The orphan transporter Rxt1/NTT4 (SLC6A17) functions as a synaptic vesicle amino acid transporter selective for proline, glycine, leucine, and alanine.

Abstract

Rxt1/NTT4 (SLC6A17) belongs to a gene family of "orphan transporters" whose substrates and consequently functions remain unidentified. Although Rxt1/NTT4 was previously thought to function as a sodium-dependent plasma membrane transporter, recent studies localized the protein to synaptic vesicles of glutamatergic and GABAergic neurons. Here, we provide evidence indicating that Rxt1/NTT4 functions as a vesicular transporter selective for proline, glycine, leucine, and alanine. Using Western blot, immunoprecipitation, immunocytochemistry, and polymerase chain reaction approaches, we demonstrate that PC12 cells express the Rxt1/NTT4 gene and protein. Small interfering RNA (siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells resulted in selective reductions in uptake levels for proline, glycine, leucine, and alanine. Likewise, gas chromatography analysis of amino acid content in an enriched synaptic vesicle fraction from wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that proline, glycine, leucine, and alanine levels were decreased in siRNA-treated cells compared with wild-type cells. Furthermore, Rxt1/NTT4-transfected Chinese hamster ovary (CHO) cells exhibited significant uptake increases of these amino acids compared with mock-transfected CHO cells. Finally, proline uptake in both PC12 cells and Rxt1/NTT4-transfected CHO cells was dependent on the electrochemical gradient maintained by the vacuolar-type H(+)-ATPase. These data indicate that the orphan Rxt1/NTT4 protein functions as a vesicular transporter for proline, glycine, leucine, and alanine, further suggesting its important role in synaptic transmission.

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The Orphan Transporter Rxt1/NTT4 (SLC6A17) Functions
as a Synaptic Vesicle Amino Acid Transporter Selective
for Proline, Glycine, Leucine, and Alanine
Leonardo A. Parra, Tracy Baust, Salah El Mestikawy, Marisol Quiroz, Beth Hoffman,1
Jack M. Haflett, Jeffrey K. Yao, and Gonzalo E. Torres
Departments of Neurobiology and Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (L.A.P.,
T.B., M.Q., G.E.T.); Institut National de la Sante ´ et de la Recherche Me ´dicale, U513, Universite ´ Pierre et Marie Curie, and
Universite ´ Pierre et Marie Curie Universite ´ Paris 06, Neurobiologie et Psychiatrie, Paris, France (S.E.M.); and Douglas Hospital
Research Center, Department of Psychiatry, McGill University, Montre ´al, Quebec, Canada (S.E.M.); Department of
Neuroscience, Amgen, Inc., Thousand Oaks, California (B.H.); Medical Research Service, VA Pittsburgh Healthcare System,
Pittsburgh, Pennsylvania (J.M.H., J.K.Y.); and Department of Psychiatry, Western Psychiatric Institute and Clinic, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (J.K.Y.); and Department of Pharmaceutical Sciences, University of
Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania (J.K.Y.)
Received June 25, 2008; accepted September 3, 2008
ABSTRACT
Rxt1/NTT4 (SLC6A17) belongs to a gene family of “orphan trans-
porters” whose substrates and consequently functions remain
unidentified. Although Rxt1/NTT4 was previously thought to func-
tion as a sodium-dependent plasma membrane transporter, re-
cent studies localized the protein to synaptic vesicles of glutama-
tergic and GABAergic neurons. Here, we provide evidence
indicating that Rxt1/NTT4 functions as a vesicular transporter
selective for proline, glycine, leucine, and alanine. Using Western
blot, immunoprecipitation, immunocytochemistry, and polymer-
ase chain reaction approaches, we demonstrate that PC12 cells
express the Rxt1/NTT4 gene and protein. Small interfering RNA
(siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells re-
sulted in selective reductions in uptake levels for proline, glycine,
leucine, and alanine. Likewise, gas chromatography analysis of
amino acid content in an enriched synaptic vesicle fraction from
wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that pro-
line, glycine, leucine, and alanine levels were decreased in
siRNA-treated cells compared with wild-type cells. Further-
more, Rxt1/NTT4-transfected Chinese hamster ovary (CHO)
cells exhibited significant uptake increases of these amino ac-
ids compared with mock-transfected CHO cells. Finally, proline
uptake in both PC12 cells and Rxt1/NTT4-transfected CHO
cells was dependent on the electrochemical gradient main-
tained by the vacuolar-type H?-ATPase. These data indicate
that the orphan Rxt1/NTT4 protein functions as a vesicular
transporter for proline, glycine, leucine, and alanine, further
suggesting its important role in synaptic transmission.
Neurotransmitter transporters play a crucial role in the
regulation of neuronal transmission by removing transmit-
ters from the synaptic cleft and loading synaptic vesicles for
subsequent release. Based on their subcellular localization,
neurotransmitter transporters can be classified as either
plasma membrane or vesicular transporters. Plasma mem-
brane transporters mediate high-affinity uptake of released
transmitters into pre- and/or postsynaptic sites and glia cells
(for review, see Torres and Amara, 2007). Molecular cloning
techniques have further divided these plasma membrane
transporters into two major groups based on amino acid
homology. The first group is made up of transporters for
dopamine (DA), norepinephrine, serotonin, GABA, and gly-
cine, all of which contain 12 transmembrane putative span-
ning domains and transport their substrates in a sodium-
and chloride-dependent manner (Nelson, 1998). The second
group includes glutamate transporters, for which five genes
This work was funded in part by Amgen research agreement 200620123.
1Current affiliation: Vertex Pharmaceuticals, Inc., San Diego, CA.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.108.050005.
ABBREVIATIONS: FITC, fluorescein isothiocyanate; HEK, human embryonic kidney; CHO, Chinese hamster ovary; FBS, fetal bovine serum; PBS,
phosphate-buffered saline; PCR, polymerase chain reaction; siRNA, small interfering RNA; PNS, postnuclear supernatant; NaGluc, sodium
gluconate; RT, reverse transcription; DA, dopamine; GFP, green fluorescent protein; VMAT, vesicular monoamine transporter; VAChT, vesicular
acetylcholine transporter; VGLUT, vesicular glutamate transporter; VIAAT, vesicular inhibitory amino acid transporter; V-ATPase, vacuolar-type
H?-ATPase; NMDA, N-methyl-D-aspartic acid; GlyT, glycine transporter; WT, wild type.
0026-895X/08/7406-1521–1532$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 74:1521–1532, 2008
Vol. 74, No. 6
50005/3404937
Printed in U.S.A.
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have been identified (Seal and Amara, 1999). In contrast,
vesicular transporters package neurotransmitters into syn-
aptic vesicles through a mechanism involving a proton elec-
trochemical gradient (Chaudhry et al., 2008a,b). To date,
molecular cloning approaches have identified vesicular
transporters for monoamines (Erickson et al., 1992; Liu et
al., 1992), acetylcholine (Roghani et al., 1994), GABA (Mc-
Intire et al., 1997; Sagne ´ et al., 1997), and glutamate (Ni et
al., 1994; Aihara et al., 2000; Bellocchio et al., 2000; Taka-
mori et al., 2000, 2001, 2002; Bai et al., 2001; Fremeau et al.,
2001, 2002; Herzog et al., 2001; Gras et al., 2002; Scha ¨fer et
al., 2002).
In addition to these transporters with established substrates,
several groups have cloned a family of putative transporter
proteins whose substrates and consequently functions remain
unidentified (Uhl et al., 1992). These proteins, referred to as
“orphan transporters,” include Rxt1/NTT4 (Liu et al., 1993; el
Mestikawy et al., 1994), XT2/ROSIT (Wasserman et al., 1994),
XT3/rB21a (Smith et al., 1995), NTT5 (Farmer et al., 2000), and
v7-3/NTT7 (UhI et al., 1992; Sakata et al., 1999; Farmer et al.,
2000). Although these orphan transporters share approxi-
mately 30 to 65% homology among themselves and approxi-
mately 30 to 45% homology to the sodium- and chloride-depen-
dent plasma membrane transporters, their tissue distributions
andsubcellularlocalizationshavebeenfoundtobeverydiverse.
XT2/ROSIT, XT3/rB21a, and NTT5 are found predominantly in
peripheral tissues (Wasserman et al., 1994; Smith et al., 1995;
Farmer et al., 2000), whereas Rxt1/NTT4 and v7-3/NTT7 are
expressed predominantly in brain (UhI et al., 1992; Liu et al.,
1993; el Mestikawy et al., 1994; Masson et al., 1996; Farmer et
al., 2000). Indeed, Rxt1/NTT4 is present primarily in glutama-
tergic and GABAergic neurons (el Mestikawy et al., 1994, 1997;
Masson et al., 1995). Furthermore, biochemical and immunocy-
tochemical studies revealed an unexpected synaptic vesicle lo-
calization for Rxt1/NTT4 in glutamatergic neurons (Fischer et
al., 1999; Masson et al., 1999). Based on these observations, it
has been suggested that Rxt1/NTT4 might function as a vesic-
ular transporter, or alternatively, reside in intracellular vesi-
cles as a reserve pool from which they need to be translocated to
the plasma membrane to become functional. In this study, we
provide evidence supporting the contention that the orphan
Rxt1/NTT4 transporter exists and functions as a vesicular
amino acid transporter with selectivity for proline, glycine,
leucine, and alanine. The physiological significance of our find-
ings is discussed.
Materials and Methods
Materials. The polyclonal antibody against Rxt1/NTT4 was gen-
erated by immunizing rabbits with a fusion protein derived from the
carboxyl-terminal domain of the rat Rxt1/NTT4 (amino acids 304–
322) and affinity purified as described in Masson et al. (1995). The
monoclonal anti-synaptophysin antibody was obtained from BD Bio-
sciences (San Jose, CA), whereas antibodies against calnexin and the
transferrin receptor were from Millipore Corporation (Billerica, MA)
and Zymed Laboratories (South San Francisco, CA), respectively.
Peroxidase- and FITC-conjugated secondary antibodies were pur-
chased from GE Healthcare (Chalfont St. Giles, Buckinghamshire,
UK), whereas Texas Red-conjugated antibody was purchased from
Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).
Sprague-Dawley rats were supplied by Hilltop Laboratory Animals,
Inc. (Scottsdale, PA), and C57BLC mice were supplied by The Jack-
son Laboratory (Bar Harbor, ME). Cell culture media were from
Invitrogen (Carlsbad, CA), and protease inhibitors were from Pierce
Chemical (Rockford, IL). Radiolabeled substrates were from Perkin-
Elmer Life and Analytical Sciences (Waltham, MA). All other
materials were from Sigma-Aldrich (St. Louis, MO) unless stated
otherwise.
Cell Culture. Human embryonic kidney (HEK) 293, the human
neuroblastoma cell line SKNSH, PC12, and Chinese hamster ovary
(CHO) cells were purchased from American Type Culture Collection
(Manassas, VA). HEK293 cells were cultured in minimal essential
medium supplemented with 10% fetal bovine serum (FBS), 1 mM
glutamine, and 50 ?g/ml each penicillin and streptomycin at 37°C in
a humidified, 5% CO2incubator. SKNSH and PC12 cells were main-
tained in Dulbecco’s modified Eagle’s medium supplemented with 5%
FBS, 5% horse serum, 1 mM glutamine, and 50 ?g/?l each of peni-
cillin and streptomycin at a 37°C in a humidified, 10% CO2incuba-
tor. CHO cells were cultured in F-12 ? Glutamax, 5% FBS, and 50
?g/?l each penicillin and streptomycin at a 37°C in a humidified, 5%
CO2incubator. If necessary, cells were transfected with 5 ?g of DNA
using Transfectin (Bio-Rad Laboratories, Hercules, CA) according to
the manufacturer’s recommendations. Transfected cells were al-
lowed to grow for an additional 48 h before further studies.
Preparation of Brain and Cell Lysates. Cells, striata, or whole
brain from rats or mice were homogenized in radioimmunoprecipi-
tation assay buffer (100 mM Tris, 150 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 1% deoxycholate, and 0.1% SDS) containing protease
inhibitors and incubated with rotation for 1 h at 4°C. Brain lysates
were centrifuged at 16,000g for 10 min and at 20,000g for 60 min at
4°C, whereas cell lysates were centrifuged at 13,000g for 10 min at
4°C. The final supernatants were then collected, and protein concen-
trations were measured using the DC Protein Assay (Bio-Rad) and
used in subsequent experiments.
Immunoprecipitations. PC12 cell lysates were incubated with
either preimmune serum or anti-Rxt1/NTT4 (1:200) antibody over-
night at 4°C. Then, 40 ?l of protein A-Sepharose (GE Healthcare)
was added to all samples. Samples were incubated again for 1 h at
4°C and centrifuged at 13,000g for 1 min. The resulting pellets were
washed twice with radioimmunoprecipitation assay buffer and twice
with PBS. The immunoprecipitated proteins were eluted using
Laemmli sample buffer (Bio-Rad) containing 10% ?-mercaptoetha-
nol. Then, samples were heated at 37°C for 30 min and resolved by
polyacrylamide gel electrophoresis and Western blot.
Western Blot Analysis. Samples were separated by polyacryl-
amide gel electrophoresis on 10% Tris-HCl polyacrylamide gels and
TABLE 1
Primers used to amplify Rxt1/NTT4 in RT-PCR
Position Primer SequenceExpected Size
bp
475–2658
Forward
Reverse
475–2648
Forward
Reverse
475–2112
Forward
Reverse
475–2112
Forward
Reverse
718–2112
Forward
Reverse
2401–2658
Forward
Reverse
475–678
Forward
Reverse
2184
5?-ATGCCGAAGAACAGCAAGGTG-3?
5?-TCACAGCTCTGACTCAGGGGT-3?
2174
5?-ATGCCGAAGAACAGCAAGGTG-3?
5?-GACTCAGGGGTGCTGGCCAAG-3?
2174
5?-ATGCCGAAGAACAGCAAGGTG-3?
5?-GACTCAGGGGTGCTGGCCAAG-3?
1638
5?-ATGCCGAAGAACAGCAAGGTG-3?
5?-AATCCAGGCCACAGCGATGTT-3?
1395
5?-AACATCTGGAGGTTCCCCTAC-3?
5?-AATCCAGGCCACAGCGATGTT-3?
258
5?-TTGCTCTCCGACGGGTCCAAC-3?
5?-TCACAGCTCTGACTCAGGGGT-3?
204
5?-ATGCCGAAGAACAGCAAGGTG-3?
5?-CTGCAGCTTGCTATTCCAGGC-3?
bp, base pairs.
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transferred to nitrocellulose membranes using the Bio-Rad system.
Whole brain or striata lysate (50 ?g) was used as a positive control in
all experiments. Nitrocellulose membranes were first blocked for 1 h
in Tris-buffered saline buffer (50 mM Tris-HCl, 150 mM NaCl, and
0.2% Tween 20) containing 5% dry milk, followed by incubation with
the primary antibody for 1 h in blocking buffer. After washing the
membrane three times for 10 min each in Tris-buffered saline buffer,
a horseradish peroxidase-conjugated secondary antibody was added
in blocking buffer for 1 h. The membrane was washed a final three
times, and West Pico chemiluminescence (Pierce Chemical) was used
to visualize protein bands.
Immunocytochemistry. Wild-type PC12 cells were grown on
poly-D-lysine-coated coverslips overnight and then fixed using 4%
paraformaldehyde for 10 min, followed by washing with PBS. After
permeabilization with 0.05% Triton X-100 for 10 min, the samples
were incubated with blocking solution (5% goat serum and 1% bovine
serum albumin) for 30 min. Preimmune or anti-Rxt1/NTT4 was
incubated at a 1:200 dilution in blocking solution for 1 h, followed by
three washes in PBS, and an additional 1-h incubation with FITC-
conjugated secondary antibody at a 1:1000 dilution in blocking solu-
tion. For colocalization experiments, samples were also incubated
with synaptophysin, calnexin, or transferrin receptor antibodies all
at 1:200 dilution, followed by Texas Red-conjugated secondary anti-
body at a 1:500 dilution for 1 h. Immunofluorescent experiments
were also performed on CHO cells transfected with Rxt1/NTT4
tagged with GFP (GFP-Rxt1/NTT4). In all cases, cells were mounted
with Fluoro mounting medium (MP Biomedicals, Irvine, CA) and
visualized using confocal microscopy (TCS SL; Leica Microsystems,
Inc., Deerfield, IL).
RNA Isolation, cDNA Synthesis, and Cloning. Total RNA from
rat brain, PC12, or CHO cells was isolated using TRIzol reagent
(Invitrogen) as recommended by the manufacturer. Reverse tran-
scription was performed using SuperScript III (Invitrogen). The
cDNA was purified using the QIAquick polymerase chain reaction
(PCR) purification kit (QIAGEN, Valencia, CA), and the full coding
sequence for Rxt1/NTT4 was amplified using iTaq DNA polymerase
(Bio-Rad) and PCR. The primers used for the PCR and nested PCR
are listed in Table 1. PCR products were purified by agarose gel
electrophoresis, isolated using the QIAquick gel extraction kit (QIA-
GEN), subcloned using the pcDNA3.1/V5-His TOPO vector (Invitro-
gen), and sequenced by automated DNA sequencing.
Generation of siRNA Stable Clones. The pSilencer 4.1-CMV
Puro vector was obtained from Ambion (Austin, TX) and used to
produce siRNAs against Rxt1/NTT4. According to the manufactur-
er’s instructions, 21 pairs of annealed DNA oligonucleotides were
inserted into this vector between the BamHI and HindIII restriction
sites to construct the siRNAs. The Rxt1/NTT4 siRNA sequence was
5?-AAACAGGACAATAACTGCCAC-3? corresponding to nucleotides
1444 to 1464 of rat Rxt1/NTT4. The sequence 5?-AAACTACCGTT-
GTTATAGGTG-3? was used as a scramble control. Inserted se-
quences were confirmed by DNA sequencing. To isolate siRNA-
containing clones, PC12 cells were transfected with pSilencer
4.1-CMV-Rxt1/NTT4 siRNA using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instructions. Twenty-four hours af-
ter transfection, 200 ng/ml puromycin was maintained in the me-
dium for selection. Western blot analysis for Rxt1/NTT4 was per-
formed as described under Western blot analysis.
Postnuclear Supernatant Preparation and Vesicle Isola-
tion. Media were removed from confluent 10-cm plates of either
PC12 or CHO cells. After rinsing the plate with PBS, cells were
Fig. 1. Rxt1/NTT4 is expressed in PC12 cells. A, Western blot analysis of
Rxt1/NTT4 using four different cell lines. Positive controls from an en-
riched synaptic vesicle preparation (SV) and whole brain lysates from rat
(RB) and mouse (MB) are also shown. Blots were also probed with
preimmune serum as a specificity control. B, immunoprecipitation of
Rxt1/NTT4 from PC12 cells. Immunoprecipitations using rat brain (RB)
lysate were also used as a positive control. Samples were incubated with
preimmune serum (?) or Rxt1/NTT4 antibody (?). C, immunocytochem-
ical analysis of Rxt1/NTT4 in PC12 cells. Fixed cells were immunostained
with the anti Rxt1/NTT4 (top; scale bar, 20 ?m) or preimmune serum
(bottom; scale bar, 18 ?m) and labeled with a FITC-conjugated antibody.
Contrast phase controls also shown (right).
Fig. 2. Rxt1/NTT4 colocalizes with synaptophysin in PC12 cells. Fixed
PC12 cells were immunostained with Rxt1/NTT4 (green; left), synapto-
physin, calnexin, or transferrin receptor antibodies (red; middle). The
merged image shows an almost complete colocalization of Rxt1/NTT4
with synaptophysin (right, top; scale bar, 20 ?m). In contrast, the endo-
plasmic reticulum marker calnexin (right, middle; scale bar, 18 ?m) and
the endosomal marker transferrin receptor (right, bottom; scale bar, 8
?m) showed little overlap with Rxt1/NTT4.
Rxt1/NTT4 Functions As a Vesicular Amino Acid Transporter
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scraped from dishes and pelleted at 305g for 3 min. The resulting
pellet was resuspended in buffer A (320 mM sucrose, 0.1 MgCl2, 0.5
EGTA, and 10 HEPES, pH 7.4) containing protease inhibitors. After
homogenization using 25 strokes in a Wheaton homogenizer
(Wheaton Science Products, Millville, NJ) followed by 25 passages
through a 25-gauge needle, the sample was centrifuged at 1000g for
5 min at 4°C, resulting in the postnuclear supernatant (PNS) prep-
aration. Further isolation of synaptic vesicles was obtained by addi-
tional centrifugations at 27,000g for 35 min and 180,000g for 2 h at
4°C. The resulting pellet contained an enriched synaptic vesicle
fraction and was called P3.
Determination of Vesicular Amino Acids by Capillary Gas
Chromatography. P3 fractions from PC12 cells (wild type, siRNA-
Rxt1/NTT4, and siRNA-scramble) were resuspended and sonicated
in 300 ?l of water. Aliquots of lysed cells were used for amino acid
analysis according to the procedure established by the EZ:faast free
amino acid kit (Phenomenex, Torrance, CA). In brief, 100 ?l of
Norvaline solution (20 nmol) was added to each sample as an inter-
nal standard. The mixture was slowly passed through a sorbent tip,
and the filtrate was discarded. The sorbent tip was washed with 200
?l of 2-propanol/H2O. The amino acids were then recovered by elut-
ing the sorbent tip with 200 ?l of a sodium hydroxide and n-propanol
mixture [3:2 (v/v)]. Finally, the recovered amino acids were deriva-
tized by adding 50 ?l of chloroform, 100 ?l of isooctane, and 100 ?l of
1 N HCl solvents consecutively with vigorous vortexing in between
the solvent addition. Then, 100 ?l of the upper layer was used for
amino acid determination using capillary gas chromatography. The
amino acid derivatives were separated on a 10-m Zebron ZB-AAA
column (0.25 mm i.d.) with helium as the carrier gas (1.5 ml/min)
using a Hewlett-Packard capillary gas chromatograph model 5890
series II, equipped with a hydrogen flame ionization detector. The
sample was injected under a split ratio of 15:1. Oven temperature
was initially set at 110°C and then programmed to 280°C at 32°C/
min. The oven temperature remained at 280°C for another 5 min
before the end of the run. Total run time was approximately 10.3
min. Peaks were identified by comparing the retention times of the
samples with those of standard mixtures and calculated by Chem-
Station (rev. A.09.03; Agilent Technologies, Santa Clara, CA) using
an internal standard mode. These results were normalized using
protein concentration.
Vesicular Uptake. PNS (200 ?g) in 50 ?l was added to 200 ?l of
uptake buffer (20 mM HEPES, 100 mM NaCl, mM 2.5 MgSO4, and
2 mM ATP, pH 7.4) warmed to 29°C, followed by the addition of
radiolabeled substrate (50 nM). After incubation for 6 min, the sam-
ples were filtered through a 0.2-?m SUPOR membrane filter (Pall
Corporation, East Hills, NY). The reaction was stopped by washing
twice with 1.5 ml of ice-cold uptake buffer. The filter was then added
to 4 ml of Biosafe II scintillation liquid (Research Products Interna-
tional, Mt. Prospect, IL), and radioactivity was counted in an LS
6500 scintillation counter (Beckman Coulter, Fullerton, CA). In some
cases, the uptake buffer was altered to contain 100 mM LiCl or 100
mM sodium gluconate (NaGluc) instead of NaCl; depleted of ATP; or
supplemented with 10 ?M bafilomycin A1, 20 ?M valinomycin, 5 ?M
nigericin, or 1 mM ouabain. To determine kinetic constants for
[3H]Pro and [3H]Gly transport, we used 200 ?g of PNS from Rxt1/
NTT4-transfected CHO cells using normal uptake conditions. The
substrate [3H]Pro or [3H]Gly (at 50 nM) and increasing concentra-
tions of unlabeled Pro or Gly ranging in concentration from 1 ?M to
3 mM were used. Nonspecific uptake was determined in the presence
of 50 mM Pro or Gly.
Data Analysis. Results are presented as mean ? S.E. Significant
differences between means were determined by Student’s t test, with
p ? 0.05 considered statistically significant.
Results
Rxt1/NTT4 Is Expressed in PC12 Cells. Although pre-
vious in vivo electron microscopy studies have indicated that
Rxt1/NTT4 is localized to synaptic vesicles of glutamatergic
and/or GABAergic neurons (Fischer et al., 1999; Masson et
al., 1999), we were unaware of any cell lines endogenously
expressing Rxt1/NTT4. Thus, in an effort to find a suitable
cell system to use for the identification of the substrate(s) for
Rxt1/NTT4, we screened several cell lines for endogenous
expression of this protein. Tested cell lines included HEK293,
SKNSH, MN9D, and PC12 cells, whereas whole brain lysates
and synaptic vesicle preparations from mouse and rat were
used as positive controls. Western blot analysis using an
affinity-purified polyclonal antibody against Rxt1/NTT4 re-
vealed the presence of an approximately 75-kDa band in both
PC12 cells and in the positive controls (Fig. 1A). No bands
were detected in HEK293, SKNSH, or MN9D cells. As a
control for specificity, blots were also incubated with preim-
mune serum, and no bands were detected in any of the cell
lines or positive controls (Fig. 1A). As an additional approach,
we performed immunoprecipitation on PC12 cells using ei-
ther the Rxt1/NTT4 antibody to detect the transporter. Con-
sistent with our previous data, the transporter successfully
coprecipitated with the Rxt1/NTT4 antibody but not with the
preimmune serum in both PC12 cells and rat brain lysates
(Fig. 1B). Next, we examined the subcellular localization of
Rxt1/NTT4 in PC12 cells using confocal microscopy. PC12
cells exhibited extensive Rxt1/NTT4 staining throughout the
cell only in those samples incubated with the antibody
against Rxt1/NTT4 but not in those cells incubated with the
preimmune serum (Fig. 1C). Furthermore, to test whether
Rxt1/NTT4 was present in synaptic vesicles, we also con-
ducted colocalization experiments using synaptophysin as a
Fig. 3. PC12 cells express the Rxt1/NTT4
gene. A, RT-PCR analysis of Rxt1/NTT4
mRNA from PC12 cells reverse tran-
scribed into cDNA. Rxt1/NTT4 cDNA
from rat brain was used as a positive
control (?), whereas the negative control
contained no cDNA (?). B, to confirm the
amplification of the desired sequence,
nested PCR followed first-round amplifi-
cation. The isolated PCR fragment was
initially cloned using pcDNA3.1/V5-His-
TOPO cloning kit, and its sequence was
confirmed by DNA sequencing.
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synaptic vesicle marker. Merged images show an almost
complete overlap of synaptophysin and Rxt1/NTT4 (Fig. 2,
top). In contrast, little colocalization was found between
Rxt1/NTT4 and the ER marker calnexin (Fig. 2, middle) or
the endosomal marker transferrin receptor (Fig. 2, bottom).
Our findings are consistent with the synaptic vesicle local-
ization of Rxt1/NTT4 in neurons as reported by Masson et al.
(1999).
Because approaches using antibodies cannot confirm the
identity of a given protein, we sought to isolate the Rxt1/
NTT4 gene from PC12 cells. To do this, we performed RT-
PCR using flanking primers against the transporter se-
quence. As shown in Fig. 3A, a band of approximately 2200
base pairs corresponding to the size of the full-length Rxt1/
NTT4 was detected in PC12 cell samples. This band was
purified and used as a template for nested PCR. Using five
sets of internal primers (Table 1), we were able to visualize
bands of the expected sizes (Fig. 3B). Finally, the identity of
Rxt1/NTT4 was confirmed by DNA sequencing (data not
shown). Taken together, these data demonstrate that Rxt1/
NTT4 is expressed in PC12 cells, which represent a suitable
model system to be used in the identification of the trans-
porter’s substrate(s).
Rxt1/NTT4-MediatedVesicular
Cells. Previous studies failed to identify the substrate(s) for
Rxt1/NTT4 because it was assumed that the transporter was
targeted to the plasma membrane. However, more recent
findings show that this transporter is actually targeted to
synaptic vesicles, suggesting the possibility that Rxt1/NTT4
functions as a vesicular transporter. To test this hypothesis,
we developed a strategy in which vesicular content and up-
take were measured in wild-type PC12 cells and compared
with cells in which the expression of Rxt1/NTT4 was reduced
by siRNA. In our hands, overexpressing an siRNA construct
against nucleotides 1444 to 1464 in Rxt1/NTT4 results in
reduction of protein levels by approximately 50% compared
with wild-type cells or cells overexpressing a scramble-siRNA
sequence (Fig. 4, A and B). Under the same conditions, the
synaptic vesicle proteins SV2, synaptophysin, and synapto-
brevin were not affected (Fig. 4A). Immunofluorescent exper-
iments further confirmed this effect of siRNA on Rxt1/NTT4
expression in PC12 cells (Fig. 4C). Next, we tested multiple
substrates in an effort to find an uptake activity that would
be decreased in siRNA cells compared with wild-type or
scramble-siRNA cells. The tested substances included nor-
epinephrine, epinephrine, Asp, Lys, Tyr, Met, dopamine, se-
rotonin, Trp, Cys, Arg, Glu, GABA, Ser, Ala, Pro, Gly, and
Leu. All were used at a concentration of 50 nM. Under these
experimental conditions, the transport of Pro, Gly, and Leu
were all decreased by approximately 70%, and Ala transport
was decreased 50% in Rxt1/NTT4 knockdown cells compared
with wild-type or scramble-siRNA cells (Fig. 5A). In contrast,
vesicular transport for all other tested substances remained
unchanged in all three cell lines (Fig. 5A). To complete test-
ing of the remaining amino acids as potential substrates for
Rxt1/NTT4, we used unlabeled substrates as potential com-
petitors for [3H]Pro uptake. Using this rationale, we used 50
mM unlabeled GABA, His, Glu, Gln, Phe, Thr, Val, Ile, Ala,
Leu, Gly, and Pro. The addition of unlabeled Pro resulted in
a 35% decrease of [3H]Pro uptake (Fig. 5B). In agreement
with our previous findings, Leu, Gly, and Ala also signifi-
cantly inhibited [3H]Pro uptake. In contrast, we did not ob-
Uptake inPC12
served significant changes in [3H]Pro uptake in the presence
of any of the remaining substances (Fig. 5B). Although it is
intriguing that [3H]Pro uptake was reduced only 35% when
competing with 50 mM unlabeled Pro, similar reduction lev-
els of [3H]Pro uptake were observed when unlabeled Pro was
used at 0.5 or 5 mM (Fig. 5C). This indicates that the [3H]Pro
uptake that remains after competition is most likely due to
background unrelated to Rxt1/NTT4 function. Taken to-
gether, these data suggest that Rxt1/NTT4 can transport
Pro, Gly, Leu, and Ala in PC12 cells.
Rxt1/NTT4-Mediated Vesicular Content in PC12 Cells.
Because our first experiments were performed using exogenous
substrates, we sought to determine whether in fact these sub-
strateswerepresentinPC12vesiclesandwhethertheircontent
was reduced in cells expressing siRNA against Rxt1/NTT4.
Thus, we obtained an enriched synaptic vesicle fraction (called
P3) from PC12 cells. Our data confirmed that Rxt1/NTT4 and
other synaptic vesicle proteins including SV2 and synaptophy-
sin were enriched in our P3 fraction compared with the initial
homogenate (Fig. 6A). Next, we analyzed the amino acid con-
tent of the P3 fraction by gas chromatography in wild-type,
scramble-siRNA, and siRNA-Rxt1/NTT4 PC12 cells. Consistent
withtheuptakedata,thevesicularcontentofPro,Gly,Leu,and
Ala were significantly reduced in siRNA cells compared with
wild-type or scramble cells (Fig. 6, B and C). No significant
differences in vesicular content of Val, Ile, Thr, Met, Phe, Trp,
His, Tyr, or Lys were detected (Fig. 6C). These findings are
consistent with the notion that Rxt1/NTT4 functions as a vesic-
ular transporter selective for Pro, Gly, Leu, and Ala.
Rxt1/NTT4 Confers Pro, Gly, Leu, and Ala Uptake in
CHO Cells. To provide definitive proof that Pro, Gly, Leu,
and Ala are substrates of Rxt1/NTT4, we next determined
whether in fact the coding sequence of Rxt1/NTT4 is suffi-
cient to transport these amino acids. CHO cells, which do not
express Rxt1/NTT4 as demonstrated by RT-PCR (Fig. 7A)
Fig. 4. Rxt1/NTT4 expression is decreased by 50% when knocked down by
siRNA in PC12 cells. A, representative immunoblots of wild-type (WT),
scramble-siRNA (Scr), and Rxt1/NTT4-siRNA PC12 cells. Rxt1/NTT4
expression is reduced in the siRNA-transfected cells, whereas the synap-
tic vesicle markers synaptobrevin, synaptophysin, and SV2 are not al-
tered. B, densitometry analysis of Rxt1/NTT4 expression normalized to
arbitrary densitometry units of ?-tubulin (n ? 3; ?, p ? 0.05). C, immu-
nofluorescence analysis of Rxt1/NTT4 in wild-type PC12 cells (top; scale
bar, 20 ?m) and Rxt1/NTT4 siRNA-treated PC12 cells (bottom; scale bar,
24 ?m). Fixed cells were immunostained with the anti-Rxt1/NTT4 and
labeled with a FITC-conjugated secondary antibody.
Rxt1/NTT4 Functions As a Vesicular Amino Acid Transporter
1525

Page 6

and immunoblotting (Fig. 7B), were transfected with a GFP-
tagged Rxt1/NTT4, allowing detection by Western blot and
confocal microscopy (Fig. 7, B and C). Next, we performed
uptake experiments using a postnuclear preparation (PNS)
from mock and GFP-Rxt1/NTT4-transfected CHO cells.
Mock-transfected cell uptake was considered to be 100% in
each individual condition and increases were expressed rel-
ative to this percentage. Uptake levels were significantly
increased for Pro (10,492%), Gly (611%), Leu (160%), and Ala
(206%) in cells transfected with GFP-Rxt1/NTT4 compared
with mock cells (Fig. 7D). Similar levels of uptake were
obtained in cells transfected with an untagged Rxt1/NTT4
construct (data not shown). These percentages are dramati-
cally high, especially for Pro and Gly, because the uptake of
these substrates is almost undetectable in nontransfected
cells. No significant differences in uptake levels were ob-
served for GABA or DA. These data demonstrate that the
coding sequence of Rxt1/NTT4 is sufficient to confer Pro, Gly,
Leu, and Ala uptake in CHO cells. Pro uptake was saturated,
with an estimated Kmvalue of 0.86 ? 0.17 mM and a Vmax
value of 172 ? 12.9 pmol/min/mg protein, whereas Kmand
Vmaxvalues for Gly were 1.72 ? 0.3 mM and 199 ? 16.6
pmol/min/mg protein, respectively (Fig. 8). The Kmvalues
obtained for Pro and Gly in the low millimolar range are
consistent with what has been described for other vesicular
transporters (Table 2). In fact, with the exception of VMAT2,
all the vesicular transporters, including VAChT, VGLUTs,
and VIAAT, have Kmvalues in the low millimolar range. This
is in contrast to Kmvalues of plasma membrane transporter,
which are usually in the low micromolar range. This supports
the idea that plasma membrane transporters are high-affin-
ity carriers that primarily accumulate substrates in the ter-
minal at concentrations high enough to allow the vesicular
transporter with lower affinity to sequester these substrates.
Rxt1/NTT4 Uptake Mechanism. Because Rxt1/NTT4
shares approximately 30 to 45% homology with the sodium
chloride-dependent plasma membrane transporters, we
wanted to explore the ion dependence of this transporter’s
novel function. Thus, we measured Pro uptake in PNS from
wild-type PC12 cells and CHO cells transfected with Rxt1/
NTT4 because our previous data revealed that Rxt1/NTT4
expression correlated the highest level with Pro uptake. The
use of both wild-type PC12 and transfected CHO cells en-
abled this study to be conducted using native and recombi-
Fig. 5. Rxt1/NTT4 can transport Pro, Gly, Leu, and Ala. A, amino acid uptake (50 nM) in 200 ?g of a PNS preparation from wild-type (black),
scramble-siRNA-transfected (gray), and Rxt1/NTT4-siRNA-transfected PC12 cells (white). Uptake was determined for a period of 6 min. Nor,
norepinephrine; Epi, epinephrine; DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin). B, [3H]Pro uptake (50 nM) in PNS from wild-type PC12 cells
was performed in the presence or absence of 50 mM unlabeled amino acids (n ? 9; ?, p ? 0.05). C, [3H]Pro uptake (50 nM) in the presence of unlabeled
Pro at 0.5, 5, or 50 mM.
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Page 7

nant Rxt1/NTT4, respectively. Specifically, NaCl in the up-
take buffer was substituted for either NaGluc or LiCl. Our
results showed that Pro uptake was unaffected by the use of
NaGluc in both PC12- and CHO-transfected cells, indicating
that Rxt1/NTT4 function is not chloride-dependent (Fig. 9A).
Likewise, both cell lines exhibited approximately 70% of the
control uptake when buffer containing LiCl was used, sug-
gesting a partial dependence on sodium (Fig. 9A). Thus, it
seems that the function of Rxt1/NTT4 is not consistent with
the signature plasma membrane carrier characteristic of so-
dium and chloride-dependent transport (for review, see Nel-
son, 1998). Therefore, we next explored whether the function
of Rxt1/NTT4 is affected by the vacuolar-type H?-ATPase
(V-ATPase) that is typically coupled to vesicular transport-
ers, including VMAT2, VGLUT, VIAAT, and VAChT (Naito
and Ueda, 1985; Moriyama and Futai, 1990; Hell et al., 1991;
Eiden et al., 2004). In these experiments, uptake buffer with
and without ATP or the specific inhibitor of V-ATPase bafilo-
mycin A1 was used to measure Pro uptake in both wild-type
PC12 and Rxt1/NTT4-transfected CHO cells. In the presence
of bafilomycin A1 or in the absence of ATP, Pro uptake was
almost abolished in both wild-type PC12 cells and Rxt1/
NTT4-transfected CHO cells (Fig. 9B). The electrochemical
gradient of protons maintained by V-ATPase consists of two
components, a proton gradient and the membrane potential
(Moriyama et al., 1992). To further dissect the contribution of
Fig. 6. Rxt1/NTT4 mediates vesicular
content of Pro, Gly, Leu, and Ala in
PC12 cells. A, Rxt1/NTT4 and the
synaptic vesicle marker proteins syn-
aptophysin and SV2 are enriched in
the P3 fraction compared with the
original homogenate (H). B, represen-
tative gas chromatograph analyses of
P3 lysates from wild-type PC12 cells
(left) andRxt1/NTT4-siRNA-trans-
fected PC12 samples (right). C, amino
acid vesicular content levels were nor-
malized to protein levels. Changes are
represented as a percentage of amino
acid vesicular content for scramble-
siRNA transfected PC12 cells (gray)
andRxt1/NTT4-siRNA-transfected
PC12 cells (white) compared with
wild-type PC12 cells (black) (n ? 3; ?,
p ? 0.05). Ileu, isoleucine.
Rxt1/NTT4 Functions As a Vesicular Amino Acid Transporter
1527

Page 8

these two components in Pro uptake, we used the ionophores
nigericin and valinomycin. Pro uptake was reduced by ap-
proximately 75% in the presence of nigericin, which selec-
tively dissipates ?pH. In contrast, valinomycin, an ionophore
more selective to dissipate membrane potential, reduced Pro
uptake by only 50% (Fig. 9B). In contrast, ouabain, an agent
known to inhibit plasma membrane Na?,K?-ATPase, did not
affect Pro uptake. Collectively, these results suggest that
Rxt1/NTT4-mediated uptake of Pro is coupled to an ATP-
dependent electrochemical gradient that is influenced mostly
by the proton gradient and is less affected by the membrane
potential, further demonstrating that Rxt1/NTT4 acts as a
vesicular transporter.
Discussion
In this report, we have used multiple approaches to iden-
tify the orphan transporter Rxt1/NTT4 as a vesicular trans-
porter selective for Pro, Gly, Leu, and Ala. Our results dem-
onstrate that PC12 cells endogenously express Rxt1/NTT4
and, thus represent a suitable cell model to identify the
substrate of this transporter. Three lines of evidence are
consistent with the contention that Rxt1/NTT4 exists and
functions as a transporter for Pro, Gly, Leu, and Ala. First,
uptake for Pro, Gly, Leu, and Ala was demonstrated in a PNS
preparation from PC12 cells and was decreased in Rxt1/
NTT4 knockdown cells. Second, an enriched fraction of ves-
icles from PC12 cells express Rxt1/NTT4 and contain Pro,
Gly, Leu, and Ala as measured by gas chromatography. Fur-
thermore, the vesicular content of these amino acids was
selectively and significantly decreased in cells where Rxt1/
NTT4 expression was down-regulated by siRNA. Finally,
overexpression of Rxt1/NTT4 in CHO cells allowed Pro and
Gly transport and increased Leu and Ala uptake in a PNS
preparation. Thus, these findings demonstrate that the pres-
Fig. 7. Rxt1/NTT4 confers Pro, Gly, Leu, and Ala
uptake in CHO cells. A, RT-PCR analysis of Rxt1/
NTT4 mRNA from CHO cells reverse transcribed
into cDNA. Rxt1/NTT4 cDNA from rat brain (?) and
PC12 cells were used as a positive controls, whereas
the negative control contained no cDNA (?). B,
Western blot analysis in GFP-Rxt1/NTT4-trans-
fected CHO cells, PC12 WT cells, and CHO WT cells
using both the Rxt1/NTT4 (top) and GFP (bottom)
antibodies. C, confocal microscopy images showing
CHO cells transfected with a GFP-tagged Rxt1/
NTT4 cDNA (scale bar, 15 ?m). D, amino acid up-
take using 200 ?g of PNS from mock (black) and
GFP-Rxt1/NTT4-transfected (white) CHO cells. Up-
take levels were significantly increased for Pro
(10,492%), Gly (611%), Leu (160%), and Ala (206%)
in cells transfected with GFP-Rxt1/NTT4 compared
with mock cells. No significant differences were ob-
served for GABA (100%) and DA (102%) uptake be-
tween the mock-transfected and GFP-Rxt1/NTT4-
transfected CHO cells.
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Page 9

ence of Rxt1/NTT4 is sufficient to confer transporter function
for these amino acids.
The relatively moderate degree of sequence homology be-
tween Rxt1/NTT4 and members of the sodium- and chloride-
dependent plasma membrane transporters led to the assump-
tion that Rxt1/NTT4 was also a plasma membrane carrier (Liu
et al., 1993; el Mestikawy et al., 1994). However, electron mi-
croscopy studies revealed an unexpected vesicular localization
for Rxt1/NTT4 in both brain slices and neurons in culture
(Fischer et al., 1999; Masson et al., 1999). Based on these
findings, two potential scenarios for the role of Rxt1/NTT4 were
proposed: either Rxt1/NTT4 functions as a bona fide vesicular
transporter; or; alternatively, Rxt1/NTT4 proteins reside as a
reserve pool in intracellular vesicles from which they need to be
translocated to the plasma membrane to become functional.
Our results strongly support the contention that Rxt1/NTT4
resides and functions as a vesicular transporter selective for
Pro, Gly, Leu, and Ala. Our data showed that the transporter
activity was not dependent on sodium or chloride, a signature
characteristic of plasma membrane carriers from this family
(for review, see Nelson, 1998). In addition, as seen with other
vesiculartransportersystems,theRxt1/NTT4-mediateduptake
was ATP-dependent and sensitive to agents that disrupt the
vesicular proton gradient (bafilomycin A1 and nigericin) and
the membrane potential (valinomycin). A similar phenomenon
was reported for the vesicular glutamate transporter (Belloc-
chio et al., 2000), which was originally described as a brain-
specificplasmamembraneNa?-dependentinorganicphosphate
transporter (Ni et al., 1994).
Our findings demonstrating that Pro, Gly, Leu, and Ala are
substrates of Rxt1/NTT4 and stored in synaptic vesicles leads
to questions regarding the physiological role of a vesicular
transporter selective for these amino acids. Indeed, several
lines of evidence already suggest that Pro might function as
a neurotransmitter. Pro is biosynthesized in the brain through
the action of the enzyme pyrroline 5-carboxylate reductase
(Yoneda and Roberts, 1982). Furthermore, Pro produces elec-
trophysiological actions in the spinal cord and brain (Felix and
Ku ¨nzle, 1976). Previous studies have shown that potassium
stimulation of neurons in brain and spinal cord slices leads to
release of radiolabeled Pro (Mulder and Snyder, 1974). In ad-
dition, a plasma membrane Pro transporter has been identified
and is widely distributed in rat brain (Fremeau et al., 1992).
Finally, immunohistochemistry analysis with a selective Pro
antibody demonstrated that Pro is localized within terminals in
several groups of neurons (Takemoto and Semba, 2004). Thus,
our data showing that Rxt1/NTT4 mediates vesicular uptake of
Pro will stimulate further studies exploring the possibility that
Pro has a role in synaptic transmission.
Regarding Gly, its actions as an inhibitory neurotrans-
mitter as well as a necessary coagonist for activation of the
ionotropic glutamate N-methyl-D-aspartate (NMDA) receptor
are well established (Johnson and Ascher, 1987; Kemp and
Leeson, 1993). Heteromeric NMDA receptors are ubiqui-
tously distributed throughout the brain, participating in a
variety of functions, including neuronal development, synap-
tic plasticity, learning, and memory (Scheetz and Constan-
tine-Paton, 1994; Kato et al., 1999; Albensi, 2007; Lau and
Zukin, 2007). NMDA receptor activation requires not only
glutamate but also glycine (Johnson and Ascher, 1987; Kleck-
ner and Dingledine, 1988; Kemp and Leeson, 1993). Indeed,
Fig. 8. Kinetic analysis of proline and glycine uptake through Rxt1/NTT4
in CHO cells. A, PNS from CHO cells transfected with Rxt1/NTT4 were
resuspended in buffer containing 50 nM [3H]Pro and various concentra-
tions of unlabeled Pro. Uptake was performed for 6 min at 37°C. Line-
weaver-Burk analysis (inset) showed a Kmvalue of 0.86 ? 0.17 mM and
a Vmaxvalue of 172 ? 12.9 pmol/min/mg protein. The values represent the
means of triplicate experiments. B, similar experiments were conducted
using 50 nM [3H]Gly and increasing concentrations of unlabeled Gly. A
Kmvalue of 1.72 ? 0.3 mM and a Vmaxvalue of 199 ? 16.6 pmol/min/mg
protein were obtained.
TABLE 2
Apparent affinity of vesicular neurotransmitter transporters
Substrate Transporter
Km
References
GlutamateVGLUT1
VGLUT2
VGLUT3
VGAT/VIAAT
VAChT
VMAT1
VMAT2
?1 mM
?1.1–4.7 mM
?0.5–1.5 mM
?1 mM
?1 mM
1.56 ?M
0.32 ?M
Bellocchio et al., 2000
Bai et al., 2001; Fremeau et al., 2001; Herzog et al., 2001
Fremeau et al., 2002; Gras et al., 2002; Schäfer et al., 2002
McIntire et al., 1997
Varoqui et al., 1996; Kim et al., 1999
Erickson et al., 1992
Liu et al., 1992
GABA
Acetylcholine
Serotonin
Dopamine
Rxt1/NTT4 Functions As a Vesicular Amino Acid Transporter
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Page 10

Rxt1/NTT4 is expressed in glutamatergic neurons in which
glycine could be coreleased with glutamate (Johnson and
Ascher, 1987; Kemp and Leeson, 1993). Therefore, changes in
extracellular Gly concentrations may lead to alterations in
NMDA receptor function (Martina et al., 2004). Such changes
in receptor function have been implicated in neurodegenera-
tion and neuropsychiatric disorders, including schizophrenia
(Krystal et al., 1998; Newcomer et al., 1999). Thus, it is
interesting that although glycine transporters (GlyTs) are
localized to inhibitory synaptic terminals, GlyT1 is broadly
expressed in regions of the brain not known to have glycin-
ergic inhibition (cortex, hippocampus, and thalamus), sug-
gesting that this transporter influences excitatory synaptic
signaling by regulating glycine concentrations. In fact,
knockout of GlyT1 in mice causes neonatal death (Gomeza et
al., 2003a), and mice deficient of GlyT2 died within 2 weeks
of birth (Gomeza et al., 2003b). These findings illustrate the
crucial role of glycine in brain function. In addition, admin-
istration of either the GlyT1 inhibitor N[3-(4?-aurophenyl)-3-
(4?-phenylphenoxy)propyl] sarcosine or the GlyB site agonist
D-serine potentiated NMDA-mediated responses in the pre-
frontal cortex (Chen et al., 2003). These results suggest that
GlyB binding sites on NMDA receptors are likely to be un-
saturated. In this context, Gly release from glutamatergic
neurons might represent another regulatory component in
the activation of NMDA receptors. A novel NMDA receptor
subunit, NR3B, has been cloned from rat brain and shown to
form a novel excitatory Gly receptor (Ciabarra et al., 1995;
Sucher et al., 1995). Analysis of the NR3B subunit tissue
distribution revealed that this protein is primarily expressed
in motor neurons of the spinal cord and brainstem (Nishi et
al., 2001; Matsuda et al., 2002). These new findings provide a
novel mechanism to study the actions of Gly. In this context,
it will be interesting to examine whether Rxt1/NTT4-medi-
ated Gly release plays a role in the activation of this novel
excitatory Gly receptor. Finally, we are not aware of any
studies suggesting that Leu or Ala functions as a neurotrans-
mitter. Further studies will be required to distinguish
whether Leu and Ala exist as true neurotransmitters or
alternatively uptake of these amino acids might represent a
pharmacological property of this transporter.
Two groups have identified the substrate for another or-
phan transporter, v7-3/NTT7, as a plasma membrane neu-
tral amino acid transporter for Pro, Leu, Met, and Ala (Bro ¨er
et al., 2006). Furthermore, in situ hybridization studies have
revealed that v7-3/NTT4 and Rxt1/NTT4 have extensive
overlapping expression patterns, especially in the olfactory
bulb, cerebral cortex, hippocampus, and cerebellum (Inoue et
al., 1996; Luque et al., 1996; Masson et al., 1996). Therefore,
it is tempting to speculate that v7-3/NTT7 and Rxt1/NTT4
might function in concert to transport neutral amino acids
through the plasma membrane and load them into synaptic
Fig. 9. Proline uptake in both PC12
cells and Rxt1/NTT4-transfected CHO
cells was dependent on the electro-
chemical gradient maintained by the
vacuolar-type H?-ATPase. A, Pro up-
take in 200 ?g of PNS from wild-type
PC12 cells (left) and Rxt1/NTT4-trans-
fected CHO cells (right) were measured
using control uptake buffer, buffer in
which LiCl was substituted for NaCl, or
bufferinwhichNaGlucwassubstituted
for NaCl. B, Pro uptake in 200 ?g of
PNS from wild-type PC12 cells (left)
and Rxt1/NTT4-transfected CHO cells
(right) in which ATP was omitted
(?ATP) or in the presence of 10 ?M
bafilomycin A1, 20 ?M valinomycin, 5
?M nigericin, or 1 mM ouabain.
1530
Parra et al.

Page 11

vesicles. Further studies will be necessary to test this hy-
pothesis and uncover the physiological role of these novel
transporter systems.
Acknowledgments
We thank members of the Torres and Amara laboratories for
helpful discussions. We are also grateful to Daniela Requena and
Loreto Egan ˜a for technical support.
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Address correspondence to: Dr. Gonzalo E. Torres, Department of Neuro-
biology, Room 6061, BST3, University of Pittsburgh School of Medicine, 3501
Fifth Ave., Pittsburgh, PA 15261. E-mail: gtorres@pitt.edu
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