Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells.

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.

Full text

Page 1

Int. J. Biol. Sci. 2007, 3
47
International Journal of Biological Sciences
ISSN 1449-2288 www.biolsci.org 2007 3(1):47-56
© Ivyspring International Publisher. All rights reserved
Research Paper
Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive
sodium channel (ENaC) currents in HT-29 colonic epithelial cells
Sunil K Saxena, Madhurima Singh, Simarna Kaur, and Constantine George
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology,
Hoboken, NJ 07030, U.S.A.
Correspondence to: Sunil K Saxena, Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens
Institute of Technology, 314 McLean Hall, 5th and River Street, Hoboken, NJ 07030, Telephone: 201-216-8956; Fax: 201-216-8240;
E-Mail: ssaxena@stevens.edu
Received: 2006.08.16; Accepted: 2006.10.30; Published: 2006.11.06
The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium
reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of
the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid
bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a
human cell line that is physiologically relevant as it expresses both components and also responds to
aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with
natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs.
Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A
full-length construct (1-288) in this cell line. Both γENaC and neutralizing syntaxin1A antibodies blocked native
expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect.
The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane
domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC
interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional
modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed
with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple
mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that
syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which
critically determines the overall ENaC functionality/regulation under distinct physiological conditions.
Key words: ENaC, Syntaxin1A, HT-29 colonic epithelial cells
1. Introduction
The regulation of sodium reabsorption and
excretion in the kidney is central to the control of
blood pressure and extra-cellular fluid volume.
Regulation is chiefly mediated by the adrenal
mineralocorticoid hormone,
effects the activity of the sodium channel. The
molecular target for this
amiloride-sensitive epithelial sodium channel, a
heteromultimer consisting of α, β and γ subunits 1. In
aldosterone targeted epithelia, this amiloride-sensitive
epithelial sodium channel (ENaC) represents the rate
limiting step for sodium reabsorption 2, 3. Mutations in
the human genes of β and γ subunits of ENaC cause a
form of salt-sensitive hypertension described more
effectively as Liddle’s syndrome 4-7. These mutations
either introduce frame-shifts, premature stop codons,
or truncations 8, 9.
Several mechanisms are known to influence and
tightly regulate Na+ entry through the apical
membrane of epithelial cells. These mechanisms
include changes in protein expression and the relative
distribution of ENaC protein between intracellular
vesicular pools and the plasma membrane 10, 11.
Liddle’s syndrome mutations lead to both: an increase
in channel density at the cell surface and an increase
aldosterone, which
regulation is the
in open channel probability 12 . Another mechanism
described for ENaC regulation is through a group of
proteins that could directly interact with ENaC and
regulate its function. These are syntaxins, plasma
membrane localized t-SNARES that are hypothesized
to mediate vesicle trafficking. We 13 and others 14-16
have shown that SNAREs like syntaxin1A interact
with and functionally regulate the amiloride-sensitive
sodium channel (ENaC) in the Xenopus oocyte
expression system and artificial lipid bilayer
Syntaxin1A also reportedly inhibits several other ion
channels including Ca+2 channels 18, 19, CFTR chloride
channels 20, 21, K+ channels 22 and GABA transporters 23
in the Xenopus oocyte expression system.
All these studies were performed in a system
that lacks either ENaC or syntaxin de novo expression.
In this communication, we are reporting that
syntaxin1A over-expression
function in HT-29 colonic epithelial cells that natively
express both ENaC
up-regulation is the net result of the differential effect
of multiple syntaxin motifs that involves a complex
regulatory mechanism.
2. Materials and Methods
17.
up-regulates ENaC
24, 25 and syntaxin1A. This
Materials and reagents
HT-29 cells were purchased from American type

Page 2

Int. J. Biol. Sci. 2007, 3
48
culture
Syntaxin1A constructs were a kind gift from Dr. Kevin
Kirk, University of Alabama at Birmingham,
(Birmingham, AL). ΔC syntaxin1A and Munc
constructs were kindly donated by Dr. A. Naren,
University of Tennessee at Memphis, (Memphis, TN).
Munc18 antibodies were procured from Transduction
Laboratories, (Lexington,
peroxidase–conjugated (HRP-conjugated) secondary
antibodies (anti-rabbit and anti-mouse) were from
Pierce Chemical Co., (Rockford, IL). Goat anti-mouse
IgG-Alexa 488 and Alexa 594 were purchased from
Molecular Probes, (Eugene, OR). The anti-syntaxin1A
monoclonal antibody (HPC-1), and other antibodies
and reagents were obtained from Sigma Chemicals,
(St. Louis, MO). In order to confirm our findings,
ENaC antibodies available from various sources were
used from time to time. The ENaC antibodies were
raised by Research Genetics, (Huntsville, AL) and
Genemed Synthesis,
Alternatively, ENaC antibodies from other sources
were also used to confirm the findings. αENaC
antibody was a kind gift from Dr. Peter Smith,
Department of Physiology and Biophysics, University
of Alabama at Birmingham, (Birmingham, AL). The
ENaC subunit specific antibodies were a generous gift
by Dr. Bernard Rossier, University of Lausanne,
(Lausanne, Switzerland).
purchased from Invitrogen
(Carlsbad, CA). Chariot® protein delivery system was
available from Active Motif, (Carlsbad, CA). RIPA
buffer contained 50 mM Tris-Cl pH 7.4, 1% Triton
X-100, 0.2% Sodium deoxycholate, and 0.2% Sodium
dodecyl sulfate (SDS) with protease inhibitor.
Cell line
HT-29 cells were cultured in McCoy's 5a medium
with 1.5 mM L-glutamine and 10% fetal bovine serum
in 5% CO2 at 37°C. The cells were grown on Falcon 12
or 24 well inserts for all experiments and maintained
to determine the amiloride-sensitive component of the
Isc 26, 27.
Measurements of short circuit currents (Isc)
Amiloride-sensitive currents were recorded
two-ways. The Isc were recorded with EVOMTM
epithelial voltohmeter using STX2 electrode World
Precision Instruments, (Sarasota, FL) as described
before 24. Alternatively, some of the measurements
were made in a modified Ussing chamber (Trans-24
mini perfusion chamber), Warner Instruments,
(Hamden, CT). Apical and basolateral chambers were
continuously bathed with medium and the Isc were
measured with transepithelial voltage clamped at 0
mV with a DVC-1000 dual voltage clamp. Voltage
pulses (10 mV) were applied every 3 min to monitor
the transepithelial resistance. After the initial
measurements, 10 µM amiloride were added to the
apical side, and sodium currents expressed as the
amiloride-sensitive component of the Isc.
Plasmids and transfection
Fusion proteins were made as described 28.
Oligonucleotide-directed mutagenesis was used to
produce truncations and deletions. All the constructs
were confirmed by
Abbreviations and the description of syntaxin1A
collection (ATCC), (Manassas, VA).
KY). Horseradish
(San Francisco, CA).
Lipofectamine
Life
was
Technologies,
nucleotide sequencing.
constructs: syn1A-TMD, full length syntaxin, amino
acid (aa) 1-288; syn1A∆c, syntaxin1A construct lacking
the transmembrane domain, aa 1-266, syn1AH3-TMD,
the H3 domain of syntaxin1A including TMD (aa
188-288); syn1AH3, the H3 domain of syntaxin1A (aa
188-266); syn1A∆H3-TMD, truncated syntaxin1A
lacking aa 188-266 (aa 1-194); syntaxin1A∆C,
syntaxin1A lacking TMD (aa 1-266). The cells were
transfected with plasmid DNA constructs in
lipofectamine according to the manufacturer's
protocol. The expression of each protein was
confirmed by SDS-PAGE, Western blot analysis, and
detection of the protein with concomitant antibody.
Bacterially expressed fusion proteins
GST-fusion proteins
Escherichia coli DH5a. RIPA cell lysates were
centrifuged at 6000 rpm for 30 min, and the clarified
supernatant was mixed with glutathione-sepharose
beads and rocked overnight at 4 °C. The beads were
then washed three times with wash buffer (150 mM
NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol)
supplemented with the protease inhibitors leupeptin
(10 µg/mL), aprotinin (1% v/v final concentration),
and phenylmethylsulfonyl fluoride (1 mM final
concentration) before use. The protein bound to the
beads was then used for coprecipitation experiments.
For activity assays and immunoprecipitation, the
fusion proteins were eluted with 25 mM glutathione
solution in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl,
1 mM EDTA, pH 8.0, and 0.5% Nonidet P-40 (NET-N)
supplemented with these same protease inhibitors.
For Chariot® experiments, the GST- was released by
thrombin cleavage and dialysis against PBS wash
buffer. Both bound and eluted proteins were
quantitated by comparing their band intensities with
those of known amounts of bovine serum albumin on
silver-stained polyacrylamide gels.
Cell surface biotinylation, intracellular pool and ENaC
detection
The biotinylation of cell surface proteins was
performed using the kit and according to the protocol
described by the manufacturer Pierce Biotechnology
Inc., (Rockford, IL). In short, the proteins in the intact
cells were surface-labeled with cell impermeant
Sulpho-NHS-SS-biotin (0.5 mg/mL) at 4°C for 30 min.
After washing three times with ice-cold quenching
buffer, the cells were solubilized on ice in the presence
of protease inhibitors. Surface, biotinylated proteins
were adsorbed on 50% streptavidin–agarose bead
slurry by rotating for 2 hr at 4°C. After brief
centrifugation, supernatants
intracellular pool were collected and processed
accordingly. Proteins were quantified by BCA method,
separated by SDS–PAGE, and transferred to
nitrocellulose for immunoblotting. The blots were
analyzed for ENaC expression by Western blot
analysis using subunit specific ENaC antibodies. The
blots were raised using ECL and the films were
developed using autoradiography.
Electrophoresis, immunoblotting and characterization of
proteins
The proteins were solubilized at 70°C for 15 min
in Laemelli sample buffer and run through SDS-PAGE
in 10% polyacrylamide gels. The proteins were
were produced in
representing the

Page 3

Int. J. Biol. Sci. 2007, 3
49
transferred
membrane in Towbin buffer. After blocking with 5%
non-fat dry milk in TBS-Tween (Tris-buffered saline,
0.05% Tween-20, pH 7.4), the membranes were probed
with polyclonal or mono-specific affinity purified
anti-peptide antibodies. Blots were developed using
enhanced chemiluminescence (ECL) and visualized
by light-sensitive imaging
Quantification was carried out with densitometry.
Pull down assay
HT-29 cells were lysed in RIPA buffer and
pooled lysates from 12 or 24 well plates were mixed at
4°C for 15 min on a rotary mixer. The lysates were
centrifuged at 15,000xg for 10 min at 4°C. GST and
GST-syntaxin1AΔC were added to clear supernatant
and incubated for 30-60 min on a rotary shaker, after
which glutathione sepharose beads (20 μL of 50%
slurry in lysis buffer) were added and continue to mix
at 4°C for 3 hr. At the end of 3 hr, beads were spun at
800xg for 2 min and the supernatant was discarded.
The beads were washed 3 times with lysis buffer and
the proteins were eluted with 20 μL of 5X sample
buffer for 30 min at 37°C and subjected to Western
blotting.
Delivery of antibodies
The Chariot® protein delivery system was
utilized to target antibodies in HT-29 cells. Antibodies
were complexed with the Chariot® reagent at a ratio
of 1 ng IgG: 2 μL Chariot® in 100 μL PBS for 30 min.
These IgG: Chariot® complexes were overlaid onto
cultured cells in the presence of fresh serum free
culture medium for 3 hr and amiloride-sensitive
currents were recorded.
Inhibitor studies
electrophoretically to the PVDF
film (Kodak).
The 60-70% confluent HT-29 cells were
transiently expressed with syntaxin1A. Forty-eight
hours later, the cells were exposed to different
inhibitors. The following inhibitors (final conc. in
parentheses) PI3 kinase inhibitor LY294002 (50 μM);
MAP kinase inhibitor PD98059 (50 μM); protein kinase
A inhibitor RpCPTcAMP (30 μM); phospholipase C
(PLC) inhibitor U73122 (50 nM); and protein kinase C
(PKC) activator phorbol myristate acetate PMA (200
nM) were diluted into protein-free medium just prior
to use. Inhibition or activation in intact cells was
measured by incubating HT-29 cells with the
indicated concentration of reagents or dimethyl
sulfoxide carrier (0.1%, v/v) for 45 min at 37°C. The
amiloride-sensitive currents
described before.
Statistical analysis
A paired test or analysis of variance for multiple
comparisons was used for statistical analysis. A p
value less than 0.05 was considered significant.
3. Results
were recorded as

Effect of syntaxin1A on amiloride-sensitive currents
To test the hypothesis
functionally modulates
amiloride-sensitive currents were recorded in HT-29
cells over-expressing
comprising of specific domains (Fig 1). We observed
that basal currents were augmented by the expression
of full-length syntaxin1A in HT-29 cells, though the
truncated constructs enumerate differential effect on
amiloride-sensitive currents. For example, a modest
stimulation was recorded with the syntaxin construct
having the H1-H2 domains (1-194), while the
expression of cytosolic H3 domain (194-266) inhibited
amiloride-sensitive basal currents. Surprisingly, the
inclusion of the transmembrane domain (TMD) to the
H3 motif (194-288) augmented currents. These data
suggest that expression of both H1-H2 domains (1-194)
or H3-TMD (194-288)
amiloride-sensitive currents and the enhanced
currents observed with full length syntaxin1A
expression might represent the cumulative effect of
H1-H2 with H3-TMD combined. Interestingly, the H3
domain, which has dominant coiled-coiled domain
and the TMD are reported to impart an inhibitory
effect on ENaC function in Xenopus oocytes 16 and
artificial-lipid bilayer system 17.
that
ENaC
syntaxin1A
activity,
syntaxin1A constructs
stimulate basal
Fig 1 - Syntaxin1A augments currents -
HT-29 cells grown on cell inserts were
transfected with wild-type syntaxin1A
(Syn1A) and its truncated constructs.
Inset- Structure of syntaxin1A. TMD
represents the transmembrane domain. 48
hours later the
currents were recorded as described in the
text. The data represents a mean of five
individual experiments. Experimental
conditions that resulted in a significant
change (p < 0.05) from the relevant
control values (Bar one) are denoted by
multiple asterisks. Three asterisks denote
higher statistical significance.

Dose-dependent stimulation of
amiloride-sensitive currents
Our preliminary observations were contrary to
the expected results as our initial results in the oocyte
expression system indicated an inhibition of sodium
currents 13. In order to confirm our findings, we
over-expressed syntaxin1A at different concentrations
(0.1 - 4.0 μg) and recorded amiloride-sensitive
currents. As evident from (Fig 2), we observed an
augmentation of amiloride-sensitive currents in all
doses from 0.1 to 4.0 μg/well. Surprisingly, the
stimulation was least with the highest dose (4.0 μg)
used in the study, suggesting that higher expression
amiloride-sensitive

Page 4

Int. J. Biol. Sci. 2007, 3
50
might down-regulate channel function as a consequence of overdose.
Fig 2 - Syntaxin1A up-regulates amiloride-sensitive currents in a dose-dependent manner - HT-29 cells grown on cell
inserts were transfected with wild-type syntaxin1A (Syn1A) at different concentrations. 48 hours later amiloride-sensitive
currents were recorded (A) as described in the text. (B) The protein extracts from the transfected cells were solubilized and
analyzed by Western blot analysis. The blots were developed with syntaxin1A antibody. The data represents a mean of three
individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control
values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.


Fig 3 - Syntaxin1A antibody inhibits amiloride-sensitive currents - The HT-29 cells were targeted with anti-syntaxin, or
anti-ENaC antibodies or irrelevant IgG using the Chariot® protein delivery system. Antibodies were complexed with the
Chariot® reagent at a ratio of 1 ng IgG: 2 μL Chariot® in 100 μL PBS for 30 min. Then the IgG: Chariot® complexes were
overlaid onto cultured cells in the presence of fresh serum free culture medium for 3 hr and amiloride-sensitive currents were
measured. Data represent a mean of three individual experiments. Experimental conditions that resulted in a significant
change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks.


Effect of antibody delivery in the permeabilized cells
In order to further substantiate our observations,
we introduced syntaxin1A monoclonal antibody by
permeabilizing the cells with the Chariot® delivery
system and recorded amiloride-sensitive currents.
HT-29 cells natively express the proteins against
which neutralizing antibodies were introduced in
these cells (data not shown). As indicated in (Fig 3),
the ENaC activity was completely abolished by the
introduction of syntaxin1A and not with syntaxin2.
Moreover, the complete elimination of sodium
currents in the presence of γENaC antibody suggests
that amiloride-sensitive currents are the consequence
of ENaC function in these cells. Moreover, the
syntaxin2 antibody failed to impart any effect on

Page 5

Int. J. Biol. Sci. 2007, 3
51
amiloride-sensitive currents while the antibody
against syntaxin1A binding
produced up-regulation of ENaC currents suggesting
that the effect of syntaxin1A is specific and also
stimulatory in HT-29 colonic epithelial cells. This
observation is further supported by the introduction
of munc18-1 antibody in the cells.
partner munc18-1
Fig 4 - ENaC interacts with different syntaxin1A
domains - Endogenous ENaC interacts with syntaxin in
HT-29 cells - GST-syntaxin proteins were used to pull down
proteins bound to natively expressed ENaC in HT-29 cell
lysates. The immune complex was adsorbed on glutathione
beads and then separated by SDS-Polyacrylamide gel
electrophoresis and transferred to PVDF membrane. The
blots were probed with affinity purified subunit specific
γENaC antibody. The data shows interaction of multiple
syntaxin1A domains with ENaC and points to its
involvement and physiological significance in the regulation
of the amiloride-sensitive epithelial sodium channel in
native cells.

Pull down of ENaC with syntaxin1A
In order to understand if the functional result in
the form of amiloride-sensitive currents is corollary to
the physical association between ENaC and syntaxin
under physiologically defined HT-29 cells, we
performed co-immunoprecipitation studies in HT-29
cell lysates and also utilized pull down assay with
GST-syntaxins as described in the experimental
procedures (Fig 4). As expected, we observed that
multiple syntaxin1A domains including H1-H3
interact with ENaC. As expected, the maximum
binding was observed with the H3 domain of
syntaxin1A. These observations lend support to the
proposal that syntaxin1A, a protein with multiple
coiled-coiled and binding domains, is capable of
interacting with other proteins with variable affinity
but with variable efficacy.
Biotinylation studies
Our physical and functional observations
implied that the modulation of amiloride-sensitive
currents might be a consequence of net ENaC
expression on the plasma membrane. In order to
explore this possibility, we performed biotinylation
studies on HT-29 cells transiently expressing
syntaxin1A (Fig 5). We observed increased ENaC
density at the plasma membrane in cells expressing
full-length syntaxin1A.
biotinylation experiments with multiple syntaxin
constructs point to the moderate enhancement of
αENaC expression in cells transfected with H3-TMD.
However, ENaC density decreased considerably at the
cell surface in cells expressed with H3 domain (Fig 5),
otherwise it remained unaltered in other conditions.
In addition to this,
Fig 5 - Syntaxin1A domains modulate ENaC expression at the cell surface - HT-29 cells were transfected with wild-type
syntaxin1A and its truncated constructs. Cell surface proteins were biotinylated with Sulpho-NHS-SS-biotin, pulled down
with streptavidin-agarose separated by SDS-PAGE and transferred to PVDF membrane. The blots were probed with γENaC
antibody (A). The protein was analyzed by densitometry (B). The data reflect increased expression of ENaC in HT-29 cells
transfected with syntaxin1A, which is reflected in enhanced amiloride-sensitive currents reported in the text. Data represent
three individual experiments each performed with different batches of HT-29 cells.

Intracellular (cytosolic) pool
In order to further confirm that the changes in
the apical expression of ENaC coincide with the
decrease/increase of internal or cytosolic ENaC pools,
we used the unbiotinylated pool as internal ENaC
proteins since it could not get exposed on the cell
surface29. The analysis of the internal pool by Western
blotting using γENaC antibody (Fig 6) suggested that
the cytosolic ENaC concentration(s) follows the
pattern opposite to the ENaC expression at the plasma