From the Department of Pharmacology, Yale University
School of Medicine, New Haven, Connecticut 06520-8066, § Center for Molecular Recognition, Departments of
Psychiatry and Pharmacology, Columbia University College of Physicians
and Surgeons, New York, New York 10032, and the ¶ Department
of Applied Biological Sciences, Nihon University, 1866 Kameino,
Fujisawa-shi, Kanagawa 252-8510, Japan
Received for publication, July 2, 2002, and in revised form, December 30, 2002
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ABSTRACT |
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The tnaT gene of
Symbiobacterium thermophilum encodes a protein homologous
to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in
Escherichia coli conferred the ability to accumulate
tryptophan from the medium and the ability to grow on tryptophan as a
sole source of carbon. Transport was Na+-dependent and highly selective. The
Km for tryptophan was ~145 nM, and
tryptophan transport was unchanged in the presence of 100 µM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with KI values of
200 and 440 µM, respectively. By using a T7
promoter-based system, TnaT with an N-terminal His6 tag was
expressed at high levels in the membrane and was purified to
near-homogeneity in high yield.
Transporters responsible for reuptake of neurotransmitters across
the plasma membrane of neurons and glia fall into two gene families
(1). The majority of small neurotransmitters, including glycine,
Among the sequences found to be homologous to the NSS family of
transporters are a number of "orphan" transporters, for which no
function is known. These orphans include v7-3 (18), NTT4 (19, 20),
inebriated (21), blot (22), and NTT5 (23), among
others. The largest number of orphan sequences in this family is found
in prokaryotic organisms. Although these orphan sequences are highly
similar to those encoding functional transporters, it is possible that
these proteins fulfill other functions. For example, within the
ATP-binding cassette family of transporters are the sulfonylurea
receptor (24) and the cystic fibrosis transmembrane regulator chloride
channel (25). In the dicarboxylate/amino acid:cation symporters
neurotransmitter transporter family is EAAT4, a ligand-gated ion
channel (26); SGLT3, a member of the sodium:solute symporter (SSS)
sugar transporter family, also is not a transporter but rather a
glucose-gated ion channel.2
Moreover, some proteins, such as adenylate cyclase (28) and patched (29) also have 12 transmembrane segments but no known transport function. For the orphan transporters in the NSS family, it
is important to know if any of the newly discovered prokaryotic sequences actually encode functional transporters.
Symbiobacterium thermophilum is a symbiotic thermophile, the
growth of which is dependent on co-culture with an associated Bacillus strain (30, 31). The 16 S rDNA-based taxonomy
showed that S. thermophilum occupies a novel phylogenetic
branch in the Gram-positive group without clustering with any other
genus (30). This bacterium produces a thermostable tryptophanase
directed by the tna1 gene which, when cloned (32), was found
to be part of a tna operon with an unusual gene
organization. The operon differs from the conserved structure among
enterobacteriaceae in that it consists of three open reading frames
(33). Furthermore, in the region downstream from the tryptophanase
gene, this unique bacterium appears to encode a transporter, TnaT, that
belongs, based on sequence homology, to the NSS
family.3 In this
communication, we demonstrate that the tnaT gene encodes a
Na+-dependent tryptophan transporter and that
this transporter can be expressed in the cell membrane and purified in
high yield.
Plasmid Preparations--
The nucleotide sequence of the
tna gene cluster of S. thermophilum was submitted
to the DNA Data Bank of Japan under accession number AB010832. For the
construction of the pET26b(+) expression plasmid (Novagen, Madison,
WI), the tnaT sequence was amplified by standard PCR
with the following primers: TnaT-N
(5'-CATCATATGGAGGCACAGCGC (corresponding to 5465-5485 of
the AB010832 sequence; underlines indicate an NdeI
restriction site)) and TnaT-C
(5'-CGGAAGCTTCAGCCCACCTCCCCGCCGG (corresponding to
nucleotides 6984-6957 of the AB010832 sequence; underlines
indicate a HindIII restriction site)). The product was
digested with NdeI and HindIII and ligated to
NdeI-HindIII-digested pET26b(+).
For expression of TnaT in the Escherichia coli strain
CY15212 (mtr
For inducible expression of TnaT in CY15212, an
EcoRI/NheI fragment encoding a 6xHis-TnaT (see
below) was subcloned into pQE82 (Qiagen), a plasmid with an inducible
T5 promoter. CY15212 cells were transformed with this plasmid and
selected with ampicillin and kanamycin. A pool of colonies was grown
and induced as described below, except that 0.15 mM IPTG
was used for 2 h. After induction, cells were prepared for uptake
or membranes were prepared as described below. For immunoblotting,
samples were resolved by SDS-PAGE, transferred to nitrocellulose, and
blotted with an anti-His6 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) followed by a horseradish peroxidase-conjugated secondary antibody. Chemiluminescence was detected and quantitated on a FluorChem 8000 (Alpha Innotech Corp., San
Leandro, CA) after incubation with SuperSignal West Femto substrate (Pierce).
For high level expression of TnaT in E. coli, a
His6-tagged construct was prepared using the Gateway system
(Invitrogen). The tnaT cDNA was amplified by PCR from
S. thermophilum chromosomal DNA using the following primers:
GGGGACAAGTTTGTACAAAAAAGCAGGCTCCGAGGCACAGCGCGATCAGTGG (sense) and
GGGACCACTTTGTACAAGAAAGCTGGGTACTAGCCCACCTCCCCGCCGG (antisense). By
using the manufacturer's methods, we moved the PCR product into
PDONR201 to create an entry clone and subsequently into pDEST17,
thereby generating a His6 fusion construct under control of
the T7 promoter. The resultant expression clone, pDEST17-6His-TnaT, was
used to transform E. coli BL21(DE3)/pLysE (Novagen). 150 ml of LB medium, containing ampicillin (100 µg/ml), chloramphenicol (34 µg/ml), and 1% glucose, was inoculated from a glycerol stock and
grown overnight at 37 °C. Cells were pelleted, resuspended in fresh
media, inoculated into 2 liters of LB containing ampicillin, chloramphenicol, and glucose, and grown at 30 °C to an
A600 of 0.6. Cells were induced with 0.4 mM IPTG and grown at 30 °C for 3 h. Membranes were
prepared by 3 passes through an EmulsiFlex-C5 homogenizer (Avestin
Inc., Ottawa, Canada), and the resulting membrane pellet was
solubilized in 20 mM Tris, 200 mM NaCl, pH 7.4 (Tris buffer), containing 20 mM dodecyl maltoside. The
supernatant was incubated with nickel nitrilotriacetic acid-agarose
(Qiagen, Valencia, CA) and washed with Tris buffer containing 2 mM dodecyl maltoside (wash buffer). Bound proteins were
successively eluted with wash buffer containing 20, 40, and 250 mM imidazole. Fractions were resolved by 12% SDS-PAGE and
stained with Coomassie Blue.
Transport Assay--
Transport of
L-[5-3H]tryptophan (Amersham Biosciences, TRK
460), L-[U-14C]proline (Amersham Biosciences,
CFB.71), and L-[2,5-3H]histidine (Amersham
Biosciences, TRK 199) into intact cells was measured as follows:
CY15212 cells were grown overnight from a 15% glycerol stock in LB
broth in the presence of both ampicillin (100 µg/ml) and kanamycin
(25 µg/ml) at 37 °C with shaking. In the morning, the culture was
diluted 100-fold into the same medium, and the incubation was continued
until the culture reached an A600 of 0.6.
200 µl of cell suspension (corresponding to 30 µg of protein) was
added to each well of a Multiscreen-FB 96-well filtration plate
(Millipore, Bedford, MA) that was previously soaked in 0.1% polyethyleneimine for 1 h. The membranes were washed twice by filtration with 200 µl of M9 minimal medium buffer
(NaH2PO4, 88.4 mM;
KH2PO4, 21.6 mM; NaCl, 8.4 mM; NH4Cl, 18.3 mM) including 0.4% glucose (35) at room temperature, and transport was initiated by the
addition of 200 µl of the same medium containing ~100,000 cpm of
radiolabeled substrate (see above).
The cells were incubated with substrate for 20 s (for tryptophan
and proline) or 10 min (for histidine) at room temperature with gentle
agitation, and then the reaction was terminated by washing the cells
three times with 200 µl of buffer. The filters from each individual
well were removed and placed in scintillation vials containing 3 ml of
Optifluor scintillation fluid (Packard Instrument Co.). The filters
were allowed to soak for 2 h and were then counted.
Because histidine nonspecifically bound to the filters contributing to
high background values, transport reactions with radiolabeled histidine
were carried out in microcentrifuge tubes with cells that were
previously washed two times with M9 minimal buffer + 0.4% glucose.
Transport was initiated by addition of radiolabeled histidine and
terminated 10 min later by a 30-s centrifugation to remove
nontransported substrate and a subsequent filtration step through glass
microfiber filters (Whatman catalogue number 934-AH) that were
previously soaked in 0.1% polyethyleneimine for 1 h.
Data Analysis--
Nonlinear regression fits of
experimental and calculated data were performed with Origin (Microcal
Software, Northampton, MA), which used the Marquardt-Levenberg
nonlinear least squares curve fitting algorithm. Each figure shows a
representative experiment that was performed at least twice. The
statistical analysis given in the text was from multiple experiments.
Unless indicated otherwise, data with error bars represented the
mean ± S.D. for four samples from two separate experiments.
S. thermophilum is a symbiotic thermophile (30) that
produces thermostable tryptophanase and
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid
(GABA),1 dopamine,
norepinephrine, and 5-hydroxytryptamine (5-HT, serotonin), are
transported by proteins belonging to the family designated the
neurotransmitter:sodium symporter (NSS) family 2.A.22 by Saier (2).
Glutamate, however, is transported by a family of mono- and
dicarboxylic amino acid transporters, the dicarboxylate/amino acid:cation symporters family (2). Proteins in both families play
important roles in brain function as indicated by the profound behavioral effects of drugs that influence their activity, such as
cocaine and amphetamines, which interact with amine transporters in the
NSS family (3-12), and many antidepressant drugs that inhibit serotonin and norepinephrine transporters (13-17).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
aroP
, tnaB
), a construct
was prepared that would allow constitutive expression of the
transporter proteins. The constitutively active promoter for the
-lactamase gene (Ampr) was amplified by PCR from a
pBluescript II KS(+) plasmid (Stratagene, La Jolla, CA) using the
following primers: TATAAGATCTAGGTGGCACTTTTCGGGGAAATG, which
contains a BglII site, and
ATATCTAGAACTCTTCCTTTTTCAATATTATTG, which contains an
XbaI site. The PCR product was inserted into the pET-22b(+)
plasmid (Novagen catalogue number 69744-3) between the single
restriction sites for BglII and XbaI.
tnaT was removed from the pET26b(+) vector and inserted
downstream of the promoter, using restriction sites for NdeI
and HindIII.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-tyrosinase enzymes (32, 36). In cloning the tryptophanase gene from this organism, we discovered a downstream sequence with high homology to mammalian neurotransmitter transporters. The sequence was deposited as AB010832 and identified as a putative tryptophan transporter by virtue of its
location in the tryptophanase operon and its similarity to other
transporters in the sodium-dependent neurotransmitter transporter family. Fig. 1 shows an
alignment of the sequence with the full consensus sequence of the NSS
family (pfam00209) from the Conserved Domain data base (37) and with
the sequence of rat serotonin transporter (SERT), whose substrate,
5-HT, is closest to tryptophan among the known neurotransmitter
transporters.
View larger version (54K):
[in a new window]
Fig. 1.
Alignment of consensus NSS, rat SERT, and
tnaT polypeptide sequences. The consensus NSS sequence was
obtained from the conserved domains data base (37) as pfam00209. The
three sequences were aligned using ClustalW (34) with some manual
adjustment and shaded with the BOXSHADE program with a black
background for identity and a gray background for
similarity. Transmembrane domain assignment was based on output from
MEMSAT2 (47) with some manual adjustment.
Wild type E. coli cells express endogenous
transporters capable of catalyzing tryptophan influx. Fig.
2 shows a time course of tryptophan
accumulation by E. coli K12 (open circles). To
analyze tryptophan transport resulting from expression of the S. thermophilum tnaT gene, we used E. coli strain CY15212,
obtained from the Yale E. coli Stock Center (CGSC 7672).
This strain is inactivated in the three genes, mtr,
tnaB, and aroP, that encode tryptophan
transporters (38). As shown in Fig. 2 (squares), this strain
is incapable of accumulating [3H]tryptophan. However,
transformation of CY15212 with an expression plasmid encoding the
S. thermophilum tnaT gene led to robust
[3H]tryptophan uptake, as shown in Fig. 2 (filled
circles).
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A consequence of tryptophan uptake by cells expressing TnaT is that the
transporter facilitated growth of cells on tryptophan-containing minimal media. Fig. 3 shows the dramatic
increase in growth by BL21 cells transformed with pET26-TnaT over the
first 12 h of incubation (filled circles) in contrast
to the relatively slow growth in Trp-free medium or by control cells
lacking the tnaT insert. The increase in cell growth
indicates that tryptophan taken up by cells is accumulated within the
cell where it can be metabolized and is not merely bound to the cell
surface.
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Trp transport by TnaT was saturable, as shown in Fig.
4. Under the conditions used, the
Vmax for transport was 242 ± 9 pmol per
min per mg of cell protein, and the Km was 145 ± 14 nM. The inset of Fig. 4 shows an
Eadie-Hofstee (39) transformation of the transport rate data. The rate
shows simple saturation with tryptophan concentration.
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To test the specificity of tnaT-encoded tryptophan
transport, we measured the initial rate of transport in the presence of 100 µM concentrations of the 20 naturally occurring amino
acids and also cystine, trans-proline, tryptamine, and serotonin. The results are shown in Fig. 5. Aside from
tryptophan, none of the amino acids tested significantly inhibited
tryptophan influx. A small inhibition was observed with tryptamine and
serotonin, and the concentrations of these amines that inhibited influx
by 50% were found to be 200 ± 18 and 440 ± 16 µM, respectively (not shown). With one exception,
inhibitors of mammalian biogenic amine transporters also failed to
block TnaT-mediated tryptophan transport. The following compounds
failed to inhibit at 100 µM (not shown): imipramine, desipramine, fluoxetine, citalopram, nomifensine, mazindol, GBR-12909, GBR-12935, amphetamine, and
3,4-methylenedioxymethamphetamine. Cocaine (1 mM) also did
not inhibit (not shown). Sertraline, a serotonin reuptake inhibitor
with nM affinity, inhibited tryptophan uptake with a
KI of 98 ± 3 µM (not shown).
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In TM1 of all amino acid transporters in the NSS family, there is a
glycine residue that is replaced by aspartate in the biogenic amine
transporters. In TnaT, the corresponding residue is a glycine at
position 24. Mutation of this residue to an aspartate led to a
transporter that was unable to transport tryptophan (Fig.
6A), serotonin, or
1-methyl-4-phenylpyridinium (not shown). This was not due to a
block in expression because the His6-tagged G24D mutant was
expressed in the membrane of CY15212 cells at 69 ± 15%
(n = 3) of the level of wild type
His6-tagged TnaT (Fig. 6B).
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A defining characteristic of the sodium-dependent
neurotransmitter transporter family is the requirement for sodium ions. In almost all of the family members studied, sodium is required, and
the transmembrane sodium gradient provides a driving force for solute
accumulation. In many of the transporters in this family, chloride ion
is also required, and the Cl gradient also provides part
of the driving force. The data in Fig. 7
show that TnaT absolutely requires Na+ but not
Cl
for transport. Transport was minimal below 0.1 mM Na+ and was half-maximal at ~1
mM Na+. The inset shows that influx
was essentially the same in NaCl and sodium isethionate medium but was
not detectable in medium in which
N-methyl-D-glucamine-Cl replaced NaCl. Thus,
tryptophan transport catalyzed by the tnaT gene
product was Na+-dependent but not
Cl
-dependent.
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Neurotransmitter transport utilizes transmembrane ion gradients. Within
the NSS family, the coupled inward movement, or symport, of
Na+ and neurotransmitter molecule is an almost universal
feature. However, at least one member, the K+-coupled amino
acid transporter, catalyzes substrate symport with K+ as
well as Na+ (40). Most of the known transporters in the NSS
family also require Cl, which is symported with substrate
in the cases that have been examined (41). Among bacterial transport
systems, some are energized directly by ATP, whereas others are coupled
to transmembrane ion gradients.
As an approach to determine the driving force used by TnaT to
accumulate tryptophan, we used 2,4-dinitrophenol (DNP), a proton ionophore, to dissipate the transmembrane electrochemical potential for
H+ (
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For purification of the TnaT protein, the gene was tagged at the N
terminus with 6 histidine residues and expressed in BL21(DE3)/pLysE using a T7 promoter-mediated expression system as described under "Materials and Methods." After induction by IPTG, the cells were disrupted; membranes were isolated, and the membrane proteins were
extracted with dodecyl maltoside. The His-tagged TnaT protein was
purified using nickel chromatography and analyzed by SDS-PAGE. The
purified protein migrated as a single band of ~45 kDa relative to the
predicted molecular size of the tagged construct of 57,258 kDa (Fig.
8). In prokaryotic proteins the
N-terminal Met is often cleaved, which would give a predicted mass of
57,127 kDa. Preliminary matrix-assisted laser desorption ionization
mass spectrometry analysis of the purified TnaT gave a molecular mass
of 57,012 ± 44 kDa (mean ± S.D., n = 2) or
99.8% of the predicted mass, suggesting that the protein is
full-length and unmodified. From an initial culture of 1 liter, we
obtained ~0.5 mg of highly purified protein.
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DISCUSSION |
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The tnaT gene is typical of an increasing number of prokaryotic sequences with striking homology to the NSS family of Na+-coupled neurotransmitter and amino acid transporters. To illustrate this observation, a BLAST search (46) of GenBankTM was performed using a highly conserved portion of the consensus sequence (37) for the NSS family. At the time the search was performed, it yielded, in addition to tnaT, more than 40 other sequences from bacteria and Archaea, although no significant matches were found in sequences from yeast, fungi, or plants. The sequence similarities between the predicted prokaryotic proteins, including TnaT, and mammalian members of the NSS transporter family are extensive (Fig. 1). For the 30 prokaryotic sequences with the greatest sequence similarity to the NSS family, the MEMSAT2 transmembrane topology prediction method (47) found 12 well aligned transmembrane domains (TM) in 12 of these sequences, including TnaT, 11 TMs in 16 other sequences, and 10 TMs in 2 sequences. The sequences containing fewer than 12 TMs lacked the last one or two TMs and were homologous with the 12 TM sequences through the first 10 or 11 TMs.
The apparent variability in the lengths of these transporter-like sequences highlights the uncertainty regarding their function. Without an unequivocal demonstration that these sequences encode functional transporters, we cannot rule out the possibility that they are responsible for other membrane functions. The characterization of TnaT as a functional Na+-coupled tryptophan transporter opens up this large family of orphan transporters to experimental study. We hope that these studies will shed light also on the structure and function of eukaryotic NSS neurotransmitter transporters. For example, if we find that those proteins with 10 or 11 TMs are functional as transporters, it will help to define which TMs are required for substrate binding, ion coupling, and other functions.
Like almost all of the transporters in the NSS family, TnaT required
Na+ for its function, although unlike many other NSS
transporters, we found that Cl was not required (Fig. 7).
Moreover, TnaT-mediated Trp influx saturates at relatively low
concentrations (Fig. 4) and is highly selective, similar to other NSS
transporters. This high degree of functional and sequence similarity
between TnaT and the mammalian members of the NSS family is even more
remarkable in light of the complete absence of homologous sequences in
yeast, fungi, or higher plants. This situation was found also in the
sodium:solute symporter family (2), which also has many members in
prokaryotes and animals but almost none in yeast, fungi, or higher
plants (48).
One possible reason for the restricted distribution of this family to some prokaryotes and animals lies in the Na+ dependence of the NSS family. Prokaryotic and animal cells are known to maintain transmembrane Na+ gradients that are used for driving metabolite accumulation. Yeast and plants, however, extrude Na+ but rarely use solute-Na+ symport for metabolite accumulation (49, 50). If the coupling of substrate transport to Na+ is an integral property of NSS transporters, and they exclusively function, therefore, only as Na+ symporters, the restriction of their distribution to organisms that utilize a transmembrane Na+ gradient for metabolite transport would be understandable. This may also explain the distribution among prokaryotes. For example, among bacilli, Bacillus halodulans, an alkaliphilic species that uses the Na+ gradient as a driving force, has an NSS homologue (51), whereas Bacillus subtilis, a neutralophile, does not (52). A consequence of this explanation, however, is that all bacterial NSS proteins should catalyze Na+ symport, a prediction that we are currently testing.
There is a high degree of sequence identity between TnaT and mammalian transporters for serotonin (21%), and dopamine (24%), and the consensus NSS sequence from the Conserved Domains data base (37) (30%). A similar degree of identity was found with mammalian GABA, betaine, and taurine transporters. In particular, the identity is strongest in the region beginning before TM1 and ending in TM2, where there is also strong homology among previously known members of the NSS family. The most striking difference in the primary structure is in EL2, between TM3 and TM4, which is much shorter in TnaT than in other NSS family members. Two cysteine residues thought to exist as a disulfide in SERT (53) and DAT (54) are absent. In fact, the entire TnaT sequence contains only one cysteine, near the beginning of TM5.
The TnaT sequence contains a glycine residue at position 24 in TM1. This position corresponds to the location of a glycine in all of the NSS amino acid transporters and an aspartate in all of the NSS biogenic amine transporters. In SERT, this residue has been implicated in the recognition of substrates and inhibitors (55) and was proposed to interact with the ligand amino group. Consistent with the lack of an aspartate at this position, the amines tryptamine and 5-HT (serotonin) were poor inhibitors of TnaT-mediated Trp transport (Fig. 5). Likewise, cocaine, an inhibitor of all NSS amine transporters, was ineffective as an inhibitor, as were a variety of other NSS amine transporter inhibitors. Mutation of Gly-24 to Asp as found in SERT, NET, and DAT ablated tryptophan transport but did not bestow upon TnaT the ability to transport 5-HT or 1-methyl-4-phenylpyridinium, consistent with the existence of multiple determinants of substrate specificity.
In TM3, several residues have been implicated in the binding and transport of substrates by NSS transporters. A tyrosine at position 176 in SERT, conserved as Tyr-102 in TnaT, has been implicated as a binding site residue in SERT (56) and GAT-1 (57) and is conserved in all NSS transporters. Also conserved is an isoleucine at position 105 of TnaT that corresponds to Ile-179 of SERT and Ile-155 of NET. These residues were proposed to form part of the gate that closes to prevent access to the substrate-binding site in the internal facing form of the transporter (58). An isoleucine at position 172 in SERT (proposed to be in proximity to the substrate site) is conservatively replaced by Val-98 in TnaT. This residue is a valine also in DAT but is not particularly conserved throughout the NSS family. Additional TM3 residues that are conserved between SERT, DAT, and TnaT are Ile-106, Trp-108, Leu-110, Tyr-112, and Leu-113.
The majority of ion-coupled bacterial transporters utilize
H+ symport to accumulate metabolites within the cell,
although Na+ influx is coupled to solute uptake by
some. The primary driving force for both types of transport system is
the respiratory chain that, in E. coli, pumps H+
ions out of the cell, creating a transmembrane pH difference and an
electrical potential that together comprise the electrochemical H+ potential,
The Na+ requirement for TnaT-mediated transport is likely
to reflect Na+-Trp symport, although the results presented
here do not rule out a possible H+-Trp symporter with an
external Na+ requirement. The sensitivity to uncoupling by
DNP suggests that the
As targets for major therapeutic drugs as well as drugs of abuse, neurotransmitter transporters in the NSS family are an important focus of research in neuroscience. Structural and mechanistic studies of the NSS transporters have revealed the ion coupling stoichiometry and some mechanistic aspects of these proteins (61, 62). Their topological orientation has been studied extensively (63-69). Some residues have been identified as potential sites of substrate and inhibitor binding (55-57, 70, 71), and others have been shown to change their orientation or accessibility in response to ligand binding or conformational changes that accompany transport (58, 67-69, 72, 73).
Nevertheless, many questions about the structure and mechanism of the
NSS family remain unanswered. Part of the problem lies in the
relatively low abundance of these proteins and their lability during
purification. Among transporters in this family, only the GABA
transporter has been purified in a reconstitutively active form (74),
and attempts with other members of the family have met with difficulty
(27). The discovery that a bacterial homologue of these proteins is a
functional transporter with similar characteristics to the mammalian
transporters represents an important step toward addressing questions
about their structure and mechanism. Our demonstration that TnaT can be
expressed in the cell membrane and purified in high yield (Fig. 8) will
allow biochemical and structural approaches that have not been
available previously for understanding the structure and function of
the NSS family of neurotransmitter transporters.
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ACKNOWLEDGEMENTS |
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We thank Drs. Howard Gu, Clifford Slayman, and H. Ronald Kaback for help during the execution of this work and the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the National Institute on Drug Abuse (to G. R.), a James Hudson Brown-Alexander Brown Coxe postdoctoral fellowship (to A. A.-T.), the High Tech Research Center Project of The Ministry of Education, Science, Sports, and Culture of Japan (to T. B.), and by National Institutes of Health Grants DA014942 and MH57324 (to J. A. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be
addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., P. O. Box 3333, New Haven, CT 06510. Tel.:
203-785-4548; Fax: 203-737-2027; E-mail: gary.rudnick@yale.edu.
Published, JBC Papers in Press, February 4, 2003, DOI 10.1074/jbc.M206563200
2 A. Diez-Sampedro, B. A. Hirayama, E. M. Wright, and H. Koepsell, personal communication.
3 K. Ueda and T. Beppu, GenBankTM accession number BAA24689.2.
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ABBREVIATIONS |
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The abbreviations used are:
GABA, -aminobutyric acid;
5-HT, 5-hydroxytryptamine;
NSS, neurotransmitter:sodium symporter;
DNP, 2,4-dinitrophenol;
DCCD, N,N'-dicyclohexylcarbodiimide;
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
SERT, serotonin
transporter;
TM, transmembrane;
DAT, dopamine transporter;
NET, norepinephrine transporter.
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REFERENCES |
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