(Received for publication, September 13, 1995; and in revised form, December 20, 1995)
From the
At least three high affinity Na- and
Cl
-dependent
-aminobutyric acid (GABA)
transporters are known to exist in the rat and mouse brain. These
transporters share 50-65% amino acid sequence identity with the
kidney betaine transporter which also transports GABA but with lower
affinity. The betaine transporter (BGT) is expressed on the basolateral
surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent
evidence suggests that the signals and mechanisms involved in membrane
protein sorting share many functional characteristics in polarized
neurons and epithelial cells. It was previously shown that the rat GABA
transporter GAT-1 is located in the presynaptic membrane of axons where
it plays a role in terminating GABAergic neurotransmission. When
expressed in MDCK cells by transfection, GAT-1 was sorted to the apical
membrane. In this report, we have localized the other two GABA
transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA
uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like
GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like
BGT, localized to the basolateral membrane. We have also expressed BGT
in low density cultures of hippocampal neurons by microinjection and
immunolocalized it to the dendrites. The distribution of GAT-3 in these
neurons after transfection was axonal as well as somatodendritic. These
results indicate that highly homologous subtypes of GABA transporters
are sorted differently when expressed in epithelial cells or neurons
and suggest that these two cell types share the capacity to distinguish
among these isoforms and target them to distinct destinations.
Synaptic transmission by aminergic neurons is terminated through
the uptake of the neurotransmitter by specific sodium and chloride
dependent cotransporters located in the presynaptic membranes of
neurons(1, 2) . Following transport into the cell, the
neurotransmitters accumulate in synaptic vesicles and are re-released
during the next neurotransmission. GABA ()is an amino acid
which serves as the major inhibitory neurotransmitter in the vertebrate
central nervous system(3, 4) . The cell surface
transporter responsible for GABA re-uptake was cloned (5) and
subsequently proved to be the first member of a large gene family.
Other members of this family include the transporters which function in
the presynaptic uptake of the biogenic amines
norepinephrine(6) , serotonin(7, 8) , and
dopamine(9, 10, 11) , as well as the
transporters for the amino acids taurine(12, 13) ,
glycine(14, 15, 16) , and
proline(17) . These transporters are proposed to span the
membrane 12 times, with both amino and carboxyl termini in the
cytoplasm. Another family member, the Na
- and
Cl
-dependent betaine transporter, has been cloned
from MDCK cells (18) and human brain(19) . The canine
betaine transporter is a basolateral membrane protein which protects
cells in the renal medulla from hypertonicity by mediating the uptake
of the osmolyte betaine(20) . GABA is also a substrate for the
betaine transporter. In fact, the transporter's affinity for GABA (K
100 µM) is higher
than its affinity for betaine (K
500 µM). More recently, the cDNAs for several subtypes of
GABA transporters have been isolated from mouse and rat brain, bringing
to four the number of GABA transporter isoforms which have been
identified(21, 22, 23) . Two of the high
affinity (K
10 µM) rat
GABA transporters, GAT-2 and GAT-3 (also known as GAT-B), share higher
amino acid identity (68% and 65%, respectively) with the betaine
transporter than with GAT-1 (52% amino acid identity).
Recently, we have examined the sorting behaviors of members of this family expressed by transfection in polarized epithelial cells(24) . The plasma membrane of an epithelial cell is divided into an apical membrane, which frequently faces a lumen, and a basolateral membrane which is in contact with the extracellular fluid space. The protein and lipid compositions of these two membrane domains are quite different (25, 26) . Neurons are also polarized cells whose plasma membranes can be divided into two domains: axons (including termini) and the somatodendritic surfaces (composed of the cell body and the dendrites). Axons are thin and usually extend great distances from the cell body while dendrites are thick at the base, tapered and shorter in length. The axonal and dendritic processes must be biochemically distinct in order for them to receive and transmit information in a highly regulated manner. Very little is known about the mechanism of generating and maintaining polarity in neurons. Dotti and Simons (27) proposed that the mechanisms involved in sorting proteins to the axonal and somatodendritic membranes are similar to those required to sort proteins to the apical and basolateral membranes, respectively, of polarized epithelial cells. When cultured hippocampal neurons were infected with the influenza fowl plague virus or vesicular stomatitus virus, these virus' major glycoproteins were targeted to distinct membrane domains just as they are in infected epithelial cells. The influenza hemagglutinin (HA), which is targeted to the apical membrane in MDCK cells, was predominantly sorted to the axon. In contrast, the vesicular stomatitus virus G protein, which accumulates in the MDCK basolateral membrane, was sorted to the dendrites. Furthermore, Thy-1, a glycophosphorylinositol-linked protein, was sorted to the axon in hippocampal neurons (28) whereas glycophosphorylinositol-linked proteins are generally present in the apical membranes of epithelial cells. The small GTP-binding protein rab8 (29) and the transferrin receptor (30) are endogenously expressed in the basolateral compartment of MDCK cells and in the somatodendritic domain of neurons.
In order to examine this model further, Pietrini et al.(24) studied the sorting behavior of the neuronal GABA transporter (GAT-1) in both neuronal and epithelial cells. GAT-1 was localized exclusively to the axons of hippocampal neurons in culture (24) and in situ(31) and to the apical membranes of transfected MDCK cells. The betaine transporter, expressed by transfection, was basolateral in MDCK cells, in keeping with previous studies on the polarity of the MDCK cell's endogenous complement of betaine transporters(20) . To characterize further the nature and position of the sorting information which mediates the differential targeting of these proteins, we have expressed cDNAs encoding the highly homologous GAT-2 and GAT-3 transporters in MDCK cells. We find that in stably transfected MDCK cells, GAT-2 is expressed predominantly in the basolateral membrane while GAT-3 is expressed in the apical membrane. We have also expressed the basolateral betaine transporter protein in hippocampal neurons by microinjection and found that it accumulates in the dendrites. GAT-3, on the other hand, was found in both axons and the somatodendritic membrane. These results indicate that highly homologous subtypes of GABA transporters are distributed differently when expressed in epithelial cells and suggest that these transporters require distinct subcellular distributions in order to subserve their physiologic functions. Moreover, the apical/axonal and basolateral/dendritic sorting model proposed by Dotti and Simons (27) is consistent with the behaviors manifest by this group of membrane proteins.
Figure 1:
GABA
uptake by MDCK cells stably expressing GAT-2 or GAT-3. Cells were
transfected with pCB6 vector alone, pCB6-GAT2 (GAT2), or
pCB6-GAT3 (GAT3) using the calcium phosphate method. Cells
were plated on 6.5-mm Transwell inserts 1 week before measuring GABA
uptake. PBS containing 50 nM [
H]GABA was added either to the apical (open columns) or basolateral (filled columns) side
of the cell monolayer. After 10 min, the cells were washed in ice-cold
PBS
, solubilized in 1% SDS, and counted in Optifluor.
Nonspecific uptake was determined in the presence of 0.5 mM unlabeled GABA and subtracted from the total uptake to determine
specific uptake (expressed as picomole/mg cellular protein). For pCB6,
GAT2#70, and GAT3#3 cell lines, the mean values of three separate
experiments, each done in duplicate, are shown with error bars to indicate S.D. Columns without error bars are the
averages of duplicate values from a single
experiment.
We wished to confirm the apparent polarized distributions of these transporters using a method to localize the expressed proteins which is independent of their transport capacities since functional assays only report the distribution of the active populations. We chose, therefore, to complement the transport assay with antibody based detection methods. Two polyclonal antibodies (R22 and R23) raised against peptides from GAT-1 were tested for their ability to cross-react with GAT-2 and GAT-3. In order to express high levels of transporter for antibody characterization studies, we transiently transfected COS-1 cells with GAT-2 or GAT-3 cDNAs and measured antibody binding by indirect immunofluorescence. R22 recognized GAT-2 and GAT-3 in COS-1 cells (Fig. 2, a and b); however, the intensity of fluorescence was considerably lower for the GAT-3 transfected COS-1 cells (Fig. 2b). Antibody R23 recognized neither GAT-2 nor GAT-3 (not shown). Although cell surface labeling was clearly visible, most of the transporters were localized intracellularly, probably in the endoplasmic reticulum and Golgi apparatus. This pattern of staining is often seen in COS-1 cells induced to overexpress membrane proteins. The GAT-3 specific antibody, anti-670, labeled the surface as well as intracellular membranes of COS-1 cells expressing GAT-3 (Fig. 2c). This antibody did not react with cells expressing GAT-2 (not shown).
Figure 2: An anti-GAT-1 antibody (R22) cross-reacts with GAT-2 and GAT-3 by immunofluorescence in COS-1 cells. COS-1 cells transiently expressing GAT-2 (a) or GAT-3 (b and c) were fixed in methanol, permeabilized, and stained with R22 (a and b) or anti-670 (c) followed by a fluorescein isothiocyanate-coupled anti-rabbit IgG secondary antibody. Left panels, immunofluorescence images. Right panels, corresponding phase-contrast micrographs. The distribution of the GABA transporter is mainly intracellular (endoplasmic reticulum and Golgi) as indicated by the bright staining around the cell nucleus. Cell surface labeling, which is marked by uniform staining extending to the cell boundary, is seen in a and c. Bar, 25 µm.
When stably transfected MDCK cells expressing GAT-2 were labeled with R22, a lateral staining pattern was observed (Fig. 3, A and C), similar to the pattern obtained with an antibody against the Na,K-ATPase, a known basolateral protein (Fig. 3, B and D). We discovered that MDCK cells transfected with GAT-3 did not stain with R22, so anti-670 had to be used for immunofluorescence. With the latter antibody, we observed bright staining of the apical membrane of MDCK cells transfected with GAT-3 (Fig. 3, E and G) while the Na,K-ATPase was distributed basolaterally in the same cells (Fig. 3, F and H).
Figure 3:
Differential localization of GAT-2 and
GAT-3 in transfected MDCK cells by immunofluorescence. Confluent
monolayers on 0.4-µm filters were fixed in methanol, permeabilized,
and double-labeled with anti-GABA transporter (R22 or anti-670) and
anti-Na,K-ATPase subunit (6H) antibodies. Cells transfected with
GAT-2 (A-D) were double-labeled with R22 and 6H; cells
transfected with GAT-3 (E-H) were double-labeled with anti-670
and 6H. After washing to remove excess primary antibodies, the cells
were incubated with fluorescein isothiocyanate anti-rabbit IgG (left panels) and rhodamine anti-mouse IgG (right
panels). En face (A, B, E, and F) and xz
cross-sectional (C, D, G, and H) images were obtained
using a confocal microscope. Arrows indicate the positions of
apical (ap) and basolateral (bl) surfaces. GAT-2
labeling was restricted to the basolateral membrane (A and C) while anti-GAT-3 antibody labeled the apical membrane (E and G).
Finally, we performed cell surface biotinylation experiments to confirm the polarity of GAT-2 and GAT-3 in MDCK cells. Fig. 4A shows that GAT-2, an 85-kDa protein, was accessible to biotinylation predominantly from the basolateral side of intact MDCK cells. In contrast, GAT-3 was preferentially biotinylated from the apical side. The Na,K-ATPase was biotinylated from the basolateral surface in all three cell lines (Fig. 4B), indicating that these cells are properly polarized. These data are consistent with the transport and microscopy results. We conclude, therefore, that GAT-2 behaves as a basolateral protein when expressed by transfection in MDCK cells, whereas its homologue GAT-3 accumulates in the apical plasma membrane.
Figure 4:
Steady state biotinylation of MDCK cells
expressing GAT-2 or GAT-3. Cells transfected with pCB6 alone, GAT-2, or
GAT-3 were grown on 24-mm Transwell filters (0.4 µm) and treated
with 10 mM sodium butyrate 24 h before the experiment (to
boost the expression of the exogenous proteins). NHS-biotin was added
to either the apical (ap) or basolateral (bl) media
compartments. After biotinylation, cells were solubilized and
streptavidin-agarose was used to isolate biotinylated proteins which
were then separated on an 8.5% polyacrylamide gel, transferred to
nitrocellulose, and probed with anti-GABA transporter antibody R22 (A) or anti-Na,K-ATPase monoclonal antibody 6H (B).
The positions of prestained molecular weight standards (M
10
) are shown on the left. GAT-2 was mainly biotinylated from the basolateral
surface while GAT-3 was biotinylated from the apical surface. The
Na,K-ATPase was basolaterally localized in all of these cell lines (B), demonstrating that transfection does not alter these
cells' characteristic membrane
polarity.
Figure 5: The c-Myc-tagged betaine transporter is expressed in the basolateral membrane of transfected MDCK cells. En face (top) and xz section (bottom) confocal images show that the betaine transporter is restricted to the basolateral membrane. Cells were processed for immunofluorescence as for Fig. 3except that the antibody to c-Myc was used to detect the transporter. Betaine transport activity has previously been measured at the basolateral surface of MDCK cells(20, 24) ; this distribution is confirmed by immunolocalization of the c-Myc-tagged transporter. ap, apical surface; bl, basolateral surface.
Figure 6:
Colocalization of the betaine transporter
and MAP2 in the dendrites of cultured hippocampal neurons. Neurons
after 11 days in culture were microinjected with pCB6-BGT and double-labeled with anti-c-Myc (BGT*) and anti-MAP2 (MAP2) antibodies. The betaine transporter (BGT*) was
expressed in the same processes as the dendritic marker MAP2. The arrows point to axons which are visible in the phase-contrast
micrograph (bottom panel) but do not stain with either
antibody. From this experiment, it is difficult to determine if the
axon originates from the injected cell or from its neighbors. Bar, 10 µm.
Figure 7: Expression of the betaine transporter in neurons co-injected with pCB6-BGT* and fluorescein/dextran. Neurons were injected after 8 days in culture and double-labeled for the betaine transporter (BGT*) and synapsin (SYN). Fluorescein/dextran (DEX) labeled the axon (indicated by arrows) and most dendrites (arrowheads) of the injected cell. Synapsin is found in the synaptic vesicles of axons(43) . Sites of synaptic contact between the axon and somatodendritic membrane are indicated by bright spots which are characteristic of synapsin labeling (SYN). These as well as other dendrites which are devoid of synaptic contacts expressed the betaine transporter (BGT*). The axon (arrows) did not express the transporter.
Some of the injected cells
were double-labeled with anti-c-Myc and an antibody against the
synaptic vesicle protein, synapsin(43) . Synapsin was found in
the axons (of both injected and uninjected cells) and appeared as
bright spots at sites of synaptic contact with the cell body and
dendrites (Fig. 7, SYN). The arrowheads point
to processes which express BGT. Three of the six short
BGT
-labeled processes are most likely dendrites since they
do not contain synapsin. The other three processes are labeled by
synapsin but they could be dendrites in contact with neighboring axons.
Figure 8: Expression of hemagglutinin in the axons of cultured hippocampal neurons by microinjection. Neurons after 17 days in culture were microinjected with pCB6-HA and double-labeled with anti-HA (HA) and anti-MAP2 (MAP2) antibodies. The arrow indicates the axon which is negative for MAP2. HA was found in the axon in addition to being present in the cell body and dendrites. The insets show another cell whose thin axon (arrows) runs along the edges of the main dendrite (labeled with MAP2; arrowheads). Bar, 15 µm.
Figure 9: Expression of GAT-3 in the axons of cultured hippocampal neurons. Neurons after 8 days in culture were co-injected with pCB6-GAT3 and fluorescein/dextran. The cells were double-labeled with anti-670 (top left panel) and monoclonal anti-MAP2 (bottom left panel) antibodies followed by rhodamine-conjugated anti-rabbit IgG (GAT3) and Cy5 anti-mouse IgG (MAP2). Immunofluorescence was examined by confocal microscopy. GAT-3 is expressed in the long, thin axon (arrows) originating from the injected cell marked by fluorescein/dextran (DEX). MAP-2 positive dendrites are indicated by arrowheads. Bottom right, phase-contrast micrograph of same cell shown in the confocal images.
These results suggest that certain proteins (e.g. betaine transporter) are restricted to the somatodendritic membrane while others (HA and GAT-3) are expressed in both somatodendritic and axonal membranes. Moreover, apical membrane proteins appear to be transported to axons while basolateral membrane proteins remain in the somatodendritic membrane when expressed in neurons.
We have previously shown that two members of the
neurotransmitter transporter gene family, GAT-1 and the betaine
transporter, are sorted to two different membrane domains in MDCK
cells(24) . It should be noted that the betaine transporter can
also transport GABA but with a lower affinity. In this report, we show
that the two most recently cloned GABA transporters, GAT-2 and GAT-3,
are also differentially localized in MDCK cells according to both
functional and biochemical criteria. GAT-3, like GAT-1, was expressed
on the apical surface of transfected MDCK cells. GAT-2, like the
betaine transporter, was expressed mainly on the basolateral surface of
MDCK cells. The construction of chimeric proteins consisting of
sequences from GAT-3 and GAT-2, which share 65% sequence identity,
should help us to determine the sorting signals which mediate the
subcellular targeting of members of this family of proteins.
In the context of our data on sorting, it is interesting to consider the tissue specific expression of the GABA transporter isoform mRNAs by Northern blot analysis. GAT-1 and GAT-3 are expressed in brain and retina, while GAT-2 mRNA is found in liver and kidney as well as the brain and retina(21) . It would appear, therefore, that both of the exclusively neuronal forms of the GABA transporter behave as apical proteins, while the isoforms endogenously expressed in epithelia are primarily basolateral. The functional significance of these non-neuronal GABA transporters is not known. Presumably, however, epithelial GAT-2 may function like the betaine transporter to import a solute from the serum. Thus, we expect that this protein is basolaterally disposed in the tissues which express it endogenously.
To study the sorting mechanism in neurons, we have developed a method of transfecting primary cultures of embryonic hippocampal neurons. Primary cultures of neurons are difficult to transfect with DEAE-dextran, calcium phosphate, or cationic lipids, methods commonly used to transfect other mammalian cells in culture. High efficiency transfection of neurons has been accomplished using Herpes Simplex or Semliki Forest viral vectors(44, 45) ; however, there are problems associated with introducing viral components. Hippocampal neurons do not survive for more than 8 h following viral infection (45) and may be subjected to alterations of cellular physiology which could affect the polarized sorting of neuronal proteins. For example, in cultured dorsal root ganglion and spinal cord neurons, the normally somatodendritic MAP2 appears in axons after rotavirus infection(46) . The introduction of foreign DNA into the nucleus of neurons by microinjection should cause little damage to the cells since the plasma membrane is expected to immediately re-seal by a calcium-dependent mechanism following puncture(47) .
We expressed the renal betaine transporter by microinjection of cultured hippocampal neurons and found that this basolateral membrane protein is sorted exclusively to the somatodendritic membrane. Next, we expressed influenza hemagglutinin by microinjection and found that, unlike the betaine transporter, this protein was also delivered to axons. Finally, we expressed the GABA transporter GAT-3 in hippocampal neurons and noted that, like HA, it was transported to the axon as well as being present in the cell body and dendrites. GAT-3 would be expected to reside in presynaptic membranes if its role in the brain, like that of GAT-1, involves neurotransmitter uptake from the synaptic cleft following neurotransmission. The subcellular distribution of GAT-2 in neurons has yet to be determined. However, in light of its preference for the basolateral membrane when expressed in MDCK cells, we can predict that GAT-2 will be sorted like BGT, i.e. to the dendrites.
Our results are consistent with the hypothesis that apical and basolateral sorting signals are interpreted as axonal and dendritic targeting signals, respectively, by hippocampal neurons. While the betaine transporter was expressed exclusively in the somatodendritic membrane, influenza HA and GAT-3 were less polarized. The latter two proteins were found in dendrites as well as axons. Dotti and Simons (27) also found some HA in the dendrites of influenza-infected neurons. The presence of axonal proteins in processes resembling dendrites may be explained in part by the close proximity of axons and dendrites, since axons often run along the surfaces of dendrites and may be superimposed upon these MAP2-positive processes (Fig. 8, insets). It is also possible that there may actually be expression of the HA and GAT-3 proteins in the membranes of dendrites, especially when these proteins are overexpressed in transfected neurons. It is possible, for example, that if the axonal delivery system becomes saturated, proteins with axonal sorting information would be shunted to the somatodendritic surface.
Since the Dotti and Simons (27) model has been proposed, the
sorting of several epithelial and neuronal proteins have been examined
in both cell types. At least two proteins do not abide by this model.
The amyloid protein precursor is axonal in cultured hippocampal neurons (48) but basolateral in MDCK cells(49) . The amyloid
protein precursor sequence includes a tyrosine-based internalization
signal. Recent evidence suggests that proteins containing the YXRF
motif may be targeted to the cell's most endocytically active
domain(34) . In the case of MDCK cells, the preferred domain
would be basolateral. Therefore, when expressed in MDCK cells, the
internalization signal of amyloid protein precursor may overide the
axonal/apical sorting signal and target the protein to the basolateral
surface. In neurons, both axons and dendrites participate in
endocytosis (50) and the internalization signal might not be a
dominant sorting signal. In keeping with this hypothesis the
transferrin receptor, which manifests both an autonomous basolateral
sorting signal as well as an endocytosis motif, is only present in the
somatodendritic membrane of cultured neurons(30) . The 1
and
3 isoforms of the Na,K-ATPase are found in both the axons and
dendrites of cultured hippocampal neurons (32) although these
proteins are polarized to the basolateral membranes of MDCK cells.
Further study of membrane protein dynamics in neurons and epithelia
will be required to understand fully the similarities and differences
relating sorting in these cell types.
Our data, taken together with that of Pietrini et al.(24) , demonstrate that the four members of a highly homologous family of transport proteins are differentially distributed in epithelial cells and neurons. Attainment of the proper subcellular distribution almost certainly plays an important role in ensuring the correct function of these transporters in situ. It will be important, therefore, to identify the sorting information embedded in these proteins and to determine its role in their function. Once these sorting signals are determined, the techniques presented here can be employed to ascertain whether the same signals are recognized by the neuronal and epithelial sorting machinery.