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The Journal of Neuroscience, March 1, 2003, 23(5):1697
A Regulated Interaction of Syntaxin 1A with the
Antidepressant-Sensitive Norepinephrine Transporter Establishes
Catecholamine Clearance Capacity
Uhna
Sung1, *,
Subramaniam
Apparsundaram1, *,
Aurelio
Galli2,
Kristopher
M.
Kahlig2,
Valentina
Savchenko1,
Sally
Schroeter1,
Michael W.
Quick3, and
Randy D.
Blakely1
1 Department of Pharmacology and Center for Molecular
Neuroscience, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-8548, 2 Department of Pharmacology,
University of Texas at San Antonio, San Antonio, Texas 78249, and
3 Department of Neurobiology, University of Alabama at
Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
Norepinephrine (NE) transporters (NETs) terminate noradrenergic
synaptic transmission and represent a major therapeutic target for
antidepressant medications. NETs and related transporters are under
intrinsic regulation by receptor and kinase-linked pathways, and
clarification of these pathways may suggest candidates for the
development of novel therapeutic approaches. Syntaxin 1A, a presynaptic
soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, interacts with NET and modulates NET
intrinsic activity. NETs colocalize with and bind to syntaxin 1A in
both native preparations and heterologous systems. Protein kinase C
activation disrupts surface NET/syntaxin 1A interactions and
downregulates NET activity in a syntaxin-dependent manner. Syntaxin 1A
binds the NH2 terminal domain of NET, and a deletion of
this domain both eliminates NET/syntaxin 1A associations and prevents
phorbol ester-triggered NET downregulation. Whereas syntaxin 1A
supports the surface trafficking of NET proteins, its direct interaction with NET limits transporter catalytic function. These two
contradictory roles of syntaxin 1A on NET appear to be linked and
reveal a dynamic cycle of interactions that allow for the coordinated
control between NE release and reuptake.
Key words:
catecholamine; norepinephrine; antidepressant; transport; syntaxin 1A; phorbol ester; surface trafficking
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Introduction |
The catecholamine neurotransmitter
norepinephrine (NE) modulates multiple cognitive and emotional circuits
in the mammalian brain including those subserving alertness, attention,
learning and memory, and mood (Foote et al., 1983 ). NE also regulates
autonomic function at brainstem sites and via its role as a
neurotransmitter in postganglionic sympathetic synapses (Axelrod and
Kopin, 1969). NE is released predominantly at axonal varicosities in
which the availability of extracellular NE is limited by NE
transporters (NETs), presynaptic transporters that catalyze
neurotransmitter reuptake via coupling to transmembrane
Na+ and Cl
gradients (Iversen, 1971; Graefe and Bönisch, 1988). NETs are targets of psychostimulants, including cocaine and amphetamine (Ritz et
al., 1990; Chen and Reith, 1994; Wall et al., 1995), and have
been recognized as important sites of action for tricyclic antidepressants (Schildkraut, 1965). Although more selective NET antagonists [norepinephrine serotonin reuptake inhibitors (NSRIs)] appear promising in the treatment of mood disorders (Tatsumi et al.,
1997; Burrows et al., 1998; Gorman and Sullivan, 2000), strategies for
manipulating catecholamine reuptake remain limited to the identification of agents capable of occluding NE binding or
translocation. Dysfunction of NE clearance or NET density has been
associated with attention and mood disorders (Hadley et al., 1995;
Delgado and Moreno, 2000; Arnsten, 2001), suicide (Klimek et al.,
1997), and cardiovascular disease (Esler et al., 1981; Liang et al., 1989; Merlet et al., 1992; Bohm et al., 1995; Shannon et al., 2000).
Notably, altered regulatory mechanisms that might disrupt NET function
in disease states are unknown.
NETs belong to a gene family (SLC6A) of
Na+/Cl-dependent
transporters (Pacholczyk et al., 1991), the other members of which include dopamine, serotonin, glycine, and GABA transporters
(DAT, SERT, GLYT, and GAT, respectively) (Barker and Blakely, 1995). These transporters exhibit a predicted topology of 12 transmembrane domains (TMDs) with cytoplasmic N and C termini. Increasing evidence indicates that both extrinsic signals and intracellular kinase-linked pathways regulate biogenic amine transporters, with effects seen on
transporter cell surface trafficking and/or intrinsic activity (Blakely
and Bauman, 2000; Zahniser and Doolen, 2001). For example, NE transport
capacity in noradrenergic SK-N-SH cells can be diminished rapidly by
muscarinic receptor activation (Apparsundaram et al., 1998a), a process
linked to protein kinase C (PKC) activation and a loss of carriers from
the cell surface. Direct activation of PKC with phorbol esters also
redistributes NETs in heterologous expression systems as visualized by
confocal microscopy (Apparsundaram et al., 1998b). Analogous findings
of cell surface redistribution after receptor stimulation have been
reported for DAT, SERT, and GAT1 proteins (Qian et al., 1997; Beckman
et al., 1999; Ramamoorthy and Blakely, 1999; Saunders et al., 2000) and
may involve phosphorylation of transporters as well as the coordinated
association of protein kinases, phosphatases, and scaffolding proteins
(Blakely and Bauman, 2000; Deken et al., 2001). Recently, we identified
a trafficking-independent pathway for NET regulation linked to PI-3
kinase and p38 mitogen-activated protein (MAP) kinase activation
(Apparsundaram et al., 2001), suggesting that neurons likely have
multiple pathways to modulate NE clearance capacity intrinsically. A
greater understanding of these mechanisms will clarify how the
processes of neurotransmitter release and reuptake are coordinated in
space and time and extend the range of targets for drug development.
Neurotransmitter transporters, like other membrane proteins involved in
cell signaling, appear to be organized as multiprotein complexes. For
example, several PDZ [postsynaptic density-95 (PSD-95)/Discs large
(Dlg)/zona occludens-1 (ZO-1)] domain proteins have been identified that physically interact with C termini of glutamate, GABA,
and biogenic amine transporters (Perego et al., 1999; Jackson et al.,
2001; Torres et al., 2001) and may help to establish targeted expression to discrete membrane domains, although their role in acute
transporter regulation is unclear. Several studies recently have drawn
explicit attention to the physical and functional interaction of
transporters with the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) protein syntaxin 1A
(Beckman et al., 1998; Geerlings et al., 2000; Haase et al., 2001).
These studies with the transporters for inhibitory amino acids suggest
that syntaxin 1A may control neurotransmission not only via the
regulated fusion of neurotransmitter vesicles but also via the delivery
of transporters that support neurotransmitter inactivation (Deken et
al., 2000; Geerlings et al., 2001). Whether regulated catecholamine
transporter trafficking and/or intrinsic activity are/is supported
similarly by syntaxin 1A interactions and whether these processes are
linked are unknown. In the present report we used botulinum toxin C1
(BoNT/C1) and syntaxin 1A antisense treatments to establish a tonic
requirement for the SNARE protein on NE transport capacity in neuronal
preparations. Moreover, we find that NET colocalizes, and forms stable
associations, with syntaxin 1A in vivo. NET/syntaxin
1A interactions are direct and mediated by the NET
NH2 terminus, and stimuli known to trigger NET
redistribution destabilize NET/syntaxin 1A interactions. Whereas syntaxin 1A supports surface expression of NETs, we find that the SNARE
protein limits NET catalytic activity, measured as an elimination of
NET-associated currents and a dissociation of surface NET density from
NE uptake activity. We discuss our findings in the context of a model
whereby vesicular NE release and the trafficking and intrinsic activity
of NETs are both linked to syntaxin 1A availability, allowing enhanced
coordination of catecholamine release and reuptake. We suggest that
physical interactions of syntaxin 1A with NET provide a means to
elaborate tight control of NE clearance capacity via modulation of both
surface trafficking and intrinsic activity in parallel with signals
impinging on NE release.
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Materials and Methods |
Antibodies and other reagents. Polyclonal antibody
43411, generated against the C-terminal sequences of mouse NET and
recognizing rat, mouse, and human NET (hNET) proteins, has been
described previously (Schroeter et al., 2000). Polyclonal NET antibody
43408, raised against the peptide TKYSKYKFTPAAEFY (amino acids 99-214) located in the second extracellular loop of human and mouse NET, was
used at 1:500 and will be described more fully (V. Savchenko, U. Sung,
and R. D. Blakely, unpublished data). Monoclonal anti-hNET antibody (catalog number NET 17-1, Mab Technologies, Atlanta, GA) was
used at a dilution of 1:1000 for immunoblotting. Immunoblotting of
syntaxin 1A was performed by using anti-syntaxin antibody (HPC-1, Sigma, St. Louis, MO) at a dilution of 1:2000.
Immunohistochemistry of syntaxin 1A was performed with anti-syntaxin
antibody from Chemicon (Temecula, CA) at a dilution of
1:1000. Monoclonal anti-histidine antibody (Clontech, Palo Alto, CA)
was used at 1 µg for each immunoprecipitation. Polyclonal
anti-histidine antibody (Santa Cruz Biotech, Santa Cruz, CA) was used
at a dilution of 1:200 for immunoblots. Anti-hemagglutinin (HA)
antibody (3F10) conjugated with peroxidase (Boehringer
Mannheim, Mannheim, Germany) was used at a dilution of
1:200-1:500 for immunoblots. Phorbol 12-myristate 13-acetate ( -PMA)
was from Calbiochem (La Jolla, CA) or from
Alexis (San Diego, CA); okadaic acid (OA) was also from
Alexis. Methacholine, carbachol, desipramine, and cocaine were obtained from Sigma.
Constructs. HA-tagged hNET in pcDNA3
(Invitrogen, Carlsbad, CA) has been described previously
(Bauman and Blakely, 2002). His-hNET in pcDNA3 was prepared by
inserting HHHHHHG between the translation initiation site and the
second residue (L) of hNET. Insertion of tags and deletion and point
mutations of hNET (hNET 2-42, hNET43-64, hNET2-64, hNET
D51A, D53A, E58A) in pcDNA3 were made by site-directed mutagenesis with
the Quick Change site-directed mutagenesis kit
(Stratagene, La Jolla, CA). Syntaxin 1A in pCMV5 and
Munc18-1 in pGEX KG were generous gifts from Dr. T. Sudhof (Howard
Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX). The cytoplasmic domain of syntaxin 1A in
pCMV5 (SynTM) was made by inserting a stop codon just before the
transmembrane domain. Munc18-1 was moved to pcDNA3
(Invitrogen). pGEX5X-1-SynTM (GST-syntaxin 1A bearing a
deletion of the transmembrane domain, GST-SynTM), pMAL NET-N [ maltose-binding protein (MBP) fused to the N-terminal domain of NET,
residues 1-63], and pMAL-NET-C (MBP fused to the C-terminal domain of
NET, residues 576-617) were constructed by the subcloning of PCR
products of the corresponding sequences into pGEX
(Pharmacia, Peapack, NJ) or pMAL cRI (New England
Biolabs, Beverly, MA).
Primary neuronal cultures and immunohistochemistry. C57BL/6
mouse pups, 2 d old, were anesthetized with Nembutal (50 mg/kg). Superior cervical ganglia (SCG) were dissected, treated with 0.3% collagenase and 0.1% trypsin for 30 min, plated on culture dishes, and
incubated in F-14 medium containing 5% FCS and 20 ng/ml NGF for 2 hr
at 37°C to purify SCG neurons from fibroblasts. Floating cells were
replated on poly-D-lysine and laminin-coated coverslips and
incubated further. After 24 hr the cultures were treated with 10 µM 5-fluoro-5'-deoxyuridine and grown for 5-14 d before
staining. For double staining of total NET and syntaxin 1A, SCG cells
were fixed with 3% paraformaldehyde, blocked with 2% normal donkey serum (NDS) and 0.2% NP-40 in PBS, and incubated with NET and syntaxin
1A antibodies in the same solution for 1 hr at room temperature (RT),
followed by an incubation with donkey anti-rabbit conjugated with CY3
(1:1000) and donkey anti-mouse conjugated with CY2 (1:500; Jackson ImmunoResearch, West Grove, PA). To detect surface
labeling of NET, we incubated live cells with antibody 43408 in
PBS/1% NDS for 1 hr at RT, fixed them with PBS/3% paraformaldehyde,
blocked them with PBS/2% NDS/0.2% NP-40, and incubated them with
syntaxin 1A antibody in the same buffer for 2 hr at RT. Methods for
tissue preparation and immunofluorescence localization of NET with
antibody 43411 and syntaxin were implemented on rat vas deferens as
described previously (Schroeter et al., 2000). Briefly, vas deferens
was obtained from perfused Sprague Dawley rats
(Harlan, Indianapolis, IN), cryoprotected, frozen, and
then sectioned at 14 µm with a cryostat and collected on slides.
Sections were blocked and permeabilized for 30 min in 4% normal goat
serum/0.3% Triton X-100/TBS (50 mM Tris, 90 mM
NaCl, pH 7.4) and incubated with antibodies. All specimens were
examined with a Zeiss (Oberkochen, Germany) LSM 410 confocal imaging system equipped with internal He/Ne and external Ar/Kr lasers (Vanderbilt University Medical Center Cell Imaging Core Resource). Z-series images were collected by optical
sectioning at intervals of 1 µm. Image processing and montage
assembly were performed with Adobe Photoshop.
Transport assays. NE transport assays on rat brain
synaptosomes or minced rat vas deferens (Bauman et al., 2000) and
uptake activity of cells (Apparsundaram et al., 1998a) were performed as described previously. All uptake assays were performed by using [3H]NE
(1-[7,8-3H]noradrenaline;
Pharmacia) at 50 nM final concentration,
37°C for 10 min. Nonspecific uptake was defined by using 1 µM desipramine (Sigma). Mean values for
specific uptake (pmol/mg protein ± SEM) were determined from at
least three experiments. BoNT/C1 (Calbiochem) was applied
before uptake assays by incubating the synaptosomes or minced vas
deferens with the toxin (10 ng/ml or as noted in the figures) for 1 hr
at 37°C as described previously (Beckman et al., 1998; Deken et al.,
2000). For experiments with drugs the synaptosomes or tissue slices
were incubated with vehicle, -PMA at 1 µM, or OA at 1 µM for 30 min at 37°C before transport assays. Effects
of drugs and toxin on transport activity versus vehicle controls were
evaluated with a two-tailed Student's t test, with
p < 0.05 considered significant.
Cell culture and transfection. CAD cells (Qi et al., 1997)
were a generous gift from Dr. D. M. Chikaraishi (Duke University Medical Center, Durham, NC) and were maintained in DMEM/F-12 medium supplemented with 8% fetal bovine serum (FBS), 2 mM
L-glutamine (L-Glu), 100 IU/ml penicillin, and
100 µg/ml streptomycin (pen/strep). CAD-hNET cells were generated by
stable transfection of hNET in pcDNA3 with Lipofectin
(Invitrogen, San Diego, CA) and selected/maintained in the same medium with the addition of 200 µg/ml of G418
(Mediatech, Herndon, VA). SK-N-SH cells
(ATCC, Manassas, VA) were maintained in RPMI 1640, 10%
FBS, L-Glu, and pen/strep. Human embryonic kidney-293 (HEK-293) hNET cells were described previously (Galli et al., 1995).
Chinese hamster ovary (CHO) cells and COS-7 cells were maintained in
DMEM, 10% FBS, L-Glu, and pen/strep. CHO-M3 cells were
maintained in Ham's F-12, 10% FBS, L-Glu, and pen/strep. Transfections, unless otherwise mentioned, were performed with Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's instruction. Typically, 1 µg cDNA was added to 300,000 cells in each
well of six-well plates, or 200 ng of cDNA was added to 50,000 cells
per well in 24-well plates. All cells were incubated for 24-48 hr
before assay. Amounts of syntaxin cDNAs that were transfected were
adjusted after plasmid titration experiments to diminish nonspecific
effects on NET protein synthesis (reported in the figure legends).
Transfection of oligonucleotides into CADhNET cells was performed as
described by Beckman et al. (1998) with minor modification. The sense
and antisense oligonucleotides, corresponding to bases -1 to 18 bp of
mouse syntaxin 1A (sense strand 5'-CATGAAGGACCGAACCCAG-3'), were
synthesized in the Vanderbilt University Medical Center DNA Chemistry
Core Facility. A mixture of oligonucleotides, 1% serum-containing
medium, and Lipofectamine (Invitrogen) was added on cells
plated on poly-D-lysine (Boehringer Mannheim).
Cells were incubated for 2 hr, supplemented with 5× volume of normal
media, and further incubated for 42 hr before assays.
Biochemical analysis. Glutathione S-transferase
(GST) or MBP fusion proteins were expressed in Escherichia
coli BL21pLysS (Invitrogen) and induced with 1 mM IPTG for 4 hr. Fusion proteins were purified
by one-step affinity chromatography, using glutathione beads
(Amersham Biosciences, Uppsala, Sweden) or amylose resin (New England Biolabs) according to the manufacturers'
instructions. All gel analyses were performed by using 10% SDS-PAGE.
Pull-down experiments were performed as described previously (Deken et
al., 2000). Briefly, ~150 pmol of fusion proteins was bound to 15 µl of amylose or glutathione resin before the experiments.
Protein-coated beads were incubated with GST-SynTM in PBS or with
cell lysates expressing hNET in PBS/1% Triton X-100, containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF;
Sigma) at 4°C for 1-2 hr. Immunoprecipitation of the
extracts from rat vas deferens was performed by using 43411 antisera
against NET as described by Bauman et al. (2000). For immunoprecipitation of His-hNET the transfected cells in six-well plates were washed with (in mM) 50 NaH2PO4, 10 Tris, 100 NaCl, 0.5 PMSF, pH 8.0, and incubated in 400 µl/well of lysis buffer [containing (in mM) 50 NaH2PO4, 10 Tris, 100 NaCl,
0.5 PMSF, pH 8.0, plus 1% Triton X-100] for 1 hr at 4°C. Cell
lysates were recovered by centrifugation at 20,000 × g
for 30 min at 4°C and incubated with anti-histidine antibody for 1 hr
to overnight at 4°C. Complexes were retrieved by the addition of 15 µl of protein G-Sepharose (Amersham Biosciences),
followed by three washes with the same lysis buffer. For drug
treatments the cells were preincubated in serum-free medium overnight
and further incubated with serum-free medium containing -PMA at
0.1-1 µM or OA at 1 µM
for 30 min. Cell surface biotinylation was performed as described in
detail previously (Apparsundaram et al., 1998b), using EZ-link
NHS-sulfo-S-S-biotin (Pierce, Rockford, IL), followed by
streptavidin bead capture. Bound proteins were eluted by using Laemmli
sample buffer containing 3% -mercaptoethanol. For the
immunoprecipitation of surface proteins the cells were biotinylated
with EZ-link NHS-sulfo-S-S-biotin (Pierce) and lysed as
described above. Monomeric avidin beads (15-30 µl of beads/cell
lysates from one well; Pierce) were preblocked with 10 mg/ml BSA in lysis buffer before use for the capture of biotinylated
proteins. Avidin beads were washed five times with lysis buffer, and
bound proteins were eluted by using three washes (total 600 µl) with
lysis buffer containing 2 mM biotin
(Sigma). Anti-histidine antibody was added to the eluted
proteins and processed for immunoprecipitation as described above. For
estimation of relative amounts of proteins in immunoblots, exposed
films of immunoblots were scanned with an Agfa Duoscan
T1200, and the captured images were processed in Adobe Photoshop and
quantitated with NIH Image. Multiple films were exposed for each
immunoblot to insure linearity of detection.
Electrophysiology. Cells stably transfected with the hNET
were plated at a density of 105 per 35 mm
culture dish. Before electrical recordings the attached cells were
washed three times with the bath solution of (in mM) 137 NaCl, 2.7 KCl, 1.5 KH2PO4,
and 9.7 NaHPO4 pH-adjusted to 7.4 and 276 mOsm.
The recording pipette, in the cell-detached inside-out configuration,
was filled with a solution containing the following (in
mM): 130 NaCl, 1.3 KH2PO4, 0.5 MgSO4, 1.5 CaCl2, 10 HEPES, and 34 dextrose pH-adjusted to 7.35 and 300 mOsm. NE (30 µM) was added to the patch electrode solution to activate
NET channel-like activity along with ascorbic acid (100 µM) to prevent NE oxidation. GST, GST-SynTM, and
cocaine were dissolved in the bath solution used to perfuse the
cytoplasmic face of the plasma membrane of the detached patch. Quartz
electrodes (10 M ) were pulled with a programmable puller (P-2000,
Sutter Instruments, Novato, CA). An Axopatch 200B
amplifier band-limited at 1000 Hz was used to measure NET channel
activity. Single channel events were recorded at RT with a membrane
patch potential set to -80 mV. Data were stored digitally on a VCR and
analyzed with a Nicolet Integra Model 20 oscilloscope and
a DELL computer, using instrumentation and programs written by W. N. Goolsby (Emory University, Atlanta, GA). Multiple 8 sec traces
captured for each condition (GST, GST-SynTM, cocaine) were
analyzed to calculate the mean open time
(NPo). Comparisons were performed by a
one-way ANOVA, followed by Tukey's test, with p < 0.05 taken as significant.
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Results |
NET-mediated NE transport requires syntaxin 1A
To evaluate the syntaxin 1A dependence of NET-mediated NE
transport, we established hNET stably transfected CAD cells (CADhNET) and examined whether antisense oligonucleotide-mediated suppression of
syntaxin 1A synthesis would influence NE transport. CAD cells are
catecholaminergic neuroblastoma cells (Qi et al., 1997) that natively
express syntaxin 1A protein. We found that a 2 d treatment with
syntaxin 1A antisense oligonucleotides, but not sense oligonucleotides, markedly diminished syntaxin 1A levels (Fig.
1A, top). In
parallel, we measured a significant, dose-dependent reduction in
desipramine-sensitive NE transport activity (Fig.
1A). Because CAD cells lack other pathways for NE
accumulation at the concentrations that have been used, these findings
suggest that syntaxin 1A-dependent processes linked to NET surface
expression or intrinsic activity were modified. An independent, and
more rapid, paradigm for inactivation of syntaxin 1A involves cleavage
of the SNARE protein with BoNT/C1. BoNT/C1 cleaves syntaxin 1A near the
plasma membrane and destroys its activity in mediating SNARE-dependent
vesicular fusion (Schiavo et al., 2000). Efficient cleavage of syntaxin
1A was achieved with BoNT/C1 after 1 hr incubations of rat cortical
synaptosomes, slices from rat vas deferens, human SK-N-SH cells
(express native hNET), or hNET stably transfected CAD cells, as
revealed by syntaxin 1A immunoblots (Fig. 1B). Assays
of NET activity performed on these preparations revealed a significant
50-75% loss of desipramine-sensitive NE transport activity (Fig.
1B). The reduction of NE transport mediated by
BoNT/C1 transport in SK-N-SH cells was dose-dependent and saturated at
10 ng/ml toxin (data not shown), with ~25% activity retained after 1 hr of treatment.
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Figure 1.
Toxin or antisense disruption of syntaxin 1A
diminishes NE transport activity. A, Antisense
suppression of syntaxin 1A expression reduces NE transport in CADhNET
cells. CADhNET cells were transfected with mouse sense or antisense
syntaxin 1A oligonucleotides and assayed for syntaxin content or NE
transport activity 2 d later. The cells in one well for each
transfection were lysed in PBS/1% Triton X-100, assayed for protein,
and then immunoblotted for syntaxin 1A content. Cells treated with
antisense syntaxin 1A oligonucleotides (8.6 µM) displayed
a reduction of syntaxin 1A protein, compared with the cells treated
with sense oligonucleotides. In parallel, a dose-dependent effect of
syntaxin 1A antisense oligonucleotides on NE transport activity was
observed. Results are mean values ± SEM (n = 3); *p < 0.05; Student's t test.
B, Treatments with BoNT/affect syntaxin cleavage
and reduce NE transport in native rat tissues. Minced rat vas deferens
and synaptosomes from rat brain cortex as well as SK-N-SH cells and
CADhNET cells were incubated with BoNT/C1 for 1 hr at 37°C before NE
transport assay. Aliquots of tissue extracts or cell lysates treated
with BONT/C1 (+) or vehicle ( ) were immunoblotted for syntaxin 1A
content. BONT/C1-treated tissues or cells displayed weak or no
immunoreactivity for syntaxin 1A in contrast to vehicle-treated cells.
Values reported are mean transport activities ± SEM
(n = 3); *p < 0.05; Student's
t test.
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Syntaxin 1A colocalizes and associates with NET
The effects on NE transport activity after syntaxin 1A
oligonucleotide and toxin treatments could represent long-range or indirect effects of SNARE manipulation or, alternatively, could arise
from disruptions of more intimate associations. In the latter case, NET
and syntaxin 1A proteins would be expected to colocalize in neurons.
Syntaxin 1A is expressed on axonal membranes and at membrane terminals
in the CNS (Bennett et al., 1993; Sesack and Snyder, 1995) as well as
on noradrenergic varicosities in the periphery (Brain et al., 1997).
NET proteins also are enriched on sympathetic noradrenergic axonal
membranes (Schroeter et al., 2000). To establish whether syntaxin 1A
and NET proteins are colocalized, we double-labeled cultured
sympathetic neurons prepared from mouse SCG. After differentiation the
SCG cultures elaborate neurites with periodic, varicose enlargements
that label with FM1-43, a dye taken up after synaptic vesicle recycling
(data not shown). Staining of these cultures with syntaxin 1A and NET
antibodies revealed colocalization at the varicosities (Fig.
2A-F). Using permeabilized cells, we obtained similar results with either an intracellular NET-directed antibody (43411) or a surface
epitope-directed antibody (43408). By using the surface
epitope-directed NET antibody on living cells, however, followed by
fixation and permeabilization to detect the cytoplasmically directed
syntaxin 1A epitope, we could determine that this colocalization was
evident with plasma membrane-inserted NET and not a consequence of
close apposition between NET transport vesicles and the plasma
membrane. To verify these findings in native tissues, we immunolabeled
the rat vas deferens, a peripheral preparation rich in noradrenergic
axons and high-affinity desipramine-binding sites (Raisman et al.,
1982), and found again NET and syntaxin 1A labeling to be strikingly discontinuous and colocalized, consistent with the spacing and size of
sympathetic varicosities (Fig. 2G-L).
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Figure 2.
Colocalization of NET and syntaxin 1A in
sympathetic axons. Mouse superior cervical ganglion cultures were
cultured for 5 d and stained for NET and syntaxin 1A as described
in Materials and Methods. In A-C, double staining was
performed by using permeabilized, fixed cells to reveal total NET
(antibody 43408; A) and syntaxin immunoreactivity
(B); a merged image is shown in C.
In D, labeling of live, nonpermeabilized cells was
achieved with 43408 antibody to detect surface NET protein, followed by
permeabilization to detect cytoplasmic labeling of syntaxin 1A
(E). Note the overlap in labeling
apparent as yellow fluorescence in the merged image
(F), particularly evident at
varicosities (arrows). Colocalization of NET and
syntaxin 1A in rat vas deferens is shown in G-L. Frozen
sections of rat vas deferens were double labeled with anti-NET (43411 antibody) and anti-syntaxin 1A as described in Materials and Methods,
and the immunofluorescence was detected by confocal microscopy. The
merged images (I, L) demonstrate the discontinuous and
colocalized expression of NET and syntaxin 1A along sympathetic axons
in vivo. Scale bars: A-C, 15 µm;
D-F, 7 µm, G-L, 5 µm.
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Our evidence that NET and syntaxin 1A colocalize at noradrenergic
varicosities could reflect coexpression of the two proteins at synaptic
membranes in separate complexes. However, the results of native tissue
coimmunoprecipitation experiments support a more direct interaction
(Fig. 3A,B). NET immune serum
(the same serum used for immunocolocalization studies), but not
preimmune serum, precipitated syntaxin 1A from the rat vas deferens
(Fig. 3A). In contrast, we detected no 25 kDa
synaptosome-associated protein (SNAP-25) above background (data not
shown). Similar results were found with coimmunoprecipitation
experiments that used the mouse vas deferens (Fig. 3B).
Importantly, this preparation allowed us to check the specificity of
our immunoprecipitations with tissue derived from NET knock-out mice
(Xu et al., 2000). Indeed, we recovered little or no syntaxin 1A in NET
immunoprecipitations by using homozygous (/) vas deferens despite
normal levels of syntaxin 1A protein (Fig. 3B).
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Figure 3.
Coimmunoprecipitation of NET and syntaxin 1A.
A, Syntaxin 1A coimmunoprecipitates with NET.
Solubilized rat vas deferens membranes were immunoprecipitated with NET
antisera 43411 or preimmune serum, and complexes were resolved by
SDS-PAGE, followed by immunoblotting for syntaxin 1A. An aliquot of the
total extracts was blotted in parallel. B,
Coimmunoprecipitation of NET and syntaxin 1A is diminished in vas
deferens extracts from NET knock-out mice. Extracts were prepared from
wild-type C57BL/6 (+/+) and homozygous null (/) NET knock-out mice
as described for rat preparations and were immunoprecipitated with NET
antibody 43411 before syntaxin 1A immunoblot. Total extracts were
blotted for syntaxin 1A in parallel and showed no loss of syntaxin as a
result of NET deficiency. C, Coimmunoprecipitation of
syntaxin 1A and His-NET in cotransfected CHO cells. As noted by others
(Bittner et al., 1996; Rowe et al., 1999), reduced concentrations of
syntaxin 1A cDNA were required in cotransfection studies to limit the
suppression of hNET biosynthetic progression, observed as diminished
N-glycosylated cell surface transporters (see below). CHO cells grown
in six-well plates were singly or cotransfected with His-tagged hNET
(670 ng) and full-length syntaxin 1A (45 ng). Cell lysates were
immunoprecipitated with anti-His, resolved on SDS-PAGE, and blotted for
syntaxin 1A. Immunoprecipitations also were performed with extracts
mixed from separately transfected cells (Mix). Aliquots
of total cell lysates show equal expression of syntaxin 1A in each
transfection (Total) except for lysates derived
from cells transfected with only hNET. CHO cells do not express
endogenous syntaxin 1A. D, Coexpression of Munc18
diminishes recovery of syntaxin 1A from NET immunoprecipitates. CHO
cells were transfected with syntaxin 1A alone (S, 42 ng), His-hNET (640 ng) and syntaxin 1A (42 ng) (N+S), or
His-hNET (640 ng), syntaxin 1A (42 ng), and Munc18 (318 ng)
(N+S+M). pcDNA3 was used to adjust transfections
to 1 µg of total DNA for N and N+S.
Extracts were immunoprecipitated with anti-HIS before SDS-PAGE and
syntaxin 1A immunoblots. Blots of total cell lysates reveal equivalent
expression of syntaxin 1A. Results presented in A-D are
representative of two to six experiments for each condition.
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Next we sought to reconstitute a NET/syntaxin 1A interaction in
cotransfected mammalian cells to provide a model system suitable for a
structural and functional characterization of transporter/SNARE interactions. For these studies CHO cells were transiently transfected with either, or both, syntaxin 1A and His-tagged hNET cDNAs. We found
that NH2 terminal His or HA-tagged hNET cDNAs
express equivalently to hNET cDNAs (data not shown). We found that NET
antibodies failed to immunoprecipitate syntaxin 1A from cells
transfected with either hNET or syntaxin 1A alone. In contrast,
syntaxin 1A was readily detectable in immunoprecipitates of dually
transfected cells (Fig. 3C). To control for the possibility
that our coimmunoprecipitation results arise from nonspecific
aggregation of solubilized proteins, we mixed detergent extracts
prepared from separately transfected cells and then repeated our
immunoprecipitation experiments but found no evidence of association
(Fig. 3C). As an additional test of specificity, we sought
to compete for hNET/syntaxin 1A interactions with the high-affinity
syntaxin-binding protein Munc18 (Hata et al., 1993; Pevsner et al.,
1994). Transfected Munc18 cDNA had no effect on the amount of hNET
(data not shown) or syntaxin 1A protein evident in total extracts.
However, Munc18 transfection significantly reduced the amount of
syntaxin 1A recovered from NET immunoprecipitates (Fig. 3D).
Therefore, NET and syntaxin 1A form a stable complex in intact cells
that, like the assembly of SNARE complexes engaged in vesicular fusion
(Jahn and Sudhof, 1999), can be influenced by the availability of
additional cellular syntaxin 1A binding partners.
Syntaxin 1A binds directly to the hNET N terminus
Possibly, NET/syntaxin 1A complexes observed in immunoprecipitates
of cotransfected CHO cells could reflect indirect associations. Because
we also were able to achieve similar results by using cotransfected
COS-7 and CAD cells (data not shown), we suspected that the lack of
host cell specificity to these interactions might indicate direct
interactions as observed for GAT1, CFTR, sodium, potassium, and calcium
channels (Bezprozvanny et al., 1995; Naren et al., 1997; Beckman et
al., 1998; Saxena et al., 1999; Yang et al., 1999; Fili et al., 2001).
To explore this question, we first tested a GST fusion protein
containing the cytoplasmic domain of syntaxin 1A (GST-SynTM) (Naren
et al., 1997; Deken et al., 2000) for its ability to extract NET
proteins from detergent extracts of NET transfected CHO cells. Whereas
GST protein recovered little or no NET in pull-down
experiments, GST-SynTM fusion bound NET significantly above
background (Fig. 4A).
NETs exist in transfected cells in both 60 kDa immature forms and 90 kDa mature forms, differing the extent of N-glycosylation (Melikian et
al., 1996). GST-SynTM was able to extract both forms of NET from
detergent extracts, indicating that interactions are likely independent
of transporter carbohydrate modifications.
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Figure 4.
Syntaxin 1A binds NET directly via sequences in
the NH2 terminus of the transporter. A, GST-SynTM
pull-down of NET protein. COS-7 cells were transfected with HA-tagged
hNET, and detergent lysates were incubated with glutathione beads
precoated with either GST (GST) or GST-SynTM
(Syn). Proteins bound to the beads were eluted and
subjected to SDS-PAGE, followed by immunoblotting with anti-HA. Unlike
GST beads, GST-SynTM beads retrieved NET proteins both in the
immature and mature forms. B, Direct binding of syntaxin
1A cytoplasmic domain to the hNET NH2 terminus. Amylose
resins, precoated with equimolar MBP, MBP-hNET NH2 terminal
protein (MBP-N), or MBP-hNET COOH terminal
protein (MBP-C), were incubated with GST-SynTM
as described in Materials and Methods, followed by elution of bound
material, SDS-PAGE, and immunoblotting for syntaxin 1A. Only MBP-N
retained GST-SynTM. Membranes subsequently were stained with
Coomassie brilliant blue to reveal equivalent amounts of MBP fusion proteins used in the
experiments. C, An hNET NH2 terminal
deletion disrupts NET/syntaxin 1A coimmunoprecipitation. The top
panel shows an immunoblot of hNET along with the
NH2 terminal deletion mutants that were used. CHO cells
were transfected with either His-hNET (wtNET) or
His-hNET mutants N2-42 or N43-64. Aliquots of extracts were
analyzed in SDS-PAGE and probed with polyclonal anti-His antibody to
reveal equivalent expression. The bottom panel shows
results of cotransfection of N or NET mutants (670 ng) with syntaxin 1A
(45 ng)/coimmunoprecipitation experiments, immunoprecipitating with
anti-His and probing for syntaxin 1A. The N2-42 mutant
significantly diminished syntaxin 1A recovery relative to wt hNET or
N43-64. Syntaxin 1A expression was equivalent in all samples as
assessed with syntaxin 1A immunoblots of total cell extracts. Results
presented in A-C are representative of three to six
experiments for each condition.
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We next considered whether a discrete domain of NET supported syntaxin
1A associations. Although the GAT1 NH2 terminus
has been reported to serve as a direct binding partner for syntaxin 1A
(Deken et al., 2000), this region exhibits limited conservation with
hNET and other members of the gene family. To explore the possibility
that the equivalent hNET domain harbors a structurally analogous
docking site for syntaxin 1A, we synthesized MBP fusions incorporating
either the hNET N or C terminus (MBP-N and MBP-C, respectively) and
tested their ability to bind to GST-SynTM in vitro.
MBP-N, but not MBP-C nor MBP, could be recovered in GST-SynTM pull-down assays (Fig. 4B). If the latter findings
are important for NET/syntaxin 1A interactions in intact cells, we
expect that mutation of NH2 terminal NET sequences should abolish
interactions in intact cells. A full deletion of the hNET N terminus
(2-64) fails to support normal levels of transporter protein
expression and could not be examined further. However, hNET deleted
from amino acids 2-42 (hNET2-42) or from amino acids 43-64
(hNET43-64) is expressed at levels equivalent to hNET (Fig.
4C, top). Importantly, the hNET43-64 deletion
spans the area implicated in syntaxin 1A/GAT1 interactions (Deken et
al., 2000). Remarkably, coimmunoprecipitation experiments revealed no
impact of the hNET43-64 deletion on syntaxin 1A recovery, relative
to full-length hNET (Fig. 3C, bottom). Moreover, because this region harbors several acidic residues (51D, 53D, 58E)
that could be homologous to the sites of charge-charge pairing proposed to stabilize syntaxin 1A and GAT1 interactions (Deken et al.,
2000), we mutated these sites in hNET to alanine but also failed to
disrupt hNET/syntaxin 1A associations (data not shown). In contrast,
the 2-42 mutation effectively abolished coimmunoprecipitation of
syntaxin 1A. These findings demonstrate that, although both GAT1 and
NET bind syntaxin 1A, distinct domains within the
NH2 termini of these transporters support stable
interactions with the SNARE protein.
Acute regulation of the NET/syntaxin 1A interaction
As noted previously (Bauman et al., 2000), acute (1 µM, 30 min, 37°C) treatment of rat vas deferens slices
with the phorbol ester -PMA significantly diminishes
desipramine-sensitive NE transport activity (Fig.
5A), an effect attributable to
a diminished NE transport capacity
(Vmax control = 6.9 ± 0.7 pmol/mg per min, Vmax -PMA = 4.8 ± 0.47 pmol/mg per min; Km
control = 416 ± 32 nM,
Km -PMA = 391 ± 22 nM). Given our findings of NET/syntaxin 1A
colocalization and complex formation, we next examined the sensitivity
of these associations to phorbol ester treatment and found a
significant loss of the SNARE protein in coimmunoprecipitation experiments (Fig. 5B). Recently, we also have shown that
PP1/2A phosphatase antagonists also diminish NET activity in the vas deferens in vitro (Fig. 5A) and in
vivo (Bauman et al., 2000). Okadaic acid treatments (1 µM, 30 min, 37°C) that diminish NE transport
capacity also significantly diminish syntaxin 1A content in NET
immunoprecipitates (Fig. 5B). Finally, we ascertained
whether syntaxin 1A participates in phorbol ester-mediated NET
regulation by measuring transport activity in rat synaptosomes exposed
to BoNT/C1. As shown in Figure 5C, whereas -PMA triggered
a reduction of NE transport activity in untreated synaptosomes, this
regulation was lost in BoNT/C1-treated preparations. These findings
indicate that syntaxin 1A/NET associations are responsive to phorbol
esters and that they are required to elaborate -PMA-triggered NET
downregulation.
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Figure 5.
Phorbol ester and okadaic acid modulate NE
transport and levels of NET/syntaxin 1A complexes in rat vas deferens.
A, NE transport activity of rat vas deferens. Minced vas
deferens was pretreated with DMSO (Veh), 1 µM -PMA (PMA), or 1 µM
okadaic acid (OK) for 30 min at 37°C and
subjected to evaluation of NE transport activity. Mean
Km and Vmax
values were obtained from three separate experiments; for
Vmax, control = 6.9 ± 0.7 pmol/mg per min, PMA = 4.8 ± 0.47 pmol/mg per min, and
OK = 4.1 ± 0.55 pmol/mg per min.
Vmax values of PMA and OK are different from the control value in the
analysis of one-way ANOVA, followed by Tukey's test;
p < 0.05. The mean Km
values were control = 416 ± 32 nM, PMA = 391 ± 22 nM, and OK = 364 ± 41 nM. The differences of Km values
are not statistically significant. B,
Top, Evaluation of NET/syntaxin coimmunoprecipitation
after phorbol ester or okadaic acid treatment. Minced rat vas deferens
was pretreated with DMSO (Veh), 1 µM PMA
(PMA), or 1 µM okadaic acid
(OK), as described in A, before
extraction, immunoprecipitation with anti-NET sera 43411, SDS-PAGE, and
immunoblotting for syntaxin 1A. Total extracts from all treatments were
blotted in parallel for syntaxin 1A and revealed equivalent levels.
Bottom, Average syntaxin band density ± SEM from
three different immunoprecipitation experiments conducted as in
top panel were quantitated by densitometric scanning;
the values obtained after phorbol ester or okadaic acid treatments are
expressed as a percentage of syntaxin 1A levels found in the
vehicle-treated sample. *Significant loss of syntaxin 1A from NET
immunoprecipitates as assessed by one-way ANOVA, followed by Tukey's
comparisons of group means; p < 0.05. C, Phorbol ester-induced downregulation of NET activity
in rat synaptosomes is lost after BONT/C1 pretreatment. Rat cortical
synaptosomes, prepared as described in Materials and Methods, were
pretreated with BONT/C1 (100 nM) 1 hr before treatment with
either vehicle or 1 µM PMA for 30 min, followed by assay
of [3H]NE transport as described in Materials and
Methods. Results reflect the mean of three experiments ± SEM;
*p < 0.05; Student's t test.
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Consistent with our findings of a regulated NET/syntaxin 1A complex in
native tissues, we also found that syntaxin1A/hNET complexes could be
destabilized in cotransfected CHO cells after acute treatments with
phorbol esters or okadaic acid (Fig.
6A). The PKC inhibitor
staurosporine, which blocks phorbol ester-triggered downregulation of
NET (Apparsundaram et al., 1998a,b), reverses phorbol
ester-induced dissociation of NET/syntaxin 1A complexes in this model
(data not shown). M3 muscarinic receptors are coupled to phospholipase
C and PKC activation and have been shown to trigger rapid NET
downregulation and internalization in the noradrenergic neuroblastoma
SK-N-SH (Apparsundaram et al., 1998a). We cotransfected His-hNET and
syntaxin 1A into M3-CHO cells and found that the muscarinic agonists
methacholine and carbachol destabilized NET/syntaxin 1A complexes (Fig.
6B). Together, these findings indicate that the
NET/syntaxin 1A interaction is not constitutive but, rather, can be
affected by stimuli known to alter NET trafficking.
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Figure 6.
Regulated association of NET and syntaxin 1A in
cotransfected CHO cells. A, Phorbol ester or okadaic
acid treatments diminish recovery of syntaxin 1A from NET
immunoprecipitations. CHO cells, cotransfected with His-hNET and
syntaxin 1A, were preincubated with DMSO (Veh), 1 µM -PMA (PMA), or 1 µM
okadaic acid (OK) for 30 min at 37°C before
immunoprecipitation with anti-His, SDS-PAGE, and immunoblotting for
syntaxin 1A. Total cell extracts for each condition were blotted for
syntaxin 1A in parallel. Both PMA and okadaic acid diminished recovery
of syntaxin 1A relative to vehicle-treated samples. B,
Muscarinic receptor activation diminishes recovery of syntaxin 1A from
NET immunoprecipitates. Stable M3 muscarinic receptor-transfected CHO
cells that had been cotransfected transiently with His-hNET and
syntaxin 1A were treated with the indicated concentrations (in
µM) of the muscarinic agonists methacholine or carbachol
(30 min) before extraction and immunoprecipitation of complexes with
anti-His, SDS-PAGE, and blotting for syntaxin 1A. In parallel, syntaxin
1A was blotted from total cell extracts and is evident at equivalent
levels in all conditions. C, Phorbol ester regulation of
the interaction between NET and syntaxin 1A occurs with plasma
membrane-localized complexes. CHO cells, cotransfected with His-hNET
and syntaxin 1A, were treated with DMSO (Veh) or 1 µM -PMA for 30 min at 37°C. Surface proteins were
labeled with NHS-sulfo-biotin at 4°C before cell lysis and recovery
of surface complexes (Surface) on avidin beads. Bound
proteins were eluted with 2 mM biotin, immunoprecipitated
with anti-His, resolved on SDS-PAGE, and immunoblotted for syntaxin 1A.
Nonbound (Intra) extracts were immunoprecipitated and
blotted in parallel. Phorbol ester-induced reduction in syntaxin 1A in
NET immunoprecipitates is evident in surface fractions, but not in
intracellular complexes. Blots of total cell extracts
(Total) and nonbiotinylated, intracellular
samples (Intra Total) show no impact of phorbol
ester on syntaxin 1A content. PMA increased syntaxin 1A contents in
total biotinylated pools (Surface Total). The bar
graph on the right is a quantitation of syntaxin 1A
recovery in the immunoprecipitates. D, hNET
NH2 terminal deletion that disrupts NET/syntaxin 1A
interactions diminishes phorbol ester-mediated NET downregulation. CHO cells were
transfected with His-hNET (wt) or hNET N2-42 as
described in Figure 4, followed by treatment of cells with 1 µM -PMA or vehicle for 30 min. Cells receiving hNET
N2-42 were significantly less sensitive to phorbol ester treatment
with respect to NE transport activity (n = 3;
*p < 0.05; Student's t
test).
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Access to a cell culture model supporting regulated NET/syntaxin
associations allowed us to implement biotinylation techniques to
explore whether NET/syntaxin complexes are evident on the plasma membrane and whether these complexes are regulated selectively by
-PMA. We repeated our coimmunoprecipitation experiments as above,
except that before immunoprecipitation we enriched the pool of plasma
membrane NET protein complexes by surface biotinylation and subsequent
capture on immobilized avidin (Fig. 6C). Analysis of the avidin retained and nonretained complexes demonstrates that
NET/syntaxin 1A complexes are recovered from both cell surface-enriched and intracellular fractions. However, evaluation of -PMA sensitivity of these complexes differed because only the surface pool of
NET/syntaxin 1A complexes was destabilized by -PMA treatments. These
findings suggest that the destabilization of the NET/syntaxin 1A
complex may be a step in the pathway toward cell surface NET
downregulation. In support of this idea, we found that loss of syntaxin
1A interactions incurred by the hNET 2-42 mutation limited the
ability of phorbol ester (Fig. 6D) and methacholine
(data not shown) treatments to downregulate NET activity.
Syntaxin 1A inhibits NET intrinsic activity
To this point our studies are consistent with an influence of
syntaxin 1A on NET surface expression in both the SNARE-mediated fusion
of vesicles harboring transporters as well as via direct associations
that might be disassembled to permit NET internalization. An additional
role for NET/syntaxin 1A associations is in the modulation of NET
intrinsic activity, an idea supported by multiple studies showing
direct actions of syntaxin 1A on transport and gating properties of
other ion channels and transporters (Bezprozvanny et al., 1995; Naren
et al., 1997; Beckman et al., 1998; Saxena et al., 1999; Yang et al.,
1999; Fili et al., 2001). To ascertain whether syntaxin 1A modulates
NET function, we took advantage of our ability to monitor NET-mediated
channel activity in detached patches pulled from stably transfected
HEK-293 hNET cells (Galli et al., 1995, 1998). These currents are
recorded in the inside-out configuration with patch pipettes filled
with 30 µM NE (Fig. 7) and
defined as NET-dependent through (1) their absence from nontransfected cells, (2) their blockade by desipramine or cocaine, and (3) their correlation with amperometric spikes recorded on a separate
catecholamine sensor placed beneath the patch (Galli et al., 1995,
1998). Traces were isolated with well resolved inward current
transients to focus attention on NET-dependent events separate from
background channel activity. Figure 7A presents
representative current traces of detached patches in response to
sequential application of GST, GST-SynTM (syntaxin), GST (recovery),
and cocaine. The average NPo of the
NET channel measured from four independent experiments was 10.2 ± 2.8%. Notably, the application of 3 µM
GST-SynTM decreased the channel-like activity by 83% (Fig.
7B), comparable with that achieved with local perfusion of
20 µM cocaine
(NPo reduced by 86%). Because NET
channel activity could be recovered by perfusion of the membrane patch
with GST, the interaction of syntaxin with the NET protein is a
reversible process.
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Figure 7.
Syntaxin reversibly inhibits NET-mediated single
channel currents. A, Four representative current traces
of single channel activity measured from an individual inside-out
membrane excised patch held at -80 mV with sequential application of
GST (3 µM), GST-SynTM (3 µM), and
cocaine (20 µM). The patch pipette was filled with 30 µM NE (inset) to open maximally the
NET-dependent channels. B, Amplitude histograms for each
trace were analyzed to calculate the cumulative open probability
NPo for the NET-mediated channel-like
events. Statistical analysis of the NPo determined
from four independent experiments is shown. The
NPo of the channel decreased by 83% after
perfusion of the membrane patch with GST-syntaxin 1ATM, compared
with GST alone; NPo = 10.2 ± 2.8 to 1.7 ± 0.3%, respectively. The channel-like activity was
recovered by perfusion with GST alone
(NPo = 9.2 ± 1.9%). Cocaine
sensitivity, tested after completion of the reversal experiments,
revealed the hNET origin of the channel-like activity, reducing
NPo to 1.4 ± 0.4%.
*p < 0.05 by one-way ANOVA, followed by Tukey's
test.
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Syntaxin 1A cytoplasmic domain differentially affects NET surface
expression and NE transport activity
A striking indication of the ability of syntaxin 1A to impact the
intrinsic activity of NET proteins was found in transfection studies of
the cytoplasmic domain of syntaxin 1A (SynTM) into CADhNET cells.
Although SynTM cannot support SNARE-mediated fusion (McNew et al.,
2000), we found that SynTM significantly increased NET surface
expression (Fig. 8A)
with no change in NET protein levels as measured in total cell
extracts. We suspect that this activity arises from displacement of
syntaxin 1A from host cell syntaxin-binding proteins (e.g., Munc18,
NET) that normally would limit the availability of syntaxin 1A for
membrane fusion of NET-containing vesicles (Jahn and Sudhof, 1999).
Remarkably, we found that this increase in NET surface expression was
not paralleled by a commensurate increase in NE transport activity
(Fig. 8B). In multiple experiments we achieved an
average surface recovery of NET proteins of 232 ± 22% versus
controls after SynTM cotransfection, whereas the NE transport
activity was unaffected, averaging 96 ± 2% of activity measured
in the absence of SynTM cotransfection. This discrepancy is not
attributable to saturation of surface capacities for NET function
because, when cell surface expression is titrated across the same range
simply by variation of transfected NET cDNA, an increase in NE
transport activity is evident (Fig. 8C). We also found that
cotransfection of full-length syntaxin 1A with hNET suppressed NE
transport activity (data not shown). Together with our
electrophysiological studies, these findings indicate that transport
rates of NET proteins are not governed solely by NET surface expression
and support the contention that the cytoplasmic domain of syntaxin 1A
can influence NET activity.
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Figure 8.
Fusion-incompetent form of syntaxin (SynTM)
increases NET surface expression in CAD cells without an increase in NE
transport activity. A, HA-hNET-transfected (100 ng) CAD
cells (N) were transfected additionally
with 100 ng SynTM (N+S) or 150 ng SynTM
(N+1.5S). Surface proteins of transfected cells were
labeled with NHS-sulfo-S-S-biotin, extracted, and isolated on
streptavidin beads. Bound proteins were eluted, resolved by SDS-PAGE,
and immunoblotted for NET by using anti-HA. SynTM increases recovery
of surface biotinylated NET (top). Probing for NET in
the whole extracts reveals no impact of SynTM on total NET levels
(bottom). B, Impact of SynTM on NET
activity in cells monitored for surface NET expression in parallel.
Transfection, biotinylation, and NE transport assays were performed as
described above, with one set of samples used for surface
biotinylation/immunoblot with anti-HA and the other used for NE
transport determinations. SynTM increases surface NET but does not
increase NE transport activity. C, Analysis of the
impact of SynTM on NET surface expression and NE transport activity
in CAD cells. For cotransfection of HA-NET with SynTM
(line with filled circles), CAD cells
were transfected with 100-200 ng of HA-hNET and variable amounts of
SynTM (50-300 ng). Surface NET proteins in immunoblots were
quantitated and normalized to the amount of surface NET obtained in the
absence of SynTM (set at 100%; open circle with
X). Similarly, NE transport activity in these
assays was compared with the activity of NET in the absence of SynTM
cotransfection. The dotted line represents a comparison
of surface NET density and transport activity when surface density was
varied by varying the concentration of HA-hNET cDNA. Note that, whereas
a comparable range of surface abundance is achieved via either of these
two methods, transfection with SynTM blunts the ability of
increasing hNET surface protein to result in an increase in NE
transport activity.
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Discussion |
Norepinephrine inactivation after vesicular fusion is mediated
predominantly by reuptake via cocaine- and antidepressant-sensitive NET
proteins (Axelrod and Kopin, 1969; Iversen, 1971). Like all functionally characterized members of the
Na+/Cl-coupled
neurotransmitter transporter gene family, a single cDNA suffices to
confer high-affinity NE transport sites after heterologous expression.
Recent studies suggest, however, that the surface expression and
intrinsic activity of NETs and homologs are regulated by G-protein- and
tyrosine kinase-linked receptor pathways (Apparsundaram et al.,
1998a,b, 2001), bringing into focus the presence and activities of
transporter-associated proteins. We recently have established that the
catalytic subunit of PP2A forms a phorbol ester-sensitive complex with
NETs, DATs, and SERT proteins (Bauman et al., 2000), suggesting that
the phosphatase may coordinate transporter trafficking and/or intrinsic
activity via phosphorylation-associated mechanisms (Blakely and Bauman,
2000). Torres and coworkers (2001) have shown that the PDZ domain
protein PICK1 colocalizes and coimmunoprecipitates with NET, and
 -synuclein has been reported to form a complex with the
cocaine-sensitive DAT protein (Lee et al., 2001), although their
participation in catecholamine transporter regulation is unclear.
Recently, we obtained direct evidence for the localization of NETs at
sympathetic varicosities (Schroeter et al., 2000) and were struck by
reports of a similarly restricted localization of the SNARE protein
syntaxin 1A in the same membrane domain (Brain et al., 1997). Moreover,
evidence has been gathered that syntaxin 1A forms physical complexes
with the NET homologues GAT1 and GLYT1 (Beckman et al., 1998; Geerlings
et al., 2000). In particular, the GAT1 GABA transporters interact with
syntaxin 1A in a phorbol ester-sensitive manner, with evidence that
multiple PKC-linked receptors can trigger GAT1 redistribution and
syntaxin 1A/GAT1 complex disassembly. Because syntaxin 1A is required
for vesicular release (Jahn and Sudhof, 1999), it seemed possible that
the SNARE protein might play an unrecognized role in linking the
opposing processes of catecholamine release and reuptake. We thus
sought evidence for a functional role of syntaxin 1A in establishing NE
transport capacity.
In initial tests of the syntaxin 1A requirement for NET activity, we
found that both antisense treatments and syntaxin cleavage with BoNT/C1
significantly reduced desipramine-sensitive NET activity. Based on
current models for syntaxin 1A participation in vesicular fusion (Jahn
and Sudhof, 1999), these findings appear consistent with a requirement
for syntaxin 1A to deliver NET-containing vesicles to the cell. Because
syntaxin 1A is enriched at noradrenergic varicosities, delivery of NETs
via a syntaxin 1A-mediated membrane fusion event would assist in
localization of transporters near sites of NE release for efficient
reuptake. Indeed, evidence has been provided that NET even may be
transported on a population of catecholamine secretory vesicles
(Kippenberger et al., 1999). Regardless, the existence of NET as one of
several cargo proteins in trafficking vesicles would not predict a
stable association with syntaxin 1A nor that this association should be
modulated by G-protein-coupled receptors (GPCRs) and other cellular
stimuli. Possibly, the interactions we identify could arise
artifactually via nonspecific associations that syntaxin might make
in vivo or during extraction. However, all tests that we
could perform to document specificity with native tissues, including
loss of syntaxin 1A coimmunoprecipitations from NET knock-out mice, and our ability to document and map direct interactions in vitro
support the idea that NET/syntaxin 1A interaction occurs in
vivo and supports NE uptake. Interestingly, we found no evidence
for SNAP-25 within NET/syntaxin 1A complexes, unlike observations with
calcium and potassium channels (Rettig et al., 1996; Ji et al., 2002).
These findings indicate that the composition of transporter/syntaxin 1A
complexes is distinct from the channel/syntaxin 1A complexes that
coordinate vesicular release of neurotransmitter, although it remains
possible that the two pools communicate via syntaxin 1A availability.
NET binds syntaxin 1A directly through sequences within the cytoplasmic
NH2 terminus of the transporter. Evidence to
support this contention includes the ability of GST-SynTM to pull
down full-length NET from solubilized transfected cells, findings that GST-SynTM binds selectively MBP fusions of the NET
NH2 terminus in vitro, and loss of
coimmunoprecipitation of syntaxin 1A by an hNET
NH2 terminal truncation. Although the
NH2 terminus of GAT1 also supports syntaxin 1A
associations, the GAT1 interaction is mediated by more membrane
proximal sequences (Deken et al., 2000) in which the deletion or point
mutation in NET fails to disrupt associations. Ongoing studies are
exploring the specific contact sites required for direct NET/syntaxin
1A interactions and should illuminate better how the NET
NH2 terminus participates in transport function.
Of note are studies from our lab and others that point to important
roles for TMD1 and juxta-TMD1 sequences in ligand recognition and
transport (Mager et al., 1996; Barker et al., 1999; Bennett et al.,
2000). We found that cotransfection of the high-affinity syntaxin 1A
binding protein Munc18 (Hata et al., 1993; Pevsner et al., 1994)
reduced recovery of syntaxin 1A in NET immunoprecipitates. Munc18 binds
syntaxin 1A via the cytoplasmic domain of the SNARE protein and
therefore suggests that NET/syntaxin 1A interactions in cells are
unlikely to be reliant on the syntaxin 1A transmembrane domain. The
antagonistic activity of Munc18 also highlights the idea that complexes
containing NET and syntaxin 1A can be regulated in cells by other
SNARE-associated proteins. One class of proteins of particular note is
UNC-13 homologs, for which the ability to bind phorbol esters and
modulate syntaxin 1A/SNARE associations (Betz et al., 1998) suggests
greater complexity to phorbol ester-mediated transporter regulation
than PKC activation. Regardless, phorbol ester modulation of NET
activity appears to require intact syntaxin 1A, suggesting an intimate
role of the SNARE protein in both constitutive transporter trafficking
and acute regulation.
NETs not only support antidepressant-sensitive NE uptake but also
support substantial NE-gated channel activity (Galli et al., 1995).
Amperometric recordings reveal bursts of NE translocated across the
plasma membrane that are correlated significantly with NET channel
openings (Galli et al., 1998). The existence of NET channel activity
permits the analysis of putative cytoplasmic modulators by using
inside-out patch recordings, here achieved for the first time by using
perfused peptide modulators. We found that GST-SynTM, but not GST,
effectively could eliminate NET currents with an effect resembling that
of the competitive NET antagonist cocaine. We recognize that our
detached patch experiments with heterologously expressed NET likely
represent a highly artificial situation, because other NET-associated
proteins and syntaxin-binding proteins are absent. However, as we
identified syntaxin/NET complexes in vivo, it is tempting to
speculate that a population of "inactive" carriers resides in the
plasma membrane (Ramsey and DeFelice, 2002), awaiting activation via
the elimination of syntaxin 1A associations. Such a model would be
similar to that suggested for the GAT1 GABA transporter (Deken et al.,
2000) and bears similarity to the notion that syntaxins negatively
modulate the intrinsic activities of calcium channels and other
membrane proteins (Bezprozvanny et al., 1995; Naren et al., 1997;
Saxena et al., 1999).
Transfection of the cytoplasmic domain of syntaxin 1A enhanced NET
surface expression, but this enhancement was without consequence for NE
transport activity. This is not attributable to assay limitations because we clearly were able to monitor increased transport activity by
using hNET cDNA titration across the same surface abundance range.
Rather, our findings support the contention that, like transporter-associated currents, the intrinsic transport activity of
surface NET proteins can be controlled by syntaxin 1A interactions. It
is perhaps surprising that SynTM transfection increases NET surface
density when SynTM cannot support SNARE-mediated fusion (McNew et
al., 2000). We believe this is a consequence of SynTM displacement
of endogenous syntaxin 1A from nonproductive complexes that indirectly
limit fusion capacity. Such a model underscores two modes for syntaxin
1A in establishing NE clearance capacity, one controlling transporter
trafficking and the other controlling intrinsic activity, connected by
a limited number of syntaxin 1A molecules and the opportunity for
coordination between the two modes. Physical linkages between these two
facets of the activities of syntaxin 1A would offer tighter
coordination between transmitter release and reuptake. The ability of
syntaxin 1A antisense and BoNT/C1 to diminish NE transport activity in
native cells and tissues suggests that surface trafficking of NETs is a
dominant mode by which NE clearance capacity is supported by syntaxin
1A under basal conditions. A large reserve pool of carriers may await translocation to the plasma membrane to support neurotransmitter reuptake, a surmise in keeping with electron microscopic studies that
show intracellular pools of biogenic amine transporters in vivo (Nirenberg et al., 1997; Miner et al., 2000; Schroeter et al., 2000) (L. H. Miner, S. Schroeter, Blakely, and S. R. Sesack, unpublished data). In contrast to our findings with NET,
treatment of neurons with BoNT/C1 actually elevates GABA uptake (Deken
et al., 2000 |
TOP
ABSTRACT
Introduction
