporter. High pH would dissociate a proton from histidine that could participate in a translocation of
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protons coupled to movement of Zn2 or induce a conformational change in the protein that would be associated with the translocation step. It should be noted that the predicted amino acid sequences of ZnT-1 (28), hZIP1 (15), and hZIP2 (14) all show a histidine-containing metal binding motif. Ideally, one could take advantage of the well-characterized pH dependence of plasma membrane Zn2 transport in cortical neurons to examine the pH dependence of cloned Zn2 transporters and, by comparison, develop reasonable hypotheses about the identity of the transporter in cortical neurons. As detailed in the introduction, examination of the literature reveals an incomplete picture of the pH dependence of DMT1 (17, 35) and ZnT-1, although both genes are expressed in cortical cultures. On the other hand, 65Zn2 uptake by K562 erythroleukemia cells expressing either hZIP1 or hZIP2 was clearly inhibited by lowering extracellular pH (14, 15). The pH dependence of Zn2 transport in cells expressing hZIP1 is similar to that found in the present study for cortical neurons (see Fig. 7). Unfortunately, the rat homolog of hZIP has not been identied and functionally characterized. Although we are unable to determine with certainty which Zn2 transporter(s) in cortical neurons is responsible for pHdependent Zn2 transport, it appears that the ZIP family proteins are potentially interesting candidates worthy of additional study. Evidence for extrasynaptic compartmentalization of Zn2 . In the present study, evidence is presented for the extrasynaptic compartmentalization of Zn2 in preexisting cytoplasmic organelles. These conclusions are based on the pattern of zinquin labeling observed in cortical neurons (see Fig. 8). The uniform, albeit punctate, zinquin labeling pattern and its occurrence in cellular regions apparently devoid of cell-to-cell apposition (putative synaptic contacts) suggested that zinquin labeling was not restricted to presynaptic vesicles. In light of the physical properties of zinquin (33), several concerns can be raised as to whether the presence of zinquin will itself perturb Zn2 compartmentalization. 1) This could occur rst by cytosolic zinquin acting as a sink for intracellular Zn2 . This phenomenon is very unlikely because of zinquins low afnity for Zn2 . Zinquin will not perturb the status of MT-IIIbound Zn2 in the cytosol (binding afnity of MT-III for Zn2 is much too high to be affected by zinquin), and free cytosolic Zn2 concentrations are well below the binding afnity of the zinquin-Zn2 complex. 2) It was reported that high concentrations of zinquin can act as an ionophore for Zn2 at acidic pH (33). Could zinquin load cytoplasmic compartments with Zn2 by virtue of its ionophoric properties? Probably not, because the ionophoric effects of zinquin are concentration dependent and are negligible at the concentrations used in the proposed studies ( 25 M). In addition, zinquin would only be able to affect cytoplasmic compartments when Zn2 concentrations are micromolar and pH is 5. In this study, only when pyrithione was added would cytosolic Zn2 concentrations be expected to reach miwww.ajpcell.org

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cromolar levels. Thus only the pyrithione experiments are subject to this potential artifact. In addition, it is not possible that the ionophore properties of zinquin could assist in loading cytoplasmic compartments with Zn2 after TPEN depletion. By design, these compartments were loaded with Zn2 before zinquin incubation. Could zinquin deplete acidic organelles containing high concentrations of Zn2 ? This certainly is possible but would still be expected to be a small effect at the concentrations of zinquin used in the present study. 3) One can question whether zinquin will be selective in its interaction with intracellular membranes, such that some Zn2 containing compartments would be missed. Because the Zn2 -zinquin complex can be charged, the membrane partitioning can be affected by membrane charge. However, this is a qualitative effect, because zinquin partitions into all types of lipid bilayers and shows good stability in zwitterionic bilayers. Thus it is unlikely that preferential membrane partitioning of zinquin occurred in this study. The identication of the subcellular organelles that correspond to zinquin-labeled structures in neurons is not known for certain. Glutamate-containing synaptic vesicles are known to contain chelatable Zn2 (13) and may account for a fraction of the compartmentalized Zn2 seen in resting neurons. A possible candidate for the extrasynaptic compartmentalization of Zn2 would be mitochondria, because it has been known for many years that mitochondria transport Zn2 (3). Mitochondria have a subcellular distribution in neurons that is similar to the distribution of zinquin labeling, albeit even more numerous. It seems very likely that during pyrithione-induced loading, large quantities of Zn2 enter the cell and would be shuttled into mitochondria. This has been suggested to occur when toxic levels of Zn2 are induced in neurons by depolarization and exposure to high extracellular Zn2 (4, 30). Therefore it is plausible that the increased quantity of punctate zinquin labeling seen after pyrithione treatment represents, at least in part, an increase in mitochondrial Zn2 sequestration. Biological signicance of pH-dependent Zn2 transport. The results with zinquin demonstrate that, after Zn2 depletion with TPEN, pH-dependent plasma membrane Zn2 transport can supply Zn2 directly to subcellular compartments and effect a rapid relling of these stores in the presence of an inwardly directed Zn2 gradient and physiological pH values. This suggests that the plasma membrane pH-dependent pathway may provide the means for released Zn2 to reenter neurons. Such recycling of histochemically reactive Zn2 has been observed after electrical stimulation of the mossy bers innervating the hippocampus (19). The pH-dependent pathway may also provide the primary means for Zn2 entry into neurons under resting conditions as well. Because the cortical cultures used in the present studies may contain as much as 10% glial cells, a similar pH-dependent Zn2 transport may occur in glial cells. Eventually, a better understanding of plasma membrane transport mechanisms for Zn2
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and the fate of transported Zn2 will aid in the clarication of the role that Zn2 plays in selective neuronal death after transient brain ischemia. Although our knowledge of the mechanisms of plasma membrane Zn2 transport and homeostasis is growing, there is still 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 Biomedical Sciences, Ohio University, who supplied the microscope and digital camera setup. Also contributing to this work was Tatsuhiko Kawaguchi, a summer undergraduate research fellow in cellular and molecular biology. This work was supported by National Institute on Aging Grant AG-17741. REFERENCES 1. Aizenman E, Stout A, Hartnett K, Dineley K, McLaughlin B, and Reynolds IJ. Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release. J Neurochem 75: 18781888, 2000. 2. Bevensee MO, Weed RA, and Boron WF. Intracellular pH regulation in cultured astrocytes from rat hippocampus. I. Role of HCO3 . J Gen Physiol 110: 453465, 1997. 3. Brierley GP and Knight VA. Ion transport by heart mitochondria. X. The uptake and release of Zn2 and its relation to the energy-linked accumulation of magnesium. Biochemistry 6: 38923901, 1967. 4. Canzoniero LMT, Turetsky DM, and Choi DW. Measurement of intracellular free zinc concentrations accompanying zinc-induced neuronal death. J Neurosci 19: RC31, 1999. 5. Cheng C and Reynolds IJ. Calcium-sensitive uorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons. J Neurochem 71: 24012410, 1998. 6. Colvin RA. Characterization of a plasma membrane zinc transporter in rat brain. Neurosci Lett 247: 147150, 1998. 7. Colvin RA. Zinc inhibits Ca2 transport by rat brain Na /Ca2 exchanger. Neuroreport 9: 30913096, 1998. 8. Colvin RA, Davis N, Nipper RW, and Carter PA. Evidence for a zinc/proton antiporter in rat brain. Neurochem Int 36: 539547, 2000. 9. Colvin RA, Davis N, Nipper RW, and Carter PA. Zinc transport in the brain: routes of zinc inux and efux in neurons. J Nutr 130: 1484S1487S, 2000. 10. Coyle P, Zalewski PD, Philcox JC, Forbes IJ, Ward AD, Lincoln SF, Mahadevan I, and Rofe AM. Measurement of zinc in hepatocytes by using a uorescent probe, Zinquin: relationship to metallothionein and intracellular zinc. Biochem J 303: 781786, 1994. 10a.Dineley KE, Brocard JB, Kress GJ, and Reynolds IJ. Mechanisms of [Zn2 ]i-induced acidication in cultured forebrain neurons and astrocytes. Abstracts of the Society for Neuroscience 30th Annual Meeting 26: 1014, 2000. 11. Eide D. Molecular biology of iron and zinc uptake in eukaryotes. Curr Opin Cell Biol 9: 573577, 1997. 12. Erickson JC, Hollopeter G, Thomas SA, Froelick GJ, and Palmiter RD. Disruption of the metallothionein-III gene in mice: Analysis of brain zinc, behavior, and neuron vulnerability to metals, aging, and seizures. J Neurosci 17: 12711281, 1997. 13. Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 31: 145238, 1989. 14. Gaither LA and Eide DJ. Functional expression of the human hZIP2 zinc transporter. J Biol Chem 275: 55605564, 2000. 15. Gaither LA and Eide DJ. The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells. J Biol Chem 276: 2225822264, 2001. 16. Grotz N, Fox T, Connolly E, Park W, Guerinot ML, and Eide DJ. Identication of a family of zinc transporter genes from Arabidopsis that respond to zinc deciency. Proc Natl Acad Sci USA 95: 72207224, 1998. www.ajpcell.org

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28. Palmiter RD and Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 14: 639649, 1995. 29. Sensi SL, Canzoniero LMT, Yu SP, Ying HS, Koh JY, Kerchner GA, and Choi DW. Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17: 9554 9564, 1997. 30. Sensi SL, Yin HZ, Carriedo SG, Rao SS, and Weiss JH. Preferential Zn2 inux through Ca2 -permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production. Proc Natl Acad Sci USA 96: 24142419, 1999. 31. Sheline CT, Behrens MM, and Choi DW. Zinc-induced cortical neuronal death: contribution of energy failure attributable to loss of NAD and inhibition of glycolysis. J Neurosci 20: 3139 3146, 2000. 32. Snider B, Choi J, Turetsky D, Canzoniero L, Sensi S, Sheline C, Wang X, Yu S, and Choi DW. Nitric oxide reduces Ca2 and Zn2 inux through voltage-gated Ca2 channels and reduces Zn2 neurotoxicity. Neuroscience 100: 651661, 2000. 33. Snitsarev V, Budde T, Stricker TP, Cox JM, Krupa DJ, Geng L, and Kay AR. Fluorescent detection of Zn2 -rich vesicles with zinquin: mechanism of action in lipid environments. Biophys J 80: 15381546, 2001. 34. Wensink J, Molenaar AJ, Woroniecka UD, and Vandenhammer CJA. Zinc uptake into synaptosomes. J Neurochem 50: 782789, 1988. 35. Worthington MT, Browne L, Battle EH, and Luo RQ. Functional properties of transfected human DMT1 iron transporter. Am J Physiol Gastrointest Liver Physiol 279: G1265G1273, 2000. 36. Vilella S, Ingrosso L, Lionetto MG, Schettino T, Zonno V, and Storelli C. Effect of cadmium and zinc on the Na /H exchanger present on the brush border membrane vesicles isolated from eel kidney tubular cells. Aquat Toxicol (Amst) 48: 2536, 1999.

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