THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 38, pp. 28174 –28184, September 22, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
SNAP-25/Syntaxin 1A Complex Functionally Modulates
Neurotransmitter ␥-Aminobutyric Acid Reuptake*□
S
Received for publication, February 13, 2006, and in revised form, May 30, 2006 Published, JBC Papers in Press, July 21, 2006, DOI 10.1074/jbc.M601382200
Neurotransmitter ␥-aminobutyric acid (GABA) release to the
synaptic clefts is mediated by the formation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) complex, which includes two target SNAREs syntaxin
1A and SNAP-25 and one vesicle SNARE VAMP-2. The target
SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the
putative intermediate of the SNARE complex. Neurotransmitter GABA clearance from synaptic clefts is carried out by the
reuptake function of its transporters to terminate the postsynaptic signaling. Syntaxin 1A directly binds to the neuronal
GABA transporter GAT-1 and inhibits its reuptake function.
However, whether other SNARE proteins or SNARE complex
regulates GABA reuptake remains unknown. Here we demonstrate that SNAP-25 efficiently inhibits GAT-1 reuptake function in the presence of syntaxin 1A. This inhibition depends on
SNAP-25/syntaxin 1A complex formation. The H3 domain of
syntaxin 1A is identified as the binding sites for both SNAP-25
and GAT-1. SNAP-25 binding to syntaxin 1A greatly potentiates
the physical interaction of syntaxin 1A with GAT-1 and significantly enhances the syntaxin 1A-mediated inhibition of GAT-1
reuptake function. Furthermore, nitric oxide, which promotes
SNAP-25 binding to syntaxin 1A to form the SNARE complex,
also potentiates the interaction of syntaxin 1A with GAT-1 and
suppresses GABA reuptake by GAT-1. Thus our findings delineate a further molecular mechanism for the regulation of GABA
reuptake by a target SNARE complex and suggest a direct coordination between GABA release and reuptake.
␥-Aminobutyric acid (GABA)3 is the major inhibitory neurotransmitter in the central nervous system, which is released
* This work was supported by the Ministry of Science and Technology Grants
2003CB515405 and 2005CB522406 and the National Natural Science
Foundation of China Grants 30021003 and 30325024. The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1 and 2.
1
To whom correspondence may be addressed. Tel.: 86-21-54921369; Fax:
86-21-54921762; E-mail: baolan@sibs.ac.cn.
2
To whom correspondence may be addressed. Tel.: 86-21-54921371; Fax:
86-21-54921011; E-mail: gpei@sibs.ac.cn.
3
The abbreviations used are: GABA, ␥-aminobutyric acid; SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein receptor; t-SNARE,
target SNARE; YFP, yellow fluorescent protein; GFP, green fluorescent protein; CFP, cyan fluorescent protein; HA, hemagglutinin; siRNA, small interference RNA; PMSF, phenylmethylsulfonyl fluoride; FRET, fluorescence
28174 JOURNAL OF BIOLOGICAL CHEMISTRY
from the presynaptic terminals through the docking and fusion
of synaptic vesicles with the plasma membrane. Membrane
fusion and subsequent GABA release are catalyzed by the
assembly of a ternary complex from soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins
(1). The ternary complex is composed of two plasma membrane
proteins, including syntaxin 1A and synaptosomal associated
protein of 25 kDa (SNAP-25), which are called target SNAREs
(t-SNAREs), and one vesicle-associated protein synaptobrevin
2 (VAMP-2), which is called vesicle SNARE (2, 3). The
t-SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the
putative intermediate of the SNARE complex, and offer target
sites for the vesicle SNARE VAMP-2 leading to membrane
fusion (4, 5). After membrane fusion, GABA is released from
synaptic vesicles to synaptic clefts and binds to postsynaptic
GABA receptors and thus transmits the signal to the postsynaptic terminals.
GABA is cleared away rapidly from synaptic clefts to terminate synaptic transmission through the reuptake function of its
specific, high affinity, sodium- and chloride-dependent transporters (6), which are located on presynaptic terminals and surrounding glial cells (7). GABA transporters are mainly divided
into four subtypes, including GAT-1, GAT-2, GAT-3, and
BGT-1, of which GAT-1 is the most abundant neuronal subtype (8). GAT-1, GAT-2, and GAT-3 have 12 transmembrane
regions, with both N and C termini facing intracellularly.
GAT-2 and GAT-3 display about 52% amino acid identity with
GAT-1 (8). It is generally believed that GABA reuptake from
the synaptic clefts is an important mechanism in the regulation
of GABA activity in synaptic neurotransmission (9). Specific
GABA transporter inhibitors are revealed to prolong the decay
phase of GABA, type A, receptor-mediated postsynaptic potential (10) and to increase the magnitude of responses mediated
by the G protein-coupled GABA, type B, receptor (6, 10, 11).
Neurotransmitter transporters are regulated through a variety
of signal transduction mechanisms that maintain appropriate
levels of transmitter in the synaptic clefts. Both signaling molecules, including extracellular substrate (12, 13) and intracellular second messengers such as kinases and phosphatases (14),
and the proteins directly binding to the neurotransmitter transporters are known to act on the transporters and modulate their
function. Recent reports of transporters regulated by directly
resonance energy transfer; PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; NTG, nitroglycerine; Cy3, indocarbocyanine; Cy5,
indodicarbocyanine.
Supplemental Material can be found at:
http://www.jbc.org/cgi/content/full/M601382200/DC1
VOLUME 281 • NUMBER 38 • SEPTEMBER 22, 2006
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
Hua-Ping Fan‡§, Feng-Juan Fan‡§, Lan Bao‡1, and Gang Pei‡2
From the ‡Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences and the §Graduate School of Chinese Academy of Sciences, 320 Yue Yang Road,
Shanghai 200031, China
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
EXPERIMENTAL PROCEDURES
Plasmids—The full lengths of SNAP-25 and VAMP-2 were
cloned into modified pcDNA3 vector in-frame with HA at the
N terminus. G43D-GFP plasmid was obtained from Dr. Maurine E. Linder, and G43D was cloned into pcDNA3 vector with
HA at the N terminus. Plasmid DNA of syntaxin 1A and syntaxin 1A⌬H3 were gifts from Dr. Anjaparavanda P. Naren and
Dr. Kevin L. Kirk and were cloned into the pcDNA3 vector
in-frame with FLAG at the N terminus. Syntaxin 1A⌬H3 was
also cloned into the pcDNA3 vector with CFP at the N terminus. Plasmid DNA of GAT-1, EAAC1, GLT1, and GLAST were
provided by Dr. Jian Fei. The full lengths of GAT-1, GAT-2,
GAT-3, EAAC1, GLT1, and GLAST were cloned into modified
pcDNA3 vector in-frame with HA at the N terminus. GAT-2 and
GAT-3 were also cloned into the pcDNA3 vector with YFP at the
N terminus. Constructions of RNA interference plasmids were
described in our previous paper (25). The nucleotide sequences for
the small interference RNAs (siRNA) were 5⬘-GGCCGCAAAGACCTTGTCCTTA-3⬘ for syntaxin 1A, 5⬘-GGAGCAGATGGCCATCAGTGA-3⬘ for SNAP-25, and 5⬘-GGACCAGAAGCTATSEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38
CGGAACTA-3⬘ for VAMP-2, respectively. The siRNA for
SNAP-25 was also cloned into the BS/U6-GFP vector.
Cell Culture and Transfection—Primary cultured hippocampal neurons were prepared from 1-day-old postnatal SpragueDawley rats using the method described previously (26). To
obtain purely neuronal cultures, the Dulbecco’s modified
Eagle’s medium was replaced at 4 – 6 h later with Neurobasal-A
medium containing B27 serum-free supplement (Invitrogen)
for hippocampal neuronal culture. Twenty four hours later, the
cultures were treated with 5 ␮M cytosine arabinoside for 72 h.
The neurons were transfected using the rat neuron nucleofector kit for primary rat hippocampal or cortical neurons (Program O-03 or G-13, Amaxa Biosystems). In brief, we resuspended the prepared neurons into rat neuron nucleofector
solution at room temperature to a final concentration of 4 –5 ⫻
106 cells/100 ␮l. We then mixed 100 ␮l of cell suspension with
1–3 ␮g of DNA and transferred the nucleofection sample into a
certified cuvette (Amaxa Biosystems). The cuvette was inserted
into the cuvette holder, and program O-03 or G-13 was run.
After the addition of 500 ␮l of pre-warmed Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal
calf serum, the cell was transferred into the prepared 24-well
plates and incubated in a humidified 5% CO2 incubator at 37 °C.
After 2– 4 h, we carefully replaced the medium with 750 ␮l of
fresh Neurobasal-A medium containing B27 serum-free supplement (Invitrogen) to remove cellular debris. The transfection
efficiency was 50 –70%.
PC12 cells and HEK293 cells were obtained from the American Type Culture Collection. PC12 cells were maintained in
F12K medium (Invitrogen) supplemented with 2 mM L-glutamine, 5% fetal bovine serum, 10% horse serum, 100 units/ml
penicillin, and 100 units/ml streptomycin. Cells were transfected using Lipofectamine 2000 (Invitrogen). HEK293 cells
were maintained in minimum Eagle’s medium (Invitrogen)
supplemented with standard supplements. Transient transfection of HEK293 cells was by using the calcium phosphate
method.
[3H]GABA Uptake Assay—GABA uptake in cultured neurons and transfected PC12 or HEK293 cells was performed as
described previously (27). The cells were grown in a monolayer
on 24-well plates. The cells were incubated in Krebs-Ringer/
HEPES (KRH) medium, pH 7.4, containing 120 mM NaCl, 4.7
mM KCl, 2.2 mM CaCl2, 25 mM HEPES, 1.2 mM MgSO4, 1.2 mM
KH2PO4, and 10 mM glucose at 37 °C for 30 min, and then 10 nM
[3H]GABA (Amersham Biosciences) and 30 ␮M unlabeled
GABA (Sigma) were added to initiate the uptake. Nonspecific
uptake was determined with sodium-free KRH medium in
which choline chloride was used instead of NaCl. After incubation for 5–15 min, the uptake was terminated by two ice-cold
washes with 500 ␮l of sodium-free KRH medium, followed by
immediate lysis in 200 ␮l of ice-cold 0.1 M NaOH. 100 ␮l of
lysate was loaded onto glass fiber filters and then analyzed for
radioactivity. Finally, the filters containing cellular lysate were
processed for scintillation counting (Beckman Instruments).
Under these conditions the uptake is linear with time. The protein quantity was measured using the BCA kit (Pierce). The
GABA uptake activity was measured as fmol/min/mg protein.
Data were from at least three separate experiments, two samJOURNAL OF BIOLOGICAL CHEMISTRY
28175
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
binding proteins come from the investigations that the transporters, such as the inhibitory transmitter glycine transporter
(15), the major excitatory transmitter glutamate transporter
(16), serotonin transporter (17, 18), and norepinephrine transporter (19), are functionally regulated by the t-SNARE syntaxin
1A. As to the GABA transporters, it is reported that syntaxin 1A
interacts with the GABA transporter GAT-1 and results in a
decrease of its reuptake function (20). Syntaxin 1A positively
regulates GAT-1 surface expression (21) but exerts its inhibitory actions by directly binding to GAT-1 and decreasing its
transport rate (22). These investigations demonstrate a direct
link between neurotransmitter release and reuptake. However,
it remains largely unknown whether other SNARE proteins or
the SNARE complex regulate GABA reuptake. Further coordination between neurotransmitter release and reuptake needs to
be explored.
Increasing studies from the ion channels indicate that Kv2.1
potassium channel and cystic fibrosis transmembrane regulator chloride channel, which reside on the plasma membrane to
control the intracellular or extracellular concentrations of
respective ions, are functionally regulated by the t-SNARE
complex (23, 24). Because the t-SNARE complex acts on the ion
channels, and syntaxin 1A physically interacts with and functionally regulates those neurotransmitter transporters, it is
most likely that other SNARE proteins or the SNARE complex
may modulate those transporters. In this study, we found that
SNAP-25 significantly inhibited GABA transporter GAT-1
reuptake function. The physical and functional interactions
between SNAP-25 and GAT-1 depended on syntaxin 1A, and
SNAP-25 binding to syntaxin 1A inhibited GAT-1 reuptake
function through enhancing the physical interaction of syntaxin 1A with GAT-1. Our results also provided the evidence
that the SNARE complex formation promoted the direct binding of syntaxin 1A to GAT-1 and resulted in the inhibition of
GABA reuptake by GAT-1, elucidating a coordination of neurotransmitter release and reuptake.
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
28176 JOURNAL OF BIOLOGICAL CHEMISTRY
7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 250
␮M PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 mM sodium
orthovanadate, 5 mM sodium pyrophosphate), and the supernatants were incubated with antibodies overnight at 4 °C before
Protein G-agarose beads were added. The brains were removed
from 1-day-old postnatal Sprague-Dawley rats. Brain samples
were homogenized in ice-cold buffer (4 mM Tris-Cl, pH 7.4, 1
mM EDTA, 0.32 M sucrose, 10 mM glucose, 250 ␮M PMSF, 1
␮g/ml aprotinin, and 1 ␮g/ml leupeptin) in a glass-Teflon
homogenizer. Homogenates were centrifuged, and the supernatants were spun at 4 °C. The pellets were suspended in the
lysis buffer for neurons described above at 4 °C for 1 h and then
were centrifuged. The supernatants were incubated with antibodies (rabbit anti-GAT-1/2/3; Chemicon) overnight at 4 °C
before Protein G-agarose beads were added. Proteins in the
immunoprecipitates were analyzed by standard Western
blotting.
Fluorescence Resonance Energy Transfer (FRET) Measurements—FRET measurements were performed essentially as
described (31). Image acquisition and determination of FRET efficiency by acceptor photobleaching were obtained using the Leica
TCS SP2 confocal microscope and analytical software. Briefly,
emission spectra from the cells expressing CFP-syntaxin 1A or
CFP-syntaxin 1A⌬H3 and YFP-GAT-1 or YFP-GAT-2 or YFPGAT-3 were obtained with the ␭ mode, using the 405 nm line of
laser. For measurement of FRET efficiency by this method,
Leica software application for acceptor photobleaching was
applied. The selected cell surface areas were photobleached of
YFP-GAT-1 or YFP-GAT-2 or YFP-GAT-3 with 514 nm line of
laser. Reduction of the YFP signal after photobleaching for
CFP-syntaxin 1A and YFP-GAT-1 cotransfected PC12 cells was
on average 78 ⫾ 4.8% (n ⫽ 50). FRET was resolved as an
increase in the CFP-syntaxin 1A (donor) signal after photobleaching of YFP-GAT-1 (acceptor). Relative FRET efficiency
was calculated as (1 ⫺ (CFP Ipre-bleach/CFP Ipost-bleach)) ⫻ 100%.
For control purposes, an area of the cell surface without photobleaching was also analyzed for FRET. The cells transfected
with HA-SNAP-25 plasmid were confirmed by detection of Cy5
fluorescence followed by incubation with primary antibody
against HA and a second antibody conjugated with Cy5 (Jackson ImmunoResearch), whereas the cells transfected with GFP/
SNAP-25 siRNA were identified through GFP fluorescence
detection.
Statistical Analysis—The quantification was based on at least
three independent experiments. The results were presented as
mean ⫾ S.E. Statistics differences were determined by Student’s t test for two group comparisons.
RESULTS
SNAP-25/Syntaxin 1A Are Intrinsically Involved in the Inhibition of GAT-1 Uptake Function—It has been shown that syntaxin 1A inhibits the uptake function of GABA transporter
GAT-1 in hippocampal neurons (20, 22). As shown in Fig. 1A,
10 ␮M NO-711, a selective GAT-1 blocker (32), eliminated
88.9 ⫾ 7.5% of GABA uptake in primary cultured hippocampal
neurons, confirming the previous report (7) that the GABA
uptake is mainly contributed by GAT-1 in hippocampal neurons. To investigate whether other SNARE proteins or SNARE
VOLUME 281 • NUMBER 38 • SEPTEMBER 22, 2006
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
ples or two wells per treatment per experiment. In the assay of
GABA uptake with drug treatment, NOC-18 (Calbiochem) or
NTG (Sigma) or NOC-18/PTIO (Calbiochem) or NO-711
(Sigma) was added into the uptake reaction buffer.
Cell Surface Biotinylation and Western Blotting—Cell surface biotinylation assay in transfected HEK293 cells or cultured
neurons was performed as described (28). NOC-18 was added
into culture medium without serum to stimulate the primary
cultured hippocampal neurons and then washed away before
the cell surface biotinylation labeling. The cells were incubated
with a solution containing 1 mg/ml sulfo-NHS biotin (Pierce) in
phosphate-buffered saline/Ca2⫹/Mg2⫹ for 20 min at 4 °C with
gentle shaking. The biotinylation solution was removed by two
washes in phosphate-buffered saline/Ca2⫹/Mg2⫹ plus 100 mM
glycine and quenched in this solution by incubating the cells at
4 °C for 45 min with gentle shaking. Then the cells were lysed in
1 ml of RIPA buffer (100 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1
mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
SDS, 1 ␮g/ml leupeptin, 1 ␮g/ml aprotinin, 250 ␮M PMSF) at
4 °C for 60 min. The cell lysates were centrifuged at 20,000 ⫻ g
at 4 °C for 60 min. The supernatant fractions were incubated
with Immunopure Immobilized Monomeric Avidin beads
(Pierce) at room temperature for 60 min. After efficient washing, the beads were incubated for 30 min at 50 °C in SDS-PAGE
loading buffer. Then the samples were analyzed by standard
Western blotting using rabbit anti-GAT-1 antibody (1:1000;
Abcam) and mouse anti-syntaxin 1A antibody (1:1000; Synaptic Systems). The bands were quantified with ScionImage software (Scion).
Immunocytochemistry—Immunofluorescence staining of
transfected PC12 cells was carried out as described (29, 30). The
PC12 cells cotransfected FLAG-syntaxin 1A and/or HA-SNAP-25
with GAT-1-GFP were incubated with mouse anti-FLAG
(1:500, Sigma) and/or rabbit anti-HA antibody (1:500, Sigma)
overnight at 4 °C, followed by second antibodies conjugated
with indocarbocyanine (Cy3) and indodicarbocyanine (Cy5)
(1:100; Jackson ImmunoResearch). The cells were then washed
and mounted with PermaFluor Mountant Medium (Thermo).
GAT-1-GFP was identified through GFP fluorescence detection. NOC-18 was added into the culture medium without
serum to stimulate PC12 cells transfected with GAT-1-GFP
and were washed away before immunohistochemistry. The
cells were incubated with mouse anti-syntaxin 1A (1:500; Synaptic Systems) and rabbit anti-SNAP-25 antibody (1:500;
Sigma) overnight at 4 °C and followed by second antibodies
conjugated with Cy3 and Cy5. The images were captured with
the Leica TCS SP2 confocal microscope. The cells exhibiting
distinct “ring” staining were quantified among 100 randomly
selected positive cells in each group of experiments.
Coimmunoprecipitation—Transfected HEK293 cells were
lysed in buffer (100 mM Tris-Cl, pH 7.4, 0.8% Triton X-100, 1
␮g/ml aprotinin, 1 ␮g/ml leupeptin, 250 ␮M PMSF). The supernatants were treated with protein G-agarose beads (Amersham
Biosciences) followed by incubation with the immunoprecipitated antibodies (mouse anti-syntaxin 1A, mouse anti-SNAP25, rabbit anti-GAT-1, and mouse anti-HA) at 4 °C for 2– 4 h.
Hippocampal neurons were lysed in buffer (9.1 mM dibasic
sodium phosphate, 1.7 mM monobasic sodium phosphate, pH
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
complex can modulate GAT-1 uptake function, we first examined the effects of down-regulating SNARE protein expression
on GAT-1 transport activity in primary cultured hippocampal
neurons. The suppression of syntaxin 1 or SNAP-25 expression
increased the GABA uptake in hippocampal neurons by transfection with their specific siRNAs, but suppressing VAMP-2
expression did not affect the GABA uptake. Moreover, suppressing syntaxin 1A and SNAP-25 expression simultaneously
further increased GABA uptake but suppressing the expression
of three SNARE proteins did not enhance this increase (Fig. 1B).
Western blotting analysis showed that RNA interference specifically and effectively mediated the silencing of endogenous
syntaxin 1A or SNAP-25 or VAMP-2 (Fig. 1B). The GABA
uptake in the neurons transfected with the nonspecific siRNA
(ranges from 696 to 914 fmol/min/mg protein) showed no difference from the GABA uptake in the neurons without transfection (ranges from 718 to 903 fmol/min/mg protein), indicatSEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY
28177
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
FIGURE 1. SNAP-25 and syntaxin 1A corporately inhibit GAT-1 uptake
function. A, [3H]GABA uptake in primary cultured hippocampal neurons
incubated with 1 or 10 ␮M NO-711 for 10 min. **, p ⬍ 0.01 as compared with
the control neurons without NO-711 incubation. GABA uptake in control neurons without incubation ranges from 718 to 903 fmol/min/mg protein.
B, [3H]GABA uptake in siRNA-transfected hippocampal neurons. The specific
siRNAs for syntaxin 1A, SNAP-25, and VAMP-2 were transfected in different
combinations indicated in the figure. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared
with the control neurons transfected with nonspecific siRNA or the neurons
indicated in the figure. GABA uptake in the control neurons transfected with
nonspecific siRNA ranges from 696 to 914 fmol/min/mg protein. Representative immunoblot shows that siRNA efficiently mediates the silencing of syntaxin 1A or SNAP-25 or VAMP-2 in the neurons. Actin is served as an internal
control for protein loading. C, [3H]GABA uptake in GAT-1-GFP-transfected
PC12 cells. Specific RNA interference or overexpression of FLAG-syntaxin 1A
(FLAG-Syn 1A) and HA-SNAP-25 was used to down-regulate or up-regulate the
expression of syntaxin 1A and/or SNAP-25 in GAT-1-GFP-transfected PC12
cells. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the control cells transfected with GAT-1-GFP and ␤-galactosidase or the cells indicated in the figure. GABA uptake in control cells transfected with GAT-1-GFP and ␤-galactosidase ranges from 1199 to 1598 fmol/min/mg protein. Representative
immunoblot (IB) shows that the expression of syntaxin or SNAP-25 is efficiently up-regulated or down-regulated by overexpression or siRNA. Data in
A–C are from three independent experiments, two wells per condition per
experiment.
ing that the siRNA itself has no significant effect on GAT-1
uptake function (Fig. 1, A and B). Furthermore, the GABA
uptake was detected in GAT-1-GFP transfected rat pheochromocytoma cells (PC12), a neuroendocrine cell line endogenously expressing the SNARE proteins (33) but having no
detectable [3H]GABA uptake (data not shown). The addition of
the fluoroprotein tags to the GAT-1 protein did not alter its
subcellular distribution and uptake function (27). In these
experiments specific RNA interference or overexpression was
used to down-regulate or up-regulate the expression of syntaxin 1A and/or SNAP-25. Our results showed that overexpression of syntaxin 1A or SNAP-25 significantly inhibited the
GAT-1 uptake function, and overexpression of both proteins
enhanced the inhibition (Fig. 1C). Similar to the results in hippocampal neurons, the down-regulation of syntaxin 1A and/or
SNAP-25 expression greatly increased the GABA uptake in
specific siRNA-transfected PC12 cells (Fig. 1C). These data
indicate that two target SNAREs, syntaxin 1A and SNAP-25,
intrinsically inhibit GAT-1 uptake function.
SNAP-25 Enhances the Syntaxin 1A-mediated Inhibition of
GAT-1 Uptake Function—In order to dissect the functional
relationship of syntaxin 1A and SNAP-25 on the inhibition of
GABA uptake, we cotransfected syntaxin 1A and/or SNAP-25
with GAT-1 in different combinations and different ratios into
HEK293 cells, a cell line with no endogenous expression of
GAT-1, syntaxin 1A, and SNAP-25 detected by Western blotting (Fig. 2, A and B) and no detectable [3H]GABA uptake (Fig.
3D). The cotransfection of syntaxin 1A with GAT-1 inhibited
the GABA uptake, and this inhibition was promoted by increasing syntaxin 1A expression (Fig. 2A). The cotransfection of syntaxin 1A and SNAP-25 with GAT-1 significantly enhanced the
syntaxin 1A-mediated inhibition of GABA uptake (Fig. 2, A and
B), and this enhanced inhibition was further promoted by
increasing SNAP-25 expression (Fig. 2B), whereas the cells
cotransfected SNAP-25 alone with GAT-1 showed no decrease
of GABA uptake (Fig. 2A, the ratio of transfected GAT-1 and
FLAG-syntaxin 1A plasmids is 1:0). However, the enhancement
of SNAP-25 on syntaxin 1A-mediated inhibition of GAT-1
uptake function depended on the ratio of syntaxin 1A and
SNAP-25 expression. When the ratio of cotransfected FLAGsyntaxin 1A and HA-SNAP-25 plasmids was 0.3:1 or 1:3,
SNAP-25 significantly enhanced the inhibition of GABA
uptake by syntaxin 1A (inhibition from 23.6 ⫾ 6.3% to 47 ⫾
3.9% or from 50.9 ⫾ 6.8% to 74.8 ⫾ 4.5%, respectively) (Fig. 2, A
and B). These findings indicate that SNAP-25 inhibits GAT-1
uptake function in the presence of syntaxin 1A, and SNAP-25
enhances the inhibition of GAT-1 uptake function mediated by
syntaxin 1A.
Because the inhibition of GAT-1 uptake function is mediated
by syntaxin 1A, to further identify whether the inhibition by
syntaxin 1A is special for the uptake function of GABA transporter GAT-1 subtype (599 amino acids), we examined the
effects of syntaxin 1A on the uptake function of GAT-2 (602
amino acids) or GAT-3 subtype (627 amino acids). As shown in
Fig. 2C, neither GAT-2 nor GAT-3 uptake function was
affected by syntaxin 1A in cotransfected HEK293 cells.
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
FIGURE 3. Syntaxin 1A mediates the physical and functional interactions
between GAT-1 and SNAP-25. A, coimmunoprecipitation and immunoblotting in primary cultured hippocampal neurons. Proteins were coimmunoprecipitated with GAT-1 antibody or IgG (negative control). The endogenous
syntaxin 1A and SNAP-25 are coimmunoprecipitated with GAT-1. IP, immunoprecipitated; IB, immunoblot. B, coimmunoprecipitation and immunoblotting in transfected HEK293 cells confirm the interaction of syntaxin 1A/syntaxin 1A⌬H3 with SNAP-25/G43D, syntaxin 1A/syntaxin 1A⌬H3 with GAT-1.
Proteins were coimmunoprecipitated with GAT-1 and syntaxin 1A antibodies.
C, coimmunoprecipitation and immunoblotting in transfected HEK293 cells
detect the physical interaction between GAT-1 and SNAP-25. Different combinations of GAT-1, FLAG-syntaxin 1A/FLAG-syntaxin 1A⌬H3, HA-SNAP-25/
HA-G43D plasmids were cotransfected. Proteins were coimmunoprecipitated with SNAP-25 or GAT-1 antibody. The data in A–C represent three
independent experiments. D, [3H]GABA uptake in cotransfected HEK293 cells.
Different combinations of GAT-1, FLAG-syntaxin 1A/FLAG-syntaxin 1A⌬H3,
HA-SNAP-25/HA-G43D, and HA-VAMP-2 plasmids were cotransfected in
HEK293 cells as indicated in the figure. The ratio of transfected GAT-1, FLAGsyntaxin 1A/FLAG-syntaxin 1A⌬H3, and HA-SNAP-25/HA-G43D plasmids is
1:0.3:1. Representative immunoblot shows the expression status of GAT-1,
syntaxin 1A, SNAP-25, VAMP, and their mutants in cotransfected HEK293
cells. Data are from four independent experiments, two wells per condition
per experiment. **, p ⬍ 0.01 as compared with the cells indicated in the figure.
Physical and Functional Interactions between SNAP-25 and
GAT-1 Depend on Syntaxin 1A—To explore the underlying
mechanism of the effect of SNAP-25 on syntaxin 1A-mediated
inhibition of GAT-1 uptake function, we examined the physical
interactions among them. It is reported that syntaxin 1A binds
to GAT-1 directly (20, 22) and syntaxin 1A interacts with
SNAP-25 during the SNARE complex formation (3–5). Our
coimmunoprecipitation experiment in primary cultured hippocampal neurons showed that endogenous syntaxin 1A and
SNAP-25 were coimmunoprecipitated with GAT-1 (Fig. 3A).
In rat brain tissues and cotransfected HEK293 cells, syntaxin
1A interacted with GAT-1 but not with GAT-2 or GAT-3 (sup-
plemental Fig. 1, A and B), consistent with specific inhibition of
GAT-1 uptake function by syntaxin 1A. As reported previously,
G43D is a SNAP-25 point mutant that has a mutation of the
glycine 43 residue to aspartic acid and is revealed not to interact
with syntaxin 1A (34), and syntaxin 1A⌬H3 (syn 1A⌬H3) is a
mutant of syntaxin 1A that lacks the H3 domain (194 –266 residues) and is reported not to interact with GAT-1 (22) or
SNAP-25 (35, 36). Our data confirmed that SNAP-25 but not
G43D was coimmunoprecipitated with syntaxin 1A, and both
SNAP-25 and GAT-1 interacted with syntaxin 1A, but neither
of them bound to syntaxin 1A⌬H3 (Fig. 3B). Both mutants were
introduced in the experiments using the HEK293 cells cotrans-
28178 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 281 • NUMBER 38 • SEPTEMBER 22, 2006
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
FIGURE 2. SNAP-25 enhances the syntaxin 1A-mediated inhibition of
GAT-1 uptake function. A, [3H]GABA uptake in GAT-1-transfected HEK293
cells. FLAG-syntaxin 1A and HA-SNAP-25 were cotransfected in different combinations and different ratios. The ratio of transfected GAT-1 and HA-SNAP-25
plasmids is 1:1, and the ratios of transfected GAT-1 and FLAG-syntaxin 1A
plasmids are indicated in the figure. Representative immunoblot (IB) shows
efficient augmentation of syntaxin 1A expression with the increased amount
of plasmids. **, p ⬍ 0.01 as compared with the HEK293 cells cotransfected
FLAG-syntaxin 1A and GAT-1 at the same plasmid ratio. B, [3H]GABA uptake in
GAT-1-transfected HEK293 cells. FLAG-syntaxin 1A and HA-SNAP-25 were
cotransfected in different combinations and different ratios. The ratio of
transfected GAT-1 and FLAG-syntaxin 1A plasmids is 1:1, and the ratios of
transfected GAT-1 and HA-SNAP-25 plasmids are indicated in the figure. Representative immunoblot shows efficient augment of SNAP-25 expression
with the increased amount of plasmids. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the HEK293 cells cotransfected FLAG-syntaxin 1A with GAT-1.
GABA uptake in the control HEK293 cells transfected with GAT-1 and ␤-galactosidase in A and B ranges from 1398 to 1847 fmol/min/mg protein.
C, [3H]GABA uptake in HA-GAT-1 or HA-GAT-2 or HA-GAT-3-transfected
HEK293 cells. FLAG-syntaxin 1A was cotransfected to examine the effects of
syntaxin 1A on the uptake function of GAT-2 or GAT-3. The ratio of transfected
HA-GAT and FLAG-syntaxin 1A plasmids is 1:1. Representative immunoblot
shows the expression status of HA-GAT and FLAG-syntaxin 1A. **, p ⬍ 0.01 as
compared with the control HEK293 cells transfected with HA-GAT-1 and
␤-galactosidase. The GABA uptake in the control HEK293 cells cotransfected
␤-galactosidase with HA-GAT-1 or HA-GAT-2 or HA-GAT-3 ranges from 1094
to 1857 fmol/min/mg protein. Data in A–C are from at least three independent experiments, two wells per condition per experiment.
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
SEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38
FIGURE 4. SNAP-25 increases the surface expression of GAT-1 and syntaxin 1A. A, surface biotinylation assay in HEK293 cells cotransfected FLAGsyntaxin 1A and/or HA-SNAP-25 with GAT-1. Representative immunoblot (IB)
and quantitative analysis show that the cell surface expression (Surface) of
both GAT-1 and syntaxin 1A is increased by SNAP-25, while the total expression (Total) of both proteins is not changed. Data are from three independent
experiments. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the cells cotransfected FLAG-syntaxin 1A with GAT-1. B, immunocytochemistry experiments
in PC12 cells cotransfected FLAG-syntaxin 1A and/or HA-SNAP-25 with GAT1-GFP. GAT-1-GFP (green) was identified through GFP fluorescence detection.
FLAG-syntaxin 1A (red) or HA-SNAP-25 (blue) was detected with FLAG or HA
antibodies followed by second antibodies conjugated with Cy3 or Cy5, and
then identified by Cy3 or Cy5 fluorescence detection. Panel a, transfected
with GAT-1-GFP alone. Panels b– d, cotransfected FLAG-syntaxin 1A with GAT1-GFP. Panels e– h, cotransfected FLAG-syntaxin 1A and HA-SNAP-25 with
GAT-1-GFP. Scale bars indicate 5 ␮m. The percentage of cells exhibiting distinct ring staining of GAT-1-GFP and FLAG-syntaxin 1A were quantified
among 100 randomly selected positive cells in each group of each experiment, indicating that the surface-associated labeling of GAT-1 and syntaxin
1A is increased by SNAP-25. Data are from three independent experiments.
*, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the cells cotransfected FLAGsyntaxin 1A with GAT-1-GFP.
of GAT-1 and syntaxin 1A, suggesting that the interaction of
GAT-1 with syntaxin 1A on the cell surface might be up-regulated by SNAP-25.
SNAP-25 Potentiates Surface-associated Syntaxin 1A Binding to GAT-1—The underlying mechanism of the SNAP-25enhanced syntaxin 1A-mediated inhibition of GAT-1 uptake
function was further investigated by using FRET as a noninvasive imaging method to characterize the interaction of GAT-1
with syntaxin 1A regulated by SNAP-25. FRET is a process in
which an excited donor fluorophore transfers energy to a lower
energy acceptor fluorophore via a short range (ⱕ10 nm) dipoledipole interaction, and thus FRET efficiency detection is usually
used to reflect the physical interaction between two directly
binding proteins. It has been reported that the syntaxin 1A-mediated inhibition of GAT-1 function depends on syntaxin 1A
JOURNAL OF BIOLOGICAL CHEMISTRY
28179
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
fected SNAP-25 and/or syntaxin 1A with GAT-1 to examine
the physical interaction between SNAP-25 and GAT-1. We
found that only in the presence of syntaxin 1A was GAT-1
coimmunoprecipitated with SNAP-25 (Fig. 3C). Neither the
interaction between GAT-1 and SNAP-25 in presence of syntaxin 1A⌬H3 nor the interaction between GAT-1 and G43D in
presence of syntaxin 1A was detected in the cotransfected
HEK293 cells (Fig. 3C). These data indicate that the physical
interaction between SNAP-25 and GAT-1 depends on
syntaxin 1A.
To further identify whether the functional relationship
among GAT-1, syntaxin 1A, and SNAP-25 is directly related to
their physical interactions, different combinations of their plasmids and mutants were cotransfected in HEK293 cells and the
GABA uptake was detected. We found that SNAP-25 enhanced
the syntaxin 1A-mediated inhibition of GAT-1 uptake function
but not G43D. Our data also showed that VAMP-2 did not
affect the syntaxin 1A- and SNAP-25-mediated inhibition of
GABA uptake. SNAP-25 or VAMP alone had no effect on
GABA uptake by GAT-1 (Fig. 3D). Thus, these findings indicate
that the physical interaction among GAT-1, syntaxin 1A, and
SNAP-25 is necessary for the SNAP-25-enhanced syntaxin
1A-mediated inhibition of GAT-1 uptake function.
SNAP-25 Increases the Surface Expression of GAT-1 and Syntaxin 1A—It is reported that syntaxin 1A up-regulates the cell
surface expression of GAT-1 and decreases GAT-1 transport
rate (21, 22). To identify whether SNAP-25 regulates the surface expression of GAT-1 and then results in the enhanced
syntaxin 1A-mediated inhibition of GAT-1 uptake function, we
detected the surface expression of GAT-1 in HEK293 cells
cotransfected syntaxin 1A and/or SNAP-25 with GAT-1 by
using surface biotinylation assay. The amount of GAT-1 on the
cell surface was increased by cotransfected with syntaxin 1A,
and this increase was enhanced by further cotransfection with
SNAP-25 (⬃130%, compared with cotransfected with syntaxin
1A). Meanwhile, the surface amount of syntaxin 1A was also
increased by further cotransfection with SNAP-25 (⬃145%,
compared with cotransfected with syntaxin 1A) (Fig. 4A). Furthermore, the effect of SNAP-25 on the surface expression of
GAT-1 and syntaxin 1A was investigated using immunocytochemistry experiments in GAT-1-GFP transfected PC12 cells,
for spherical PC12 cells morphologically show more distinctly
subcellular localization than cultured hippocampal neurons. As
illustrated in Fig. 4B, in PC12 cells the transfected GAT-1-GFP
was largely dispersed in the cytoplasm, and only ⬃30% of cells
exhibited the surface-associated distinct ring staining (Fig. 4B,
panel a). Following cotransfection with syntaxin 1A, the surface-associated labeling of GAT-1-GFP was increased, with
⬃50% of cells exhibiting distinct ring staining (Fig. 4B, panel b).
After cotransfection with both syntaxin 1A and SNAP-25, the
surface-associated labeling of GAT-1-GFP was further
increased, with ⬃75% of cells exhibiting distinct ring staining
(Fig. 4B, panel e). SNAP-25 transfection also elevated the surface-associated labeling of syntaxin 1A, with ⬃80% of cells
exhibiting distinct ring staining (Fig. 4B, panel f ), and about
40% of the cells showed distinct surface-associated labeling
without transfection with SNAP-25 (Fig. 4B, panel c). These
results indicate that SNAP-25 increases the surface expression
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
28180 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 281 • NUMBER 38 • SEPTEMBER 22, 2006
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
the emission of light from the
donor. Thus FRET detection can be
accomplished by comparing the
emission of light from the donor in
the same sample before and after
destroying the acceptor by photobleaching. If FRET is present, a
resultant increase in donor emission will occur on photobleaching of
the acceptor (39). We initially
examined whether the emission of
light by the donor (CFP-syntaxin
1A) became more intense after photobleaching of the acceptor (YFPGAT-1) when two proteins were
coexpressed in PC12 cells. A representative example was provided by a
Leica confocal microscope with the
mixed emission spectra of the CFPsyntaxin 1A donor and YFP-GAT-1
acceptor fluorophores (excitation,
405 nm line of laser), and the images
were taken from the photobleached
and nonphotobleached regions of
the cell before and after localized
photobleaching (Fig. 5A). The
selected regions were photobleached of YFP-GAT-1 with 514
nm line of laser. A selective increase
FIGURE 5. SNAP-25 potentiates surface-associated syntaxin 1A binding to GAT-1. A, representative exam- in the peak emission intensity correple of mixed emission spectra of CFP-syntaxin 1A donor and YFP-GAT-1 acceptor fluorophores (excitation,
405-nm laser line) are taken before (red line) and after (blue line) photobleaching (with the 514-nm laser line) in sponding to the region of CFP-syncotransfected PC12 cells. Spectra are shown for one region photobleached (left plot) and for another region taxin 1A emission was observed in
without photobleached (right plot) in the same cell. CFP donor emission increases only in the photobleached
region of the cell. B, a set of unmixed YFP-GAT-1 and CFP-syntaxin 1A (CFP-Syn 1A) images of PC12 cells are the photobleached but not nonphotaken before and after acceptor photobleaching. The region of photobleaching is indicated by the white tobleached region of the cell (Fig.
outlined box. The enlarged pseudocolored images at the bottom show the intensity of CFP emission in the 5A). These results were consistent
photobleached and nonphotobleached regions of the cell surface taken before and after bleaching. The surface-associated intensity of donor CFP-syntaxin 1A emission in PC12 cells increases after acceptor YFP-GAT-1 with a FRET signal originating from
photobleaching. Scale bar indicates 5 ␮m. C, Averaged FRET efficiencies (%) for coexpressed YFP-GAT-1 (YFP- the transfer of resonant energy from
GAT) and CFP-syntaxin 1A (CFP-Syn) under the conditions of up-regulating or down-regulating SNAP-25 CFP to YFP and the dequenching of
expression. FRET efficiency between surface-associated YFP-GAT-1 and CFP-syntaxin 1A is significantly
increased by overexpressing SNAP-25 but decreased by suppressing SNAP-25 expression through its specific this energy transfer upon photosiRNA. The numbers above the columns indicate the cells taken for experiments. Data are from three independ- bleaching of the acceptor fluoroent experiments. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the control cells cotransfected with CFP and YFP
phore. A representative set of the
or the cells indicated in the figure.
unmixed images (CFP-syntaxin 1A
binding to GAT-1 on the cell surface (22). Because our findings and YFP-GAT-1) was illustrated from the transfected PC12
showed that SNAP-25 increased the surface expression of syn- cells before and after photobleaching of the acceptor with 514
taxin 1A and GAT-1, we presumed that SNAP-25 binding to nm line of laser (Fig. 5B). The CFP-syntaxin 1A images showed
syntaxin 1A modulated the interaction of GAT-1 with syntaxin an increase in the donor emission (pseudocolored intensity
1A on the cell surface. Here the FRET efficiency between CFP- images) after photobleaching, and this increase occurred only
syntaxin 1A and YFP-GAT-1 was detected by the acceptor pho- in the region of the cell exposed to the photobleaching (Fig. 5B).
tobleaching FRET technique (31) in transfected PC12 cells. It The averaged relative FRET efficiencies between surface-assohas been reported that the expression, targeting, and function ciated CFP-syntaxin 1A and YFP-GAT-1 were provided as the
of CFP-syntaxin 1A and YFP-GAT-1 show no significant differ- capacity of their interaction near the surface region (Fig. 5C).
ence from the wild-type syntaxin 1A and GAT-1 (37, 38). We The cells expressing CFP-YFP fusion protein (FRET efficiency
also detected the GABA uptake in cotransfected PC12 cells and 26.5 ⫾ 3.5%) were the positive control, and the cells coexpressfound that the uptake function of YFP-GAT-1 was inhibited by ing CFP and YFP (FRET efficiency 5.28 ⫾ 2.0%) were the negCFP-syntaxin 1A (data not shown), confirming that the addi- ative control. FRET efficiency between surface-associated YFPtion of fluoroprotein tags to the wild-type syntaxin 1A and GAT-1 and CFP-syntaxin 1A (14.3 ⫾ 1.8%) was significantly
GAT-1 does not alter their functional interaction. In FRET, the increased by overexpressing SNAP-25 (19.8 ⫾ 1.6%) but
nonradiative transfer of donor energy to the acceptor quenches decreased by suppressing SNAP-25 expression through siRNA
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
SEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY
28181
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
SNARE Complex Formation Induces the Inhibition of GAT-1 Uptake
Function—It is well known that
SNAP-25 binds to syntaxin 1A during
SNARE complex formation (4, 5).
Because syntaxin 1A and SNAP-25
corporately inhibit GAT-1 uptake
function, we examined whether
GAT-1 uptake function could be
affected by the SNARE complex formation. Nitric oxide (NO) is reported
to promote SNAP-25 binding to syntaxin 1A and contribute to SNARE
complex formation (40). Meanwhile,
it is revealed that NO reduces GABA
uptake in hippocampal synaptosomes
(41). Here NO donors, which produce
nitric oxide, were applied to investigate the direct link between the
SNARE complex formation and
GABA transporter GAT-1 function.
We found that treatment of primary
cultured hippocampal neurons with
NOC-18, a NO donor (42), inhibited
[3H]GABA uptake in dose- and timedependent manner (Fig. 6A). The
inhibition by treatment with 10 ␮M
NOC-18 reached the highest level at
10 min (35 ⫾ 4.5% decrease, compared with the control), whereas
treatment together with 10 ␮M 2-phenyl-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide (PTIO), a NO scavFIGURE 6. SNAP-25/syntaxin 1A complex mediates NO-induced inhibition of GAT-1 uptake function. enger (42), abolished this inhibition
3
A, [ H]GABA uptake in primary cultured hippocampal neurons treated with different concentrations of NOC-18
and/or PTIO for different times. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the control neurons without (Fig. 6A). Another NO donor (43),
treatment. B, [3H]GABA uptake in primary cultured hippocampal neurons treated with 10 ␮M NOC-18 or 500 ␮M NTG, also inhibited GABA uptake in
NTG for 10 min. Specific siRNA for syntaxin 1A, SNAP-25, and VAMP-2 were transfected to down-regulate their primary cultured hippocampal neuexpression. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the cells indicated in the figure. C, [3H]GABA uptake
in GAT-1-GFP-transfected PC12 cells treated with 10 ␮M NOC-18 for 10 min. Specific RNA interference or rons (Fig. 6B). Thus we confirmed the
overexpression of FLAG-syntaxin 1A and HA-SNAP-25 was used to down-regulate or up-regulate the expres- previous report (41) that the GABA
sion of syntaxin 1A and/or SNAP-25. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared with the cells indicated in the
figure. D, [3H]GABA uptake in HEK293 cells cotransfected FLAG-syntaxin 1A and/or HA-SNAP-25 with GAT-1. uptake in hippocampal neurons was
Ten micromoles of NOC-18 were used to treat the transfected HEK293 cells for different times. The ratios of reduced by NO. Because the GABA
transfected plasmids are indicated. Representative immunoblot (IB) shows the expression status of GAT-1, uptake in hippocampal neurons is
syntaxin 1A, and SNAP-25 in the cells with different transfected combinations. **, p ⬍ 0.01 as compared with
the cells cotransfected FLAG-syntaxin 1A and HA-SNAP-25 with GAT-1 without NOC-18 treatment. Data in A–D mainly mediated by GAT-1 (Fig. 1A),
are from three independent experiments, two wells per condition per experiment.
NO inhibits the uptake function of
neuronal GABA transporter GAT-1.
We further identified whether the SNARE proteins were
(10.3 ⫾ 2.1%) (Fig. 5C). However, our data showed that the
FRET efficiency between YFP-GAT-1 and CFP-syntaxin functionally involved in the NO-induced inhibition of GAT-1
1A⌬H3 (5.94 ⫾ 1.6%) was decreased to the level of negative uptake function. As shown in Fig. 6B, both NOC-18- and NTGcontrol, indicating that the FRET efficiencies that we detected induced inhibitions of GABA uptake in neurons were rescued
between YFP-GAT-1 and CFP-syntaxin 1A represent the spe- by suppressing the expression of syntaxin 1A or SNAP-25 but
cific interaction of GAT-1 with syntaxin 1A. On the other hand, not VAMP-2 through the transfection with their specific siRthe FRET efficiencies between CFP-syntaxin 1A and YFP- NAs, respectively. Moreover, suppressing syntaxin 1A and
GAT-2 or YFP-GAT-3 were the same level as the negative con- SNAP-25 expression simultaneously further rescued these
trol, further confirming that neither GAT-2 nor GAT-3 binds inhibitions but suppressing the expression of three SNARE proto syntaxin 1A (supplemental Fig. 1C). These data suggest that teins did not enhance this rescue. The syntaxin 1A and
SNAP-25 potentiates surface-associated syntaxin 1A binding SNAP-25 involvement in the inhibition of GABA uptake
to GAT-1, resulting in enhanced syntaxin 1A-mediated inhibi- induced by NOC-18 was also confirmed in GAT-1-GFP transfected PC12 cells with overexpression or specific RNA interfertion of GAT-1 uptake function.
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
28182 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 7. NO promotes the translocation of GAT-1, syntaxin 1A, and
SNAP-25 to the cell surface. A, surface biotinylation assay in primary cultured hippocampal neurons treated with 10 ␮M NOC-18 for 10 min. Representative immunoblot (IB) and quantitative analysis show that the cell surface
expression (Surface) of endogenous GAT-1 and syntaxin 1A (Syn 1A) is
increased by NOC-18, whereas the total expression (Total) of both proteins is
not changed. Data are from three independent experiments. *, p ⬍ 0.05, and
**, p ⬍ 0.01 as compared with the neurons without treatment. B, immunocytochemistry experiments in GAT-1-GFP-transfected PC12 cells. Ten micromoles of NOC-18 were used to treat the transfected PC12 cells for 10 min.
GAT-1-GFP (green) was identified through GFP fluorescence detection.
Endogenous syntaxin 1A (red) or SNAP-25 (blue) was detected with their antibodies, respectively, followed by second antibodies conjugated with Cy3 or
Cy5 and then identified by Cy3 or Cy5 fluorescence detection. Panels a– d,
without treatment; panels e– h, after treatment with 10 ␮M NOC-18 for 10 min.
Scale bars indicate 5 ␮m. C, the percentages of cells exhibiting distinct ring
staining of GAT-1-GFP, endogenous syntaxin 1A, and SNAP-25 in GAT-1-GFPtransfected PC12 cells with or without NOC-18 treatment were quantified
among 100 randomly selected positive cells in each group of each experiment. The surface-associated labeling of GAT-1, syntaxin 1A, and SNAP-25 is
increased by NOC-18. Data are from three independent experiments. *, p ⬍
0.05, and **, p ⬍ 0.01 as compared with the cells without NOC-18 treatment.
and SNAP-25 binding to syntaxin 1A potentiates the physical
interaction of surface-associated syntaxin 1A with GAT-1,
resulting in significant inhibition of GAT-1 uptake function.
DISCUSSION
This study demonstrates that SNAP-25 binding to syntaxin
1A to form a target SNARE complex inhibits neurotransmitter
GABA transporter GAT-1 uptake function. Overexpression of
SNAP-25 inhibited the GABA uptake by GAT-1, and suppressing its expression increased GAT-1 uptake function in the presence of syntaxin 1A. The H3 domain of syntaxin 1A was
required for the physical and functional interactions between
SNAP-25 and GAT-1. SNAP-25 binding to syntaxin 1A greatly
potentiated the physical interaction of syntaxin 1A with GAT-1
VOLUME 281 • NUMBER 38 • SEPTEMBER 22, 2006
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
ence (Fig. 6C). These results indicate that two target SNAREs,
syntaxin 1A and SNAP-25, but not the vesicle SNARE VAMP-2
are functionally involved in NO-induced inhibition of GAT-1
uptake function.
Considering that in a previous report NO promotes
SNAP-25 binding to syntaxin 1A to form the SNARE complex
(40), we further investigated whether the SNAP-25/syntaxin 1A
complex formation mediates NO-induced inhibition of GAT-1
uptake function in HEK293 cells cotransfected syntaxin 1A
and/or SNAP-25 with GAT-1. The inhibition of GABA uptake
was enhanced by NOC-18 only in HEK293 cells cotransfected
both syntaxin 1A and SNAP-25 with GAT-1 but not in cells
cotransfected syntaxin 1A or SNAP-25 alone with GAT-1 (Fig.
6D). NOC-18 did not decrease GABA uptake in the cells transfected with GAT-1 alone (Fig. 6D). Our results suggest that the
syntaxin 1A/SNAP-25 complex is necessary for the inhibition
of GAT-1 uptake function during NO-induced SNARE complex formation.
SNARE Complex Formation Promotes Surface-associated
Syntaxin 1A Binding to GAT-1—Because the above studies
demonstrated the functional relationship between the SNARE
complex and GAT-1, in which syntaxin 1A and SNAP-25 are
involved, we further detected dynamic distribution of GAT-1,
syntaxin 1A, and SNAP-25 during the SNARE complex formation. In primary cultured hippocampal neurons, surface biotinylation assay showed that cell surface expression of GAT-1
(⬃132%, compared with nontreatment) or syntaxin 1A
(⬃143%, compared with nontreatment) was increased by
NOC-18 treatment (Fig. 7A). Furthermore, we transfected
PC12 cells with GAT-1-GFP and examined the localization of
GAT-1-GFP, endogenous syntaxin 1A, and SNAP-25. In GAT1-GFP-transfected PC12 cells, GAT-1-GFP, endogenous syntaxin 1A, and SNAP-25 were largely dispersed in the cytoplasm
(Fig. 7B, panels a– d), with 30 – 40% of cells exhibiting distinct
ring staining (Fig. 7C). Following treatment with 10 ␮M
NOC-18 for 10 min, the surface-associated labeling of GAT-1GFP, endogenous syntaxin 1A, and SNAP-25 was greatly elevated (Fig. 7B, panels e– h), with 70 –92% of cells exhibiting
distinct ring staining (Fig. 7C). These data indicate that the
SNARE complex formation promotes the translocation of
GAT-1, syntaxin 1A, and SNAP-25 to the cell surface, providing the possibility for their functional interaction.
The mechanism of surface-associated SNARE proteins on
the inhibition of GAT-1 uptake function was then investigated
by FRET to characterize the interaction of surface-associated
GAT-1 with syntaxin 1A affected by the SNARE complex formation. As shown in Fig. 8, FRET efficiency between surfaceassociated YFP-GAT-1 and CFP-syntaxin 1A was significantly
increased by promoting the SNARE complex formation
through treatment with NOC-18 (21.7 ⫾ 2.3%, compared with
the nontreatment 14.3 ⫾ 1.8%). The increase was enhanced by
overexpressing SNAP-25 (29.4 ⫾ 1.9%) or was reduced by suppressing SNAP-25 expression through specific siRNA (14.5 ⫾
2.0%). These data indicate that SNAP-25 participates in the
potentiation of surface-associated syntaxin 1A binding to
GAT-1 during SNARE complex formation. All the above
results suggest that the SNARE complex formation enhances
the surface expression of SNAP-25/syntaxin 1A and GAT-1,
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
and significantly enhanced the syntaxin 1A-mediated inhibition of GAT-1 uptake function. Furthermore, nitric oxide-induced SNARE complex formation resulted in the inhibition of
neurotransmitter GABA uptake, in which the SNAP-25/syntaxin 1A complex formation promoted direct binding of syntaxin 1A to GAT-1. Our findings provide strong evidence that
the SNAP-25 binding to syntaxin 1A during the SNARE complex formation functionally modulates neurotransmitter
GABA reuptake.
It has been shown that syntaxin 1A inhibits GAT-1 reuptake
function (20). Here we further demonstrate that SNAP-25
binding to syntaxin 1A to form target SNARE complex does not
attenuate the association between syntaxin 1A and GAT-1 but
instead potentiates their interaction, resulting in the enhanced
inhibition of GAT-1 reuptake function. In addition, VAMP-2
binding to the target SNARE complex does not affect the inhibition. Our results suggest that under physiological conditions
the SNARE complex is able to modulate GAT-1 reuptake
function and the target SNARE complex serves as a key player.
The various forms of regulation affect transporter function in
the following two ways: altering the number of transporters
on the cell surface or the transport process. It has been reported
that syntaxin 1A increases the cell surface expression of GAT-1
but decreases GAT-1 transport rate (21, 22). Our results indicate that SNAP-25 binding to syntaxin 1A further increases
GABA transporter GAT-1 expression on the cell surface. The
mechanism is not clear, but it is surmised that more GAT-1
SEPTEMBER 22, 2006 • VOLUME 281 • NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY
28183
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
FIGURE 8. SNAP-25 participates in the potentiation of surface-associated
syntaxin 1A binding to GAT-1 by NO. Averaged FRET efficiencies (%) for
coexpressed YFP-GAT-1 and CFP-syntaxin 1A were examined under the conditions of up-regulation or down-regulation of SNAP-25 expression, and/or
after treatment with 10 ␮M NOC-18 for 10 min. FRET efficiency between surface-associated YFP-GAT-1 (YFP-GAT) and CFP-syntaxin 1A is significantly
increased by promoting the SNARE complex formation through treatment
with NOC-18. The increase is enhanced by overexpressing SNAP-25 or
reduced by suppressing SNAP-25 expression through its specific siRNA. The
numbers above the columns indicate the cells taken for experiments. Data are
from three separate experiments. *, p ⬍ 0.05, and **, p ⬍ 0.01 as compared
with the control cells cotransfected with CFP and YFP or the cells indicated in
the figure.
may be brought to the cell surface from synaptic vesicle membranes during the membrane fusion, for SNAP-25 binding to
syntaxin 1A to form t-SNARE complex promotes membrane
fusion. Moreover, our data show that SNAP-25 binding to syntaxin 1A promotes the surface expression of syntaxin 1A and
potentiates the physical interaction of syntaxin 1A with GAT-1
on the cell surface, resulting in inhibition of GABA uptake.
Thus, SNAP-25-enhanced syntaxin 1A-mediated inhibition of
the GAT-1 transport rate might result from more syntaxin 1A
or SNAP-25/syntaxin 1A complex binding to GAT-1 on the cell
surface.
Accumulating investigations demonstrate that the target
SNARE syntaxin 1A physically interacts with and functionally
regulates those multitransmembrane proteins such as neurotransmitter transporters (15, 16, 22) and ion channels (44, 45).
It seems that syntaxin 1A broadly interacts with the membrane
proteins. To further confirm the selectivity of interaction
between syntaxin 1A and its binding membrane proteins, our
Supplemental Material provides the results that demonstrate
the specific interaction of the GABA transporter GAT-1 with
syntaxin 1A and the glutamate transporter EAAC1 with syntaxin 1A by using coimmunoprecipitation assay and FRET
detection (supplemental Fig. 1 and supplemental Fig. 2A). In
contrast, neither GABA transporter GAT-2 nor GAT-3 subtype (supplemental Fig. 1) nor glutamate transporter GLT1 nor
GLAST subtype (supplemental Fig. 2A) interacts with syntaxin
1A. Moreover, the interaction of syntaxin 1A with G proteincoupled receptor ␦-opioid receptor or ␤2-adrenergic receptor is
not detected (supplemental Fig. 2B). Thus, the interaction
between syntaxin 1A and multitransmembrane proteins could
be selective.
Syntaxin 1A is revealed to interact with the inhibitory neurotransmitter glycine transporters GLYT1 and GLYT2 (15) and
the major excitatory neurotransmitter glutamate transporter
EAAC1 (16), resulting in the inhibition of their reuptake function. This study provides the possibility that the SNARE complex may functionally modulate those transporters. Indeed, the
properties of two ion channels, Kv2.1 potassium channel and
cystic fibrosis transmembrane regulator chloride channel, are
reported to be directly affected by the target SNARE complex
(23, 24), as mediated by their direct interaction with the H3
domain of syntaxin 1A (45, 46). Moreover, accumulating evidence also reveals that syntaxin 1A interacts with other membrane proteins and regulates their functions, and its H3 domain
is the only reported binding site (45– 47). Our results also indicate that the physical interaction between SNAP-25 and GAT-1
is mediated by the H3 domain of syntaxin 1A. Therefore, we can
speculate that the function of those syntaxin 1A-binding proteins might be modulated by the SNARE complex, but it
remains to be further investigated.
Previous studies demonstrate that the stimulation with nitric
oxide, which promotes the SNARE complex formation (40) and
induces neurotransmitter release (48, 49), inhibits the neurotransmitter GABA reuptake (41). Here we demonstrate that the
nitric oxide-induced inhibition of GABA reuptake is rescued by
attenuating the formation of the SNARE complex by suppressing the SNARE protein expression in primary cultured hippocampal neurons and PC12 cells. Thus attenuation of the
SNAP-25/Syntaxin 1A Complex Inhibits GAT-1 Reuptake Function
Acknowledgments—We thank Dr. Jian Fei for providing GAT-1, GAT1-GFP, EAAC1, GLT1, and GLAST plasmids; Dr. Anjaparavanda P.
Naren for providing syntaxin 1A plasmid; Dr. Kevin L. Kirk for providing syntaxin 1A⌬H3 plasmid; Dr. Maurine E. Linder for providing
G43D-GFP plasmid; Dr. Harald H. Sitte for providing YFP-GAT-1
plasmid; Dr. Yuechueng Liu for providing CFP-syntaxin 1A plasmid;
and Dr. Cheng He for providing CFP-YFP plasmid. We also thank Drs.
Nanjie Xu, Yaqiang Li, Yongxin Yu, Peng Xia, and Yutin Li for critical
comments and Yalan Wu and Yawei Tang for technical assistance.
REFERENCES
1. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H.,
Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature 362,
318 –324
2. Hua, Y., and Scheller, R. H. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,
8065– 8070
3. Mochida, S. (2000) Neurosci. Res. 36, 175–182
4. An, S. J., and Almers, W. (2004) Science 306, 1042–1046
5. Fasshauer, D., and Margittai, M. (2004) J. Biol. Chem. 279, 7613–7621
6. Dingledine, R., and Korn, S. J. (1985) J. Physiol. (Lond.) 366, 387– 409
7. Jursky, F., Tamura, S., Tamura, A., Mandiyan, S., Nelson, H., and Nelson,
N. (1994) J. Exp. Biol. 196, 283–295
8. Borden, L. A. (1996) Neurochem. Int. 29, 335–356
9. Soudijn, W., and van Wijngaarden, I. (2000) Curr. Med. Chem. 7,
1063–1079
10. Isaacson, J. S., Solis, J. M., and Nicoll, R. A. (1993) Neuron 10, 165–175
11. Solis, J. M., and Nicoll, R. A. (1992) J. Neurosci. 12, 3466 –3472
12. Bernstein, E. M., and Quick, M. W. (1999) J. Biol. Chem. 274, 889 – 895
13. Whitworth, T. L., and Quick, M. W. (2001) J. Biol. Chem. 276,
42932– 42937
14. Quick, M. W., Corey, J. L., Davidson, N., and Lester, H. A. (1997) J. Neurosci. 17, 2967–2979
15. Geerlings, A., Lopez-Corcuera, B., and Aragon, C. (2000) FEBS Lett. 470,
51–54
28184 JOURNAL OF BIOLOGICAL CHEMISTRY
16. Zhu, Y., Fei, J., and Schwarz, W. (2005) J. Neurosci. Res. 79, 503–508
17. Haase, J., Killian, A. M., Magnani, F., and Williams, C. (2001) Biochem. Soc.
Trans. 29, 722–728
18. Quick, M. W. (2002) Int. J. Dev. Neurosci. 20, 219 –224
19. Sung, U., Apparsundaram, S., Galli, A., Kahlig, K. M., Savchenko, V.,
Schroeter, S., Quick, M. W., and Blakely, R. D. (2003) J. Neurosci. 23,
1697–1709
20. Beckman, M. L., Bernstein, E. M., and Quick, M. W. (1998) J. Neurosci. 18,
6103– 6112
21. Horton, N., and Quick, M. W. (2001) Mol. Membr. Biol. 18, 39 – 44
22. Deken, S. L., Beckman, M. L., Boos, L., and Quick, M. W. (2000) Nat.
Neurosci. 3, 998 –1003
23. Michaelevski, I., Chikvashvili, D., Tsuk, S., Singer-Lahat, D., Kang, Y.,
Linial, M., Gaisano, H. Y., Fili, O., and Lotan, I. (2003) J. Biol. Chem. 278,
34320 –34330
24. Cormet-Boyaka, E., Di, A., Chang, S. Y., Naren, A. P., Tousson, A., Nelson,
D. J., and Kirk, K. L. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12477–12482
25. Gao, H., Sun, Y., Wu, Y., Luan, B., Wang, Y., Qu, B., and Pei, G. (2004) Mol.
Cell 14, 303–317
26. Xu, N. J., Bao, L., Fan, H. P., Bao, G. B., Pu, L., Lu, Y. J., Wu, C. F., Zhang, X.,
and Pei, G. (2003) J. Neurosci. 23, 4775– 4784
27. Cai, G., Salonikidis, P. S., Fei, J., Schwarz, W., Schulein, R., Reutter, W., and
Fan, H. (2005) FEBS J. 272, 1625–1638
28. Beckman, M. L., Bernstein, E. M., and Quick, M. W. (1999) J. Neurosci. 19,
1– 6
29. Bao, L., Jin, S. X., Zhang, C., Wang, L. H., Xu, Z. Z., Zhang, F. X., Wang,
L. C., Ning, F. S., Cai, H. J., Guan, J. S., Xiao, H. S., Xu, Z. Q., He, C., Hokfelt,
T., Zhou, Z., and Zhang, X. (2003) Neuron 37, 121–133
30. Wang, P., Gao, H., Ni, Y., Wang, B., Wu, Y., Ji, L., Qin, L., Ma, L., and Pei,
G. (2003) J. Biol. Chem. 278, 6363– 6370
31. Liu, J., Ernst, S. A., Gladycheva, S. E., Lee, Y. Y., Lentz, S. I., Ho, C. S., Li, Q.,
and Stuenkel, E. L. (2004) J. Biol. Chem. 279, 55924 –55936
32. Sipila, S., Huttu, K., Voipio, J., and Kaila, K. (2004) J. Neurosci. 24,
5877–5880
33. Chamberlain, L. H., Burgoyne, R. D., and Gould, G. W. (2001) Proc. Natl.
Acad. Sci. U. S. A. 98, 5619 –5624
34. Loranger, S. S., and Linder, M. E. (2002) J. Biol. Chem. 277, 34303–34309
35. Fergestad, T., Wu, M. N., Schulze, K. L., Lloyd, T. E., Bellen, H. J., and
Broadie, K. (2001) J. Neurosci. 21, 9142–9150
36. Misura, K. M., Scheller, R. H., and Weis, W. I. (2001) J. Biol. Chem. 276,
13273–13282
37. Xiao, J., Xia, Z., Pradhan, A., Zhou, Q., and Liu, Y. (2004) J. Neurosci. Res.
75, 143–151
38. Scholze, P., Freissmuth, M., and Sitte, H. H. (2002) J. Biol. Chem. 277,
43682– 43690
39. Kenworthy, A. K. (2001) Methods (Amst.) 24, 289 –296
40. Meffert, M. K., Calakos, N. C., Scheller, R. H., and Schulman, H. (1996)
Neuron 16, 1229 –1236
41. Cupello, A., Mainardi, P., Robello, M., and Thellung, S. (1997) Neurochem.
Res. 22, 1517–1521
42. Lin, Y. F., Raab-Graham, K., Jan, Y. N., and Jan, L. Y. (2004) Proc. Natl.
Acad. Sci. U. S. A. 101, 7799 –7804
43. Tran, N. N., Spitzbarth, E., Robert, A., Giummelly, P., Atkinson, J., and
Capdeville-Atkinson, C. (1998) J. Physiol. (Lond.) 507, 163–174
44. Leung, Y. M., Kang, Y., Gao, X., Xia, F., Xie, H., Sheu, L., Tsuk, S., Lotan, I.,
Tsushima, R. G., and Gaisano, H. Y. (2003) J. Biol. Chem. 278,
17532–17538
45. Naren, A. P., Quick, M. W., Collawn, J. F., Nelson, D. J., and Kirk, K. L.
(1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10972–10977
46. Leung, Y. M., Kang, Y., Xia, F., Sheu, L., Gao, X., Xie, H., Tsushima, R. G.,
and Gaisano, H. Y. (2005) Biochem. J. 387, 195–202
47. Cui, N., Kang, Y., He, Y., Leung, Y. M., Xie, H., Pasyk, E. A., Gao, X., Sheu,
L., Hansen, J. B., Wahl, P., Tsushima, R. G., and Gaisano, H. Y. (2004)
J. Biol. Chem. 279, 53259 –53265
48. Ohkuma, S., Katsura, M., Chen, D. Z., Narihara, H., and Kuriyama, K.
(1996) Brain Res. Mol. Brain Res. 36, 137–144
49. Meffert, M. K., Premack, B. A., and Schulman, H. (1994) Neuron 12,
1235–1244
VOLUME 281 • NUMBER 38 • SEPTEMBER 22, 2006
Downloaded from www.jbc.org at Shanghai Information Center for Life Sciences, Chinese Academy of Sciences on October 7, 2006
SNARE complex contributes to the rescued inhibition of GABA
reuptake, further indicating that the SNARE complex mediates
the coupling of neurotransmitter GABA release with the inhibition of GABA reuptake.
Postsynaptic neurotransmission is highly dependent on the
levels of neurotransmitters in the synaptic clefts. Thus the inhibition of GABA reuptake by the SNARE complex during neurotransmitter release is pivotal for facilely achieving the level of
GABA in the synaptic clefts, which sufficiently triggers their
receptors on the postsynaptic membrane. Meanwhile, SNARE
complex formation promotes the translocation of GABA transporter to the cell surface. The physiological significance of
increased GABA transporter surface expression by the target
SNARE complex remains unknown. One possibility is that the
complex is used not only to keep the transporter functionally
suppressed until a time when GABA release from the synaptic
vesicles is a priority but also to increase surface expression of
the GABA transporter so that the reuptake function can be
resumed quickly until the disassembly of the complex after
GABA release. Therefore, neurotransmitter release and
reuptake are coordinated together through the assembly and
disassembly of the SNARE complex to maintain normal neurotransmission. Maladjustment of the coordination might result
in abnormal neurotransmission, and a better understanding of
the underlying mechanism may provide a great help to the
related neurological disorders of the central nervous system.