The Axonal y-Aminobutyric Acid Transporter GAT4 Is Sorted to the ...

Vol. 269, No. 6,Issue of February 11,pp. 4668-4674,

THEJOURNAL OF B I O ~ I CCHEMISTRY AL 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

1994

Printed in U.S.A.

The Axonal y-Aminobutyric Acid Transporter GAT4 Is Sorted to the Apical Membranes of Polarized Epithelial Cells* (Received for publication, July 27, 1993, and in revised form, October 21, 1993)

Grazia PietriniSI,Young J. Suhn, Lambert Edelmannn, GaryRudnickl, and Michael J. CaplanSII From the Departments of $Cellular and Molecular Physiology and Wharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 and the IConsiglio Nazionale delle Ricerche, Center for Cytopharmacology, and the Department of Pharmacology, University of Milan, Milan 20129, Italy

Recent studies suggest that epithelial cells and neu- physiologic function requires that newly synthesizedmemrons employ similar mechanisms to target proteins to brane proteinsbe differentially sorted to their appropriate sites the distinct subdomains of their polarized cell surface of functional residence. of membranes. We have examined the sorting behavior Recent evidence suggests thatsimilar mechanismsmay the neuronal y-aminobutyric acid (GABA) transporter function to generate and maintain this polarity in both sysGAT-1 expressed by transfection in the polarized epithetems. Viral glycoproteins that are sorted to the epithelial apical lial Madin-Darby canine kidney (MDCK) cell line. We and basolateralsurfaces have been shown to accumulate in the findthatthe GABA transportersendogenouslyexaxonal and dendriticdomains, respectively, of infected neurons pressed by polarized hippocampal neurons in culture (5). Furthermore, glycophospholipid-linked proteins arereare restricted to axonal plasma membranes. In transstricted to the apical surfaces of epithelial cells and to the fected MDCK cells, theGABA transporter is found to be localized primarily to the apical cell surface when ex- axolemmas of neurons (6).Thus, the signals andmechanisms responsible for apical sorting in epitheliamay result inaxonal amined by immunocytochemistry, cell surface biotinylaTo test thismodel further we have examMDCK cells exposed to hyper- targeting in neurons. tion, and transport assay. ined the sorting behavior of a n axonal protein expressed by osmoticstressexpressacloserelative of GAT-1, the betainetransporter (BGT-1). We findthat BGT-1 ex- transfection in polarized epithelial cells. pressed by transfection inMDCK cells accumulates pre- The Na,C1-dependent GABA' carrier functions to terminate dominantly at the basolateralcell surface. These obser- GABA-ergic transmission by mediating the transportof GABA from the synapticcleft into thecytoplasm of the nerve terminal vations suggest that the sorting information required identification of the cDNAencoding the GABA for axonal targeting may be similar to that which medi-(7,8). The recent carrier demonstrates that this protein is part of a large gene atesapicallocalizationinepithelia.Furthermore,it would appear that despite their high degree of homol- family that includes the systemsresponsible for serotonin, norogy, the BGT-1 and GAT-1 transporters manifest sorting epinephrine, and dopamine transport (9). Eachof these transsignals which specify their targeting to distinctsurcell porters iscomposed of a single polypeptide characterized by 12 face domains. putative transmembrane helices. Further characterization of this gene family reveals that at least four distinct isoforms of GABA transporter canbe discerned (10,111. Designated GAT-1, GAT-2, GAT-3, and GAT-B, these transporters manifestdifferThe plasma membranes of neurons and epithelial cells are ent tissue localizations and pharmacologic sensitivities (12). divided into distinctdomains characterized by markedly differ- The pharmacologic properties of the GAT-1 isoform suggest ent protein compositions (1-3). The epithelial plasmalemma is that it is expressed predominantly in neurons and not inglia composed of an apical region separated by tight junctionsfrom (13). Electron microscopic immunolocalization studies pera basolateral surface which rests upon a basement membrane. formed on rat brain suggest that the GABA transporter is conNeuronal plasma membranes aresubdivided into anaxolemma centrated in the plasma membranesof axons and axon termiand a somatodendritic membrane (4), which become function- nals of GABA-ergic neurons (14). It would appear, therefore, ally distinct at the level of the axon hillock. In the epithelial that neurons in situ are capable of targeting this protein to case, plasmalemma1 polarity is a prerequisite for vectorial sol- axonal membrane domains. ute and fluid transport. The domains of the epithelial plasma We wondered whether the sorting information that functions membrane must possess distinct classes of transport proteins t o ensure the GABA transporter's axonal localization in neuto mediate fluxes against concentration gradients. Neuronal rons would also serve to mediate apical targeting in epithelial polarity is necessary to ensure rapid andunidirectional infor- cells. To test this possibility, we have generated stably transmation flow. For both cell types, the maintenance of normal fected MDCK cell lines which express the rat GAT-1 transporter. We find that GAT-1 does, in fact, accumulatepreferen* This work was supported by a Brown-Coxe fellowship (to G. P.), a tially at the apical surface. These results suggest thataxonal fellowship from the American Heart Association, Connecticut affiliate and apical sorting mechanisms may, in fact, share common (toY. J. S.), National Institute on Drug Abuse Grant 07259 (to G. R.),a features. This observation is especially interesting in light of fellowship fromthe David and Lucille Packard Foundation(to M. J. C.), (BGT-1) and National Institutes of Health Grant GM-42136 (to M. J. C.). The the recent demonstration that the betaine transporter costs of publication of this articlewere defrayed in part by the payment endogenously expressed at the basolateral surfaces of MDCK of page charges. This article must thereforebe hereby marked "adver- cells shares -50% sequence identity with the GAT-1 system tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate (15). this fact. /ITo whom correspondence shouldbe addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar The abbreviations used are: GABA,y-aminobutyric acid; MDCK, Madin-Darby canine kidney;M A P , microtubule-associated protein. St., New Haven, CT 06510. Tel.: 203-785-7316; Fax: 203-785-4951.

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GABA Dansporter Is Sorted to Epithelial Apical Membranes MATERIALSANDMETHODS Antibodies and Immunocytochemistry-Hippocampal neurons (the kind gift of G. Banker, University of Virginia) were isolated from day 19 embryonic rats and cultured on glass coverslips according tothe methods of Goslin and Banker (16). Neurons were fured in ice-cold methanol and processed for immunofluorescence as described previously(17).The "-2 antibody was purchased from Boehringer Mannheim and used at a dilution of 1:lOO. Antibody R24, directed against the GABA transporter, was provided byR. Jahn (Yale University) and used a t a dilution of1:50 for all immunofluorescence studies. This antibody was generated against a synthetic peptide whose sequence corresponds to amino acids 571-586 of the rat brain GABA transporter. Coupling the peptide to keyhole limpet hemocyanin and immunization in rabbits was carried out according to Lerner et al. (18). Transfected MDCK cells were grown to confluence on transparent filters (Cyclopore, Becton Dickson, Lincoln Park, NJ), fixed in methanol, and processed for immunofluorescence as described in Pietrini et al. (17). The Na,K-ATPase a-subunit was localized using monoclonal antibody 6H at a dilution of 1:lO. Production and characterization of this antibody are described elsewhere (17). Immunofluorescence was observed with a Zeiss Axiophotepifluorescence photomicroscope and photographed with Kodak T"AX 100 ASA film (Eastman Kodak Co.). Confocal microscopy was carried out on a Zeiss laser scanning confocal microscope, and X Z sections were generated as described (19). Subcloning and lkansfection-The cDNA encoding the GABA transporter, provided by B. Kanner, was subcloned into the ClaI and XbaI sites of the mammalian expression vector pCB6 (20) (gift of C. Brewer and M. Roth, University of Texas, Southwestern). MDCK cells were passaged and subjected to CaP0,-mediated transfection as described (21). Transfected cell lines were selected by growth in the antibiotic G418 (0.9 mg/ml) (Life Technologies, Inc.), and expression of the GABA transporter was assayed initially by GABA uptake assay (see below). The cDNA encoding the betaine transporter subcloned into the pSPORTl vector was kindly provided by J . S. Handler (Johns Hopkins University). The insert was excised through digestion with MluI and ClaI, whose sites are present in the Sal1 adapterat theinsert's 5'-end and in the insert's 3'-untranslated sequence, respectively. The excised insert was subcloned into pCB6 at the corresponding sites. Cell Surface Biottnylation-Cells were plated at confluent density on 0.45-pm filter inserts (Transwell, Costar Co., Cambridge, MA). Seven days after seeding, GAT-1-transfected MDCK cells were biotinylated through exposure to NHS-ss-biotin present in the medium bathing the apical or basolateral surfaces. The biotinylation protocol employed was that of Sargiacomo etal. (22), with the exception that the reaction was carried out at pH 9.0 in 10 m~ triethanolamine, 2 mM CaCl,, 125 mM NaCI. We have demonstrated previously that this modification increases the efficiency of NHS-ss-biotin incorporation (23). Following biotinylation, filters were excised fromthe cups with a razor blade, and the attached monolayers were lysed in 1%Triton X-100 in 150 mM NaCl, 5 mM EDTA, 50 m~ Tris, pH 7.5. Biotinylated proteins were recovered from cell lysates by incubation with avidin-agarose beads (Pierce) as described (22). Bound proteins were eluted from the beads in Laemmli sample buffer (241, separated by SDS-polyacrylamide gelelectrophoresis and transferred to nitrocellulose. Transfers were probed with antibody R24 at a dilution of 1:200 as described (19). Specific staining was visualized by the Enhanced Chemiluminescence technique (Amersham Corp.). For quantitation of the biotinylation experiments, autoradiographs were scanned with an LKB Ultrascan XL laser densitometer. Multiple samples were analyzed, corresponding to different quantities of total protein. In this manner, the linearity of the signal with respect to sample load was verified. GABA Uptake Assay-Transfected and untransfected MDCK cells were grown to confluence for >5 days on 0.45-pm pore size Transwell filter inserts.GABAuptake was performed at room temperature according to a modification of the method of Yamauchi et al. (25). T h e filters were washed twice with uptake buffer (100 mM NaCl, 2 mM KCI, 1 m~ CaCl,, 1m~ MgCI,, 10 m~ HEPES, pH 7.5),and each well wasincubated from the apical or basolateral side with 0.2 ml ofuptake buffer containing PHIGABA (DuPont NEN) at the final concentrations indicated in the figure legends. Followinga 10-minincubation the uptake assay was terminated by aspirating the medium, and the filters were successively dipped into three beakers, each of which contained 100 ml of ice-cold uptake buffer. The filters were excised from the cups and placed into scintillation vials, where the attached epithelial cells were solubilized in 0.2 ml of 1%SDS. Protein was determined with the BCA protein assay

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kit (Pierce) as described previously (25). The cell lysates were counted in 5 ml of Atomlight scintillation fluid (DuPont NEN). RESULTS

Axonal Localization of the GABA Dansporter Endogenously Expressed in PolarizedHippocampalNeuronsinCulturePrior to examining the distribution ofGABA transporter expressed by transfection in epithelial cells, we wished to determine whether this protein is restricted to the axolemma of An antibodydipolarized hippocampal neuronsinculture. rected against a synthetic peptide derived from the carboxylterminal sequence of the GABA transporter (R24) was provided by R. Jahn. Indirect immunofluorescence employing the R24 anti-GABA transporter antibody was carried out on7- and 14-day-old cultures of rat hippocampal neurons (gift ofG. Banker). The identity of dendritic processes was establishedby double labeling with anantibody directed against the dendritic marker MAP-2 (26) (Fig. 1, a and c ) . The distribution of the GABA transporter is shown in Fig. 1, b and d . In Fig. 1,a and b, two groups of 7-day neurons, consisting of four and three cells on the the right bottom and left sides, respectively, are recognizable by their MAP-2 staining. A long, thinand branched process emanates from one of the neuronson the left side of Fig. l b (empty arrowhead). This process is brightly stained with the GABA transporter antibody (arrows) and negative for the dendritic marker (compare arrows in b with a ) . The GABA transporter-positive process follows a dendrite along its entire length and seemsramify to twice in descending branches (asterisks) which surround the group of cells on the bottom right of the figure. The process extends beyond the tipof the colinear dendrite and terminates shortly after thesecond ramification (double arrows).The process is recognizable as an axon both by its morphology (i.e. it is thinner and longer than neighboring processes) and by its lack of MAP-2 staining. In dense neuronal cultures it difficult is to find dendrites that are entirely free of axons running along their surfaces. There are, however, processes recognizable as dendrites by both their morphology and MAP-2 reactivity which are clearly negative when stained with the GABAtransporterantibody R24 (filled arrowheads, compare a with b ) . Although some of the dendrites are stained with bothMAP-2 and R24, the patternsof staining with the two antibodies are quite distinct. MAP-2 immunostaining. appears to fill the cytoplasm, as would be expected for a cytoplasmic antigen. In contrast, the GAT-1 staining pattern is fibrillar and lacy, consistent withits origin in individual axons running along and criss-crossing over the surfaces of dendrites. In older neurons (Fig. 1,c and d ) the complex network of axons and axon bundles that originate at GABA transporter-positive neurons runs along mostof the dendriticsurfaces of the neuron depicted and are clearly detected by the GABA transporter antibody. It should be noted that only a small fraction (-10%) of the cultured hippocampal neurons exhibited positive staining for GABA transporter. This observation is consistent with the fact that only a minority population of hippocampal neurons is GABA-ergic (27). No staining of the occasional glial cells present in the primary cultures of the hippocampal neurons was ever noticed, consistent with the exclusively neuronal localization of the GAT-1 transporter (data notshown). Thus, in culture as well as in situ the GABA transporter is restricted in its distribution to the axonal plasma membrane (14). Apical Expression of the GABA Dansporter in Stably Dunsfected MDCK Cells-We next determined whether the sorting signals that specify this axonal localization would function to mediate apical targeting inpolarized epithelial cells. The :DNA encoding the rat brainGABA transporter (kindgift of B. Kanner) was subcloned into the pCB6 mammalian expression vector and used t o generate stably transfected MDCK cells, as

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GABA Dansporter Is Sorted to Epithelial ApicalMembranes

FIG.1. Immunolocalizationof the GABA transporter in polarized hippocampal neurons inculture. Hippocampal neurons cultured for 7 (panels a and h ) and 14 (panels c and d ) days were double labeled with a monoclonal antibody directed against MAF"2 (panels a and c ) and the GAFJA transporter-specific polyclonal antibody R24 (panels b and d ). Long and thin processes recognizable as axons by their morphology and negativity for MAP-2 are positively stained with R24 (arrows).The double arrows indicate an axon running along the surface of a dendrite and beyond the dendrite termination.MAP-2-positive processes (dendrites) canbe found which are devoid of GABA transporter staining (arrowheads). Empty arrowheads indicate the cell body of a neuron from which a GAFJA transporter-positive axon departs. Asterisks follow an axon's ramifications. Axon bundles are denoted by ax. Bar:panels a and b , 65 pm; panels c and d , 25 pm).

described under "Materials and Methods." MDCK cells are derived from the renal distal tubule and retain their parenttissue's polarized phenotype in culture (1-3). Cell lines stably expressing the GABA transporter were initially identified in GABA uptake assays, and their identities were confirmed by Western blotting. The GABA transporter antibodyused in these studies does not cross-react with any endogenous proteins in untransfected MDCK cells, as demonstrated both by immunofluorescence and Western blot analysis. When antibody R24 was used to probe Western blots of total cellular homogenates, a broad band of the expected molecular mass (-85 kDa) was detected only in materialderived from the transfected cells (data notshown).Similarly, no immunofluorescentlabeling could be detected when R24 was used to stain untransfected cells (not shown). Thus, theantibodies employed in these studies are specific for the GABA transporter and detect thisprotein expressed exogenously in epithelial cells. The subcellulardietribution of thc CARA trnnsporter in transfected MDCK cells was established by immunologic and functional assays. Double label immunofluorescence was carried out using tho antibody directed against the GABA transporter as well as an antibody specific for the a-subunit of the Na,K-ATPase (Fig. 2).Labeled cells were examined both enface

(Fig. 2,bottom) and inX Z cross-sections generated by confocal microscopy (Fig. 2,top). As can be seen in Fig. 2u, the GABA transporter is restricted to the apical surfaces of transfected MDCK cells (up). In contrast, the Na,K-ATPase retains its normal basolateral localization (28)(Fig. 2b), demonstrating that thetransfected cells remain appropriately polarized. The same predominantly apical localization has been revealed in experiments employing R21, an antipeptide polyclonal antibody directed against the amino terminus of GAT-1 (data not shown). .The apical localization of the GABA transporter was further supported hy the cell surface biotinylation experiments and by the flux studies presented in Figs. 3 and 4, respectively. The steady-state hiotinylation experiment presented in Fig. 3 demonstrates that the GABA transporter isavailable to cell surface biotinylation predominantly from the apical side. A broad band of the molecular mass expected of the maturefully glycosylated transporter ( 85 kDa) was readily detected in Western blots of material recovered from nvidin bend precipitntions when the NHS biotin reagent was added to theapical surface (panel A ). Little if any 85-kDa protein was detected when the biotinylation was performed at the basolateral surface (panel B ) . The same pattern wasobserved when the amino-terminal R21 an~

GABA Dansporter

Is SortedEpithelial to Apical

Membranes

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FIG.2. The GABA transporter is restricted to the apical plasma membranesof transfected MDCK cells. The GABA transporter (panel a ) and theNa,K-ATPase a-subunit (panel 6 ) were localized in transfectedMDCK cells by double label immunofluorescence. Confocal enface (panels a and 6 , bottom) and X Z cross-section (panels aand b , t o p ) are presented. When viewed enface, GABA transporter staining is seenbeto distributed in a punctate pattern characteristic of apical microvillar labeling (panel a, bottom).An apical labeling pattern is confirmed when the cells are examined incross-section (panel a, top). The Na,K-ATPase is clearly limitedto the lateral membranes(panel b,top and bottom). upand bl denote the apical and basolateral surfaces,respectively.

A B

300

T

T

W MDCKA ~

MDCKB

200 GAT-1 A

GAT-1 B

100

0

FIG.4. GABA transporter activity is predominantly apical in transfected MDCK cells. GABA influx was measured in untransfected (MDCK A and B )and transfected (GAT-I A and B 1 MDCK cells. FIG.3. Cell surface biotinylation of.GABA transporter ex- Transport was measured for 10 min a t room temperature in the prespressed in MDCK cells. MDCK cells expressing the GABA trans- ence of 10.20, or 40 p~[3HIGABA. GABAuptake wasfound to be linear porter were grown to confluence for 7 days on 0.45-pm polycarbonate with time for at least 30 min. Data are presented as pmol/min/mg of f S.E. of three independent filters and biotinylated from either the apical (lane A ) or the basolateral protein and represent the mean values (lane B ) side.Biotinylatedproteinswere recovered on streptavidin experiments performed in duplicate. beads, separatedby SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The GABA transporter was detected by probing For the experimentsdepicted in Fig. 4, MDCK cells grown on transfers with the R24 antibody. The band corresponding to mature, 13H1GABA fully glycosylated GAl3A transporter migrates with an apparent mo- permeable filter supports were incubatedwith lecular mass of 85 kDa and can be readily detected only when biotin added to either their apical or basolateralmedia compartlabeling is performed at theapical surface. The positions of molecular ments. Negligible specific uptake was detected at either surface mass markers areindicated at the left. They are (from top to bottom): of untransfected cells (MDCKA and B) at each of the GABA 97,64, and 43 kDa.

tibody was used (data not shown). Previousstudies inour laboratory have shown that under these biotinylation conditions the Na,K-ATPase a- and P-subunits are only detected at the basolateral surfaces of MDCK cells (23).

concentrations employed. In contrast, thetransfected cells mediated a rapid GABA influx, which was faster at the apical versus the basolateral surface(GAT1A and B ) . It is interesting to note that the ratio of apical to basolateral GABA uptake was larger at higher GABA concentrations. Although the ratio is

GABA Dansporter Is Sorted to Epithelial Apical Membranes

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-

100

100

80

80

I

60

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(P

c

0

c

0

40

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20

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r 4

P 19 4

m

P

0

19

4

3 FIG.5. Quantitation of the polarized distribution of the GABA transporter. The GABA transporter surface distributions determined functionally (GABAuptake A and E ) or through the biotinylation assay (Biotinylation A and B ) are presented above. GABA influx measurements from the apical ( A )or the basolateral ( E ) surfaces were camed out in the presence of 40 V M GABA for 10 min at room temperature. Values are presented as the percent of total cell surface transporter activity and represent means e S.E. of three independent experiments performed in duplicate. Quantitation of the accessibility of GABAtransporter to biotinylation from the apical or the basolateral surfaces was performed as described under “Materials and Methods.”Linearity of the biotinylation assay was determined by examining several dilutions of samples from labeled cells. Bars indicate S.E.

- 1.6 when the GABA concentration is 10 PM (which is close to the transporter’s expected K,,, of 4-5 1.1~(29)),the ratio is >2 at 40 p~ GABA. It remains to be determined whether this discrepancy is the product of technical factors relating to the geometry of the assay or if it infact represents actualdifferences in the properties of transporters localized t o the two surface domains. A comparison of measurements of functional and biochemical polarity is presented in Fig. 5. The apical to basolateral polarity ratio revealed by cell surface biotinylation experiments was determined by densitometric scanning of autoradiographs of Western blots, as described under “Materials and Methods.” Although steady-state cell surface biotinylation suggests a polarity ratio of -6.6, GABA uptake measurements indicate a ratio of -2.4, In spite of the method to method variation, however, these data demonstrate that the GABA transporter behaves as a predominantly apical protein when expressed by transfection in polarized MDCK cells.Finally, it should be noted that this phenomenon is not unique t o MDCK cells. We have found that LLC-PK1 cells (derived from the pig kidney

FIG.6. Betaine transporter activityis predominantly basolateral in stably transfectedMDCK cells. GABAuptake was measured in untransfected (MDCK-A and B ) and transfected (BGT-IA and E ) MDCK cells. Valuesrepresent three different experiments performed in duplicate for 30 min at room temperature with a GABA concentration of 100 PM.The assay is linear with time for at least 40 min. The S.E. is indicated by the bars.

cortex (30))also target the GABA transporter to their apical plasma membranes (data not shown). The Betaine Tkansporter Behavesas a Basolateral Protein in Tkansfected MDCK Cells-The Na,Cl-dependent betaine transporter is endogenously expressed at the basolateral surfaces of MDCK cells that have been exposed to hyperosmotic stress (25). Recent cloning studies demonstrate that this protein belongs to the GABA transporter family and is 50% identical to GAT-1 at the amino acid level(15).It has also been shownthat the betaine transporter can utilize GABA as a substrate for transport (15). In fact, the betaine transporter’s affinity for GABA (K,,, = -100 1.1~) is higher than that for betaine ( K , = -500 p ~ ) We . have used GABA uptake assays to assess the distribution of betaine transporterexpressed by transfection in MDCK cells that have been grown under conditions of normal osmolarity.As can be seen in Fig. 6, untransfected cells express low levels of endogenous GABA transport activity (see also Fig. 4). In contrast, cells transfected with a cDNA encoding the betaine transporter (kind giR of J. Handler) express readily detectable GABA uptake, -80% of which is present at thebasolateral surface. It would appear, therefore, that when expressed by transfection as well as under conditions of hyperosmolarity, the betaine transporter behaves as a basolateral membrane protein.

GABA Dansporter Is Sorted to Epithelial Apical Membranes DISCUSSION

Previous comparisons of neuronal and epithelial membrane protein sorting have examined the distributions in cultured hippocampal neurons of proteins whose epithelial sorting behavior had been established earlier (5, 6). The studies presented here have extended this approach by examining the sorting properties of a neuronal protein, the GAT-1 isoform of the Na,Cl-dependent GABA transporter family, exogenously expressed in epithelialcells. We first determined thelocalization of the neuronal transporter in mature, fully polarized hippocampal neurons. We find that expression of the transporter is restricted to theaxonal surfaces of the cultured neurons. This result is consistent withprevious observations on the distribution of GABA transporter in intactrat hippocampus (14). To compare the sorting behavior of the GABA transporter expressed in neurons and epithelial cells, we stably transfected an epithelialcell line (MDCK) with the cDNA encoding GAT-1. The subcellular distribution of the GABA transporter was established by both immunochemical and functional methods. Our results demonstrate thatMDCK cells express GAT-1 in a polarized manner and that the protein is concentrated predominantly at theapical plasma membrane. There is a discrepancy between the apical to basolateral polarity ratios determined by functional techniques, on the one hand,and biochemical methods on the other. When measured by GABA uptake assay, the distributionof the transporter appeared to be only about half as polarized as would be inferred from the results of the immunochemical experiments. Whether thisphenomenon reflects actual differences in the kinetic behavior of the transporters inserted in the two surface domains or is simply the product of assay conditions remains to be determined. In either event, our observations demonstrate thatMDCK cells accumulate theGABA transporter at their apical surfaces, consistent with the hypothesis that the mechanisms involved in axonal targeting in neurons are functionally related to those that mediate apical sorting in epithelia. In the context of this apparent rule equating the axonal and apical sortingpathways, it is interesting to note that the Na,K-ATPase comprises a puzzling exception. We have found that cultured hippocampal neurons express two isoforms of Both of these the Na,K-ATPase a-subunit (a1 and a3) (17). proteins are restricted to the basolateralsurfaces of polarized epithelial cells. Surprisingly, they are also present in the axonal aswell as in the dendriticdomains of the neuronal plasma membrane. This apparent inconsistency might be explained by the involvement of cytoskeletal elements in determiningor stabilizing membrane protein distributions. The Na,K-ATPase has been shown to interact directly with ankyrin and, consequently, to be associated with the fodrin-based subcortical cytoskeleton (32).Although ankyrin and fodrin are restricted to the basolateral surfaces of polarized MDCK cells (33),isoforms of these proteins havebeen found in both the axom and dendrites of neurons (34, 35). Studies on the targeting of the Na,K-ATPase suggest that in athyroid epithelial cell line (FRT) (36)as well as in at leastone clone of MDCK cells (23, 28)the sodium pump is delivered vectorially to the basolateral surface. Experiments performed on a different MDCK subclone, however, indicate that in this cell line Na,K-ATPase is randomly delivered to both cell surface domains (37).In this system, the sodium pump’s steady-state polarized distribution appears tobe the product of selective stabilization at the basolateral plasmalemma produced through interactions with the cytoskeleton (38).If Na,K-ATPase sorting in neurons is similarly dependent upon selective stabilization, the presence of cytoskeletal elements in both types of neuronal processes may explain the sodium pump’s nonpolarizeddistribution. It is

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possible, therefore, thattherelationship between neuronal and epithelial sorting may extend to a number of distinct classes of membrane proteins. Finally, it is worth noting that anothermember of the GABA transporter’s gene familyis endogenously expressed by MDCK cells. The epithelialcells of the renal distal tubule are bathed in an interstitialfluid which can become extremely hyperosmolar. To adapt to this environment, renal epithelialcells import as well as manufacture osmotically active compounds which allow the cells t o hold on to cytosolic water (39).Betaine, whose structure is related to that of GABA, serves as one of these osmolytes (40). Hyperosmotic stress induces distal tubule epithelial cells to synthesize a Na,Cl-dependent betaine carrier which is -50% identical at the amino acid level t o the GABA transporter (15, 40). Recent studies reveal that MDCK cells grown under hyperosmotic conditions express thisprotein and target it to their basolateral surfaces (25).Our data demonstrate that the betaine transporter is also basolateral when expressed by transfection in cells grown under normal osmotic conditions. Thus, despite their high degree ofhomology, the GABA and betaine transport systems are differentially sorted in epithelialcells. We are in the process of generating chimeras between these two transport systems. Analysis of the sorting behavior of the resulting constructs should allow us toidentify the residues that contribute to these molecules’ sorting signals and to determine if these signals are, in fact, identical in epithelia and in neurons. Acknowledgments-We are grateful to Drs. P. de Camilli, R. Jahn, G . Banker, B. Kanner, J. Handler, and J. Ahn for helpful discussions and for providing reagents. We also thank Dr. N. Borgese (in whose laboratory a portion of this work was completed) for hospitality and helpful suggestions. REFERENCES 1. Rodriguez-Boulan,E., and Nelson, W.J. (1989) Science 246,718-725 2. Simons, K., and Fuller, S. D. (1985)Annu. Reu. Cell Biol. 1, 295-340 3. Caplan, M. J., and Matlin, K. S. (1989) in Functionul EpithelialCellsin Culture (Matlin, K. S., and Valentich, J. D.,eds) pp. 71-127, Alan R. Liss. New York 4. Black, M. M., and Baas, P. W. (1989) 2hvd.s Neurosci. 12,211-214 5. Dotti, C. G., and Simons, K. (1990) Cell 62,63-72 6. Dotti, C. G . , Parton, R. G., and Simons, K. (1991) Nature 349, 15E-161 7. Iversen, L. L. (1971) Br J . Pharmacol. 41, 571-591 8. Radian, R., and Kanner, B. I. (1983) Biochemistry 22, 12361241 9. Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Kenyan, S., Miedel, M., Davidson, N., Lester, H., and Kanner, B. (1990) Science 249,1303-1306 10. Borden, L. A,, Smith, K. E., Hartig, P. R., Branchek, T. A., and Weinshank, R. L. (1992)J . Biol. Chem. 267,2109&21104 11. Clark, J. A., Deutch, A. Y., Gallipoli, P. Z., and Amara, S. G. (1992) Neuron 9, 337448 12. Liu, Q . R., Lopez-Corcuera,B., Mandiyan,S.,Nelson, H., and Nelson, N. (1993) J . Biol. Chem. 288,21OCM?112 13. Mabjeesh, N. J., Frese, M., Rauen, T., Jeserich, G., and Kanner, B. I. (1992) FEES Lett. 299.99-102 14. Radian, R., Ottersen, 0. P., Storm-Mathisen,J., Castel, M., and Kanner, B. (1990) J. Neurosci. 10, 1319-1330 15. Yamauchi, A., Uchida, S., Kwon, H. M., Preston, A. S., Robey, R. B., Garcia-Perez,A,, Burg, M. B., and Handler, J. S. (1992) J. Biol. Chem. 267, 649-652 16. Goslin, K., and Banker, G . (1991) in Culturing Nerue Cells (Banker, G . , and Goslin, K., eds) pp. 251-281, MIT Press, Cambridge, MA 17. Pietrini, G., Matteoli, M., Banker, G., and Caplan, M. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8414-8418 18. Lerner, R. A,, Green, H., Alexander, F., Liu, T., Sutcliffe, G . , and Shinnick, T. M. (1981)Proc. Natl. Acad. Sci. U. S. A . 78, 34033407 19. Gottardi, C. J., and Caplan, M. J. (1993)J . Cell B i d . 121, 283-293 20. Brewer, C. B., and Roth, M. G. (1991)J . Cell Biol. 114, 413421 21. Puddington, L., Woodgett, C., and Rose, J. K. (1987) Proc. Natl. Acad. Sei. U. S. A . 84,2756-2760 22. Sargiacomo,M., Lisanti, M., Graeve, L., Le Bivic,A,, and Rodriguez-Boulan,E. (1989) J . Membr Biol. 107, 277-286 23. Gottardi, C. J., and Caplan, M.J. (1993) Science 260, 552-554 24. Laemmli, U. K. (1970)Nature 227,680-685 25. Yamauchi, A,, Kwon, H. M., Uchida, S., Preston, A,, and Handler, J. S. (1991) Am. J. Physiol. 261, F197-F202 26. Caceres, A,, Banker, G. A., and Binder, L. (1986)J . Neurosci. 6, 714-722 27. Mugnaini, E., and Oertel, W. H. (1985) in Handbook of Chemical Neuroanatomy (Bjorklund,A., and Hokfelt, T.,eds) Vol. IV, Part I,Elsevier Science Publishing Co., Amsterdam

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