From the Departments of Medicine and Physiology,
University of Toronto, Toronto M5S 1A8, Canada and the
¶ Departments of Physiology and Pharmacology, Sackler School of
Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
Received for publication, December 23, 2002, and in revised form, February 19, 2003
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ABSTRACT |
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Voltage-gated K+
(Kv) 2.1 is the dominant Kv channel that controls membrane
repolarization in rat islet In neurons and neuroendocrine cells, depolarization opens
voltage-dependent Ca2+ channels
(VDCC),1 and the subsequent
Ca2+ influx triggers exocytosis of neurotransmitters or
hormones. Vesicle fusion with the plasma membrane is initiated by the
sensing of Ca2+ by the vesicle protein synaptotagmin. This
is followed by, through yet unclear mechanisms, core complex formation
by SNARE (soluble N-ethylmaleimide-sensitive factor
attachment protein receptors) proteins, which involve another vesicle
protein synaptobrevin (vesicle-SNARE) and two plasma membrane SNARE
proteins SNAP-25 and syntaxin 1 (target-SNAREs) (1-3). It is proposed
that core complex formation brings the two apposing membranes together
and liberates the energy required to drive lipid re-orientation during fusion (1-3). SNARE proteins have been known to be tethered to various
VDCC, and thus such a protein complex may provide rapid release
response with SNARE proteins exposed to a high local Ca2+
concentration permeating through the VDCCs, which in turn are being
modulated by the SNARE proteins (4-8). Because of such an intimate
physical and functional coupling, the secretory vesicle-SNARE protein-Ca2+ channel complex has been termed excitosome
(6). Therefore, besides their participation in membrane fusion, SNARE
proteins appear to have a regulatory role on other components
(i.e. membrane ion channels) of the exocytotic process.
During increased glucose metabolism, a high intracellular ATP to ADP
concentration ratio ([ATP]/[ADP]) causes inhibition of pancreatic
islet Cell Culture and Transfections--
HEK293 cells were grown at
37 °C in 5% CO2 in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Invitrogen) and
penicillin-streptomycin (100 units/ml, 100 µg/ml) (Invitrogen). The
cells were transiently transfected with GFP and Kv2.1 with or without
Syn-1A using LipofectAMINE 2000 (Invitrogen). Two days after
transfection, cells were trypsinized and placed in 35-mm dishes for
voltage-clamp experiments. Transfected cells were identified by
visualization of the fluorescence of the co-expressed GFPs.
Islet Isolation--
Rat pancreatic islets were isolated by
collagenase digestion as described previously (17). Islets were
dispersed to single cells by treatment with 0.015% trypsin
(Invitrogen) in Ca2+- and Mg2+-free
phosphate-buffered saline. Islet cells were plated on glass coverslips
in 35-mm dishes and cultured in low glucose Roswell Park Memorial
Institute medium (2.5 mM glucose) supplemented with 7.5%
fetal bovine serum, 0.25% HEPES (Sigma-Aldrich Canada Ltd.), and 100 units/ml penicillin G sodium, 100 µg/ml streptomycin sulfate (Invitrogen). Islet cells were cultured for 2 days before
electrophysiological recordings.
DNA Constructs and Generation of GST Fusion Proteins--
The
vectors pcDNA3-Kv2.1, pcDNA3-Syntaxin 2, and
pcDNA3-Syntaxin 1A were generously provided by Dr. R. Joho
(University of Texas, Southwestern Medical Center, Dallas, TX) and
Richard Scheller (Stanford University, Palo Alto, CA). The coding
sequences corresponding to N-terminal region (1-182) of Kv 2.1 were
amplified by polymerase chain reaction (PCR) and cloned into pGEX-5X-1
expression vector (Amersham Biosciences Inc.) for generation of GST
fusion proteins. The primers used for PCR were 5'-ATGACGAAGCATGGCTCGC
(sense) and 5'-CACCGACGAGTTGGGCT (antisense). The plasmids
pGEX-4T-1-Kv2.1-C1 (the region corresponding to amino acids 412-633)
and pGEX-4T-1-Kv2.1-C2 (the region corresponding to amino acids
634-853) were similarly generated. These constructs were verified by
DNA sequencing. pGEX-4T-1-syntaxin-1A (wild type) is a gift from Dr. W. Trimble (The Hospital for Sick Children, Toronto, Ontario, Canada). GST
fusion protein expression and purification were performed following the
manufacturer's instructions. Syn-1A was obtained by cleavage of
GST-Syn-1A with thrombin (Sigma).
In Vitro Binding Studies--
GST (as a control) and GST-Kv2.1-N
or -C1 or -C2 (500 pmol of protein each) were bound to
glutathione-agarose beads and incubated with thrombin-cleaved Syn-1A
(500 pmol of protein) in 200 µl of binding buffer (25 mM
HEPES pH 7.4, 50 mM NaCl, 0.1% gelatin, 0.1% Triton
X-100, 0.1% bovine serum albumin, and 0.2% Electrophysiology--
HEK293 cells were voltage-clamped in the
whole-cell configuration (20) using an EPC-9 amplifier and Pulse
software (HEKA Electronik, Lambrecht, Germany) as we previously
described (7, 8). Recording pipettes were pulled from 1.5-mm
borosilicate glass capillary tubes (World Precision Instruments, Inc.,
Sarasota, FL) using a programmable micropipette puller (Sutter
Instrument). Pipettes were then fire-polished and tip resistances
ranged from 1.5-3 M Confocal Immunofluorescence Microscopy--
Laser confocal
immunofluorescence microscopy was performed as described previously
(17). Transfected HEK cells were fixed with 100% methanol on
3-aminopropyltriethoxysilane-treated glass slides. The slides were then
incubated at 4 °C overnight with primary antibodies, including mouse
monoclonal anti-Kv2.1 (1:100) (Upstate Biotechnology Inc., Lake Placid,
NY), rabbit anti-syntaxin-1 (Calbiochem, San Diego, CA) (1:100) and
rabbit anti-syntaxin-2 (1:200, kindly provided by Dr. V. Olkkonen,
National Public Health Institute, Helsinki, Finland). The slides were
rinsed four times with phosphate-buffered saline containing 0.1%
saponin and treated with secondary antibodies (FITC sheep anti-mouse
IgG 1:500 or Texas Red-labeled goat anti-rabbit IgG 1:250) for 1 h. Next, they were incubated with 0.1% p-phenylenediamine
(ICN, Cleveland, OH) in glycerol and examined using a laser scanning
confocal imaging system (LSM-410; Carl Zeiss, Thornwood, NY). FITC
signal was visualized by excitation at a wavelength of 488 nm and
emitted fluorescence was measured through a 515- to 540-nm bandpass
filter. Texas Red signal was visualized by an excitation wavelength of
568 nm and emitted fluorescence was detected through a 590-nm long-pass filter.
Cell Surface Biotinylation--
Two days after transfection
HEK293 cells were washed and harvested in phosphate-buffered saline.
The cells were further washed with borate buffer (154 mM
NaCl, 7.2 mM KCl, 1.8 mM CaCl2, 10 mM boric acid, pH 9.0) and then incubated in 5 ml of
Sulfo-NHS-SS-Biotin (Pierce Biotechnology Inc.) (0.5 mg/ml) in borate
buffer at 4 °C for 30 min. After washing three times with ice-cold
quenching buffer (192 mM glycine, 25 mM Tris,
pH 8.3), cells were solubilized on ice in 500 µl of
immunoprecipitation buffer (1% deoxycholic acid, 1% Triton X-100,
0.1% SDS, 150 mM NaCl, 1 mM EDTA, 10 mM Tris-Cl, pH 7.5) containing a mixture of protease
inhibitors (Roche Applied Science). The cell lysate was centrifuged for
20 min at 16,000 × g, and the supernatant was
retained. 50 µl of immobilized streptavidin resin (Pierce) (50%
slurry in phosphate-buffered saline containing 2 mM
NaN3) was added to the supernatant, which was then
incubated overnight at 4 °C with gentle rocking. Samples were
centrifuged for 2 min at 8,000 × g, and the resin was
washed five times with immunoprecipitaton buffer. The protein was
eluted from the resin by the addition of SDS-PAGE sample buffer
containing 5% 2-mercaptoethanol and incubation at 65 °C for 5 min.
The samples were analyzed for Kv2.1 expression by Western blotting
using anti-Kv2.1 (1:1000, Alomone Labs, Jerusalem, Israel).
Integrated density of the bands was determined using a commercial
software (Scion Image Beta 4.02; Scion Corporation, Frederick, MD).
Syn-1A Inhibits Rat Islet
We next investigated whether the interactions between Syn-1A and the
Kv2.1 C1 and C2 domains are direct by direct protein binding studies
and functional studies using a heterologous expression model system
(i.e. HEK293), which has little if any endogenous expression
of these proteins (22).
Syn-1A Inhibits Kv2.1 Channel Activity by Directly Acting on Its
Cytoplasmic C Terminus--
Binding assay and electrophysiological
data from our previous reports (16, 17) suggest that SNAP-25 inhibits
Kv1.1 and Kv2.1 current by binding to the cytoplasmic N terminus. We
have also shown that Syn-1A binds to the N terminus of Kv1.1 (19). We
therefore examined if Syn-1A bound to the cytoplasmic N or C terminus
of Kv2.1 by performing the binding assay with recombinant proteins
(Fig. 2). Syn-1A (Fig. 2, top
panel) bound very strongly with C1 and less so with C2. Syn-1A
bound only weakly with the N terminus. As a negative control, GST did
not bind to Syn-1A at all. Fig. 2, bottom panel, shows a
Ponseau S staining of the blot, which demonstrates the equal protein
loading of the C1 and C2 proteins, whereas the N-terminal protein and
GST were loaded somewhat more but nonetheless showed little and no
binding to Syn-1A, respectively. Immunostaining of this blot with
anti-GST antibodies confirmed the presence of these proteins (data not shown).
As shown in Fig. 3, dialyzing Syn-1A-GST
fusion protein through the recording pipette into Kv2.1-transfected
cell caused a reduction (14.4 ± 5.0%; p < 0.05)
of Kv2.1 current after 6-8 min. This reduction could be abolished by
co-dialysis with C1 or C2. Again, similar to the results with rat islet
Syn-1A Reduces Kv2.1 Channel Surface Expression and Current
Density--
Dialysis of the Syn-1A might either interact with a
cytosolic protein and may not be specifically targeted to the plasma
membrane where the Kv2.1 channel is situated. This could be
circumvented by overexpressing Syn-1A, which would be appropriately
targeted to the plasma membrane compartment (7). Furthermore, Syn-1A has been shown to not only affect Kv1.1 channel function but also affected surface expression of Kv.1.1 in Xenopus oocytes
(18). As shown in Fig. 4A,
expression of Syn-1A drastically reduced Kv2.1 current density
(0.21 ± 0.05 nA/pF compared with Kv2.1 alone 0.99 ± 0.18 nA/pF; p < 0.05). The reduction in Kv2.1 current
density in the presence of syntaxin-2 (Syn-2) was small and
insignificant.
To investigate whether Syn-1A reduced Kv2.1 current density by
inhibiting the trafficking of Kv2.1 protein to the plasma membrane, we
performed confocal immunofluorescence microscopy (Fig. 4B). When Kv2.1 was expressed alone, there was bright and clear fluorescence at the cell periphery, suggesting that the majority of the Kv2.1 channel protein was present at the plasma membrane (Fig. 4B,
left panel). With Syn-1A co-expression, the Kv2.1 plasma
membrane fluorescence was dimmer and diffuse (note patches of
fluorescence beneath the cell periphery) (Fig. 4B,
middle panel), indicating that the overexpressed Syn-1A
(inset) inhibited Kv2.1 from surfacing to the plasma
membrane, and a substantial proportion of Kv2.1 was retained in the
cytoplasm. Consistent with the current density data shown in Fig.
4A, overexpression of Syn-2 (inset) did not cause
significant inhibition of surfacing of Kv2.1 (Fig. 4B,
right panel).
To quantitatively determine the amount of reduction of plasma membrane
surfacing of Kv2.1 caused by the overexpression of Syn-1A and Syn-2, we
performed the following study. Transfected HEK293 cells were
biotinylated so that plasma membrane proteins can be separated from the
rest of the cells using the streptavidin resin. Fig. 4C
(upper panel) shows that the levels of plasma membrane Kv2.1
proteins pulled down by the streptavidin resin was reduced by 49% with
the Syn-1A co-expression, but only by 22% with the Syn-2 expression.
The Kv2.1 proteins in the total lysates obtained just prior to the
treatment with streptavidin resin did not change, indicating that both
syntaxins did not have any significant effect on total protein
synthesis of Kv2.1 (lower panel).
Modulation of Kv2.1 Channel Properties by Syn-1A--
A number of
reports have already shown that SNARE proteins profoundly affected Kv
channel electrophysiological properties, such as activation and
inactivation kinetics (16, 18, 19). We next explored whether Syn-1A
targeted to the plasma membrane by co-expression would modulate the
electrophysiological properties of Kv2.1 channel current. Only those
cells expressing currents greater than 4 nA were selected for analysis
because HEK293 cells express endogenous outward K+ currents
as high as 0.4 nA (data not shown). Kv2.1 had a fairly rapid activation
rate, with a
Since Syn-1A slows down Kv2.1 activation, we then examined whether
Syn-1A would affect the voltage dependence of activation of Kv2.1. To
study this, instantaneous activation curves were obtained using the
protocol in which voltage steps from
We performed the steady-state inactivation experiments to determine
channel availability for activation as a function of membrane potential. A dual-pulse protocol was used in which a test pulse step of
+70 mV was preceded by a long pre-pulse (12 s) of different potentials.
The test pulse currents are normalized to the largest test pulse
current and plotted against the pre-pulse voltages. The curves are best
fit by the Boltzmann equation (Fig. 6B). Kv2.1 currents have
a half-maximal inactivation potential (V1/2) of
Intensive work in the past decade has revealed the putative
interacting domains between VDCC and SNARE proteins (4-8). The secretory vesicle-SNARE protein-Ca2+ channel complex
("excitosome") may serve dual purposes. First, it may support very
fast secretory response; second, SNARE proteins may provide immediate
feedback to the Ca2+ channels (4-6). Kv channels provide
the repolarizing currents, subsequently causing closure of
Ca2+ channel and cessation of the release process (10-13).
Further regulation of these Kv channels by SNARE proteins would provide an additional feedback mechanism to more tightly regulate the opening
and closing of the Ca2+ channel, and therefore, the
amplitude and duration of exocytosis. Indeed, our recent reports
demonstrated that SNAP-25 and Syn-1A directly bind and modulate
neuronal Kv1.1 channels at its cytoplasmic N terminus (16, 19). This
plasticity, provided by such versatile interactions between these SNARE
proteins and Ca2+ and Kv channels, is of great importance
not only in neurotransmitter release but also in endocrine secretion,
particularly the islet We have obtained evidence that Syn-1A modulated rat islet First, in contrast to our previous work showing that Syn-1A
co-expression in Xenopus oocytes reduced the magnitude of
Kv1.1 current, but which did not affect the Kv1.1 current activation rate (18), our current study shows co-expression of Syn-1A
substantially slowed down the activation of Kv2.1 current. Both Kv1.1
and Kv2.1 currents exhibit very slow inactivation, which is not
affected at all by co-expression of Syn-1A (Ref. 18 and this work).
Expression of Kv1.1 together with the Kv Because Syn-1A inhibited Kv2.1 currents and slowed down the Kv2.1
current activation, we tested whether Syn-1A would affect the voltage
dependence of Kv2.1 activation by analyzing the tail currents. To our
surprise, Syn-1A did not cause a significant right shift of the
activation curve, indicating that Kv2.1 activated equally readily at
lower voltages in the presence of Syn-1A. Thus, Syn-1A appears to have
a selective effect on Kv2.1, namely, decelerating activation without
affecting its voltage dependence.
Next, we show that Syn-1A can affect the voltage dependence of
steady-state inactivation of Kv2.1. Thus, we show that although Syn-1A
did not cause significant left shift of the steady-state inactivation
curve, it did significantly decrease the slope factor, indicating that
Syn-1A enhances the voltage sensitivity of such steady-state
inactivation. Remarkably, the slope of the inactivation curves lies
within the depolarization ranges, implying that the physiological role
of Syn-1A might be to render Kv2.1 channel less available as the cell
becomes increasingly depolarized during stimulation. Note that
co-expression of Syn-1A has been shown to also modulate steady-state
voltage dependence of inactivation of L- and N-type VDCC expressed in
Xenopus oocytes (24).
We show here that co-expression of Syn-1A reduced Kv2.1 current
density. We showed that this is due not only to a direct inhibition of
channel activity (Fig. 3), but also to an inhibition of the surfacing
of the Kv2.1 protein to the plasma membrane (Fig. 4). The latter was
demonstrated by confocal microscopy and at the protein level. We
recently showed that Syn-1A has a biphasic effect on Kv1.1 current: low
concentration promoting while high concentration reducing current
amplitudes (18). The enhancement of current by low syntaxin expression
was not accompanied by enhanced expression of channel protein while
reduction of current by high syntaxin expression was largely due to a
reduced expression. It is noteworthy, however, that SNAP-25 could
reduce both Kv1.1 and Kv2.1 current magnitudes without affecting
channel surface expression (16, 17). More work is required to determine
the mechanism by which Syn-1A regulates Kv channel trafficking to the
plasma membrane surface.
We demonstrated recently that insulin release and L-type
Ca2+ channel activity in HIT-T15 It can be envisaged that during exocytosis SNAP-25, Syn-1A, and Kv2.1
form a complex, reminiscent of the SNARE protein-Ca2+
channel complex. Our data here show that, besides reducing current magnitude, Syn-1A has a unique inhibitory profile on Kv2.1 currents: (i) slowing activating without affecting voltage dependence of activation and (ii) increasing sensitivity of voltage dependence of
steady-state inactivation without affecting inactivation kinetics. The
inhibition of Kv2.1 by Syn-1A may be important in preventing outward
K+ currents during very early -cells and downstream insulin
exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet
-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein
syntaxin 1A (Syn-1A) binds and modulates rat islet
-cell Kv2.1 at
its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing
Kv2.1, we observed identical effects of channel inhibition by dialyzed
GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino
acids 412-633, C2: amino acids 634-853), but not the Kv2.1
cytoplasmic N terminus (amino acids 1-182). This was confirmed by
direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins.
These findings are in contrast to our recent report showing that Syn-1A
binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and
not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing
HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of
Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1
current activation and reduction in the slope factor of steady-state
inactivation, but had no affect on inactivation kinetics or voltage
dependence of activation. Taken together, SNAP-25 and Syn-1A
mediate secretion not only through its participation in the exocytotic
SNARE complex, but also by regulating membrane potential and calcium
entry through their interaction with Kv and Ca2+ channels.
In contrast to Ca2+ channels, where these SNARE proteins
act on a common synprint site, the SNARE proteins act not only on
distinct sites within a Kv channel, but also on distinct sites between
different Kv channel families.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell ATP-sensitive K+ channels (KATP)
channels, which in turn results in depolarization and consequently
insulin release (9). Outward currents carried by voltage-gated
K+ (Kv) channels in
-cells are responsible for
repolarization, which results in closure of VDCC and subsequent
termination of exocytosis (10-13). The role for these Kv channels in
-cell excitation-secretion coupling is of clinical therapeutic
importance, as blocking Kv channels with pharmacological agents can
prolong depolarization and enhance Ca2+ entry, and thereby
sustain insulin secretion in a glucose-dependent manner
(14, 15). Although SNARE protein-Ca2+ channel interaction
has been studied in great detail, little is known about SNARE
protein-Kv channel interaction. Recently, using the Xenopus
oocyte expression system and coimmunoprecipitation experiments, we have
shown that SNAP-25 and Syn-1A physically interact with Kv1.1 and Kv2.1
(16-19). Functionally, SNAP-25 inhibits Kv1.1 and Kv2.1 currents, and
such inhibition was mediated through binding of SNAP-25 to the Kv1.1
and Kv2.1 cytoplasmic N termini (16, 17). We have also shown that
Syn-1A has a concentration-dependent biphasic effect on
Kv1.1 current amplitudes: at low concentration it enhances current
without affecting surface channel expression while at high
concentration it decreases current amplitude probably by reducing
surface channel expression (18). More recently, we further demonstrated
that Syn-1A also binds to the cytoplasmic N terminus of Kv1.1, at the
T1A domain and forms a stable complex with G
subunits (19). In
this work, we surprisingly found that Syn-1A binds to the cytoplasmic C
terminus of islet
-cell Kv2.1 channel protein and modulates channel
properties. Syn-1A, when overexpressed, also inhibited Kv2.1 surface
expression and reduced Kv2.1 current density in heterologous HEK293
cells. Although SNARE protein interactions with Kv channels follow a
similar paradigm as the Ca2+ channels, the interacting
domains within and between the Kv channel families seem to be distinct
in contrast to the highly conserved synprint site (cytoplasmic II-III
loop) between the Ca2+ channel families (4-6,8).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol) at
4 °C for 2 h with constant agitation. The beads were then
washed two times with washing buffer containing 20 mM HEPES
pH 7.4, 150 mM KOAc, 1 mM EDTA, 1 mM MgCl2, 5% glycerol, and 0.1% Triton X-100. The samples were then separated on 15% SDS-PAGE, transferred to nitrocellulose membrane (Millipore, Bedford, MA), and
identified with specific primary antibody against Syn-1A (1:2000) (Sigma).
(for HEK cells) or 2.5-4 M
(for
-cells)
when filled with intracellular solution, containing (in
mM): 140 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, and 5 MgATP (pH 7.25 adjusted with KOH). Bath solution contained
(mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES (pH 7.3 adjusted with NaOH). After a
whole-cell configuration was established, membrane potential was held
at
70 mV and outward currents were triggered with depolarizing
voltage pulses (+70 mV, 250 ms). Steady-state outward currents was
determined as the mean current in the final 95-99% of the 250-ms
pulse. All experiments were performed at room temperature
(22-24 °C). Data for voltage dependence of activation and
steady-state inactivation were fit by the Boltzmann equation:
I/Imax = 1/{1 + exp[(V
V1/2)/k]}, where V1/2 is the half-maximal activation potential
(for voltage dependence of activation) or the half-maximal inactivation
potential (for steady-state inactivation), and k the slope
factor. Results are presented as means ± S.E. Unpaired Student's
t test was employed, and p < 0.05 was
considered statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Cell Kv2.1 Activity--
We had
previously shown that Kv2.1 channels account for ~60% of the outward
current in rat islet
-cells (15, 17, 21), making this cell an
excellent model to examine the effects of Syn-1A on this channel. Fig.
1A shows the current-voltage
relationships of outward K+ currents in rat islet
-cells
after an 8-min dialysis with various fusion proteins. For clarity, the
results are presented as bar graphs at two positive voltages, which
triggered outward currents (+10 and +60 mV) (Fig. 1, B and
C). GST alone was used as a control that by itself did not
cause any significant effect on
-cell outward K+
currents (data not shown). GST-Syn-1A (1 µM) caused a 26 and 22% reduction (p < 0.05) in current density at
+10 and +60 mV, respectively. We then examined whether this is mediated
via the cytoplasmic N terminus (amino acids 1-182) as we had reported with Kv1.1 (16) or with the cytoplasmic C terminus (amino acids 411-853). Since the cytoplasmic C terminus of Kv2.1 is quite large and
difficult to generate the recombinant protein, we generated two smaller
sections of this protein, C1 (412-633) and C2 (634-853). Surprisingly, we found that co-dialysis with C1 and/or C2 prevented Syn-1A from inhibiting the Kv2.1 currents, with C2 being more effective
than C1, whereas the cytoplasmic Kv2.1 N terminus had no effect on
Syn-1A actions. These data suggest that Syn-1A inhibited
-cell Kv2.1
currents by interacting with the Kv2.1 C terminus.
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Fig. 1.
Inhibition of -cell K+
currents by dialysis of Syn-1A fusion protein was attenuated by Kv2.1
C-terminal fragments but not affected by Kv2.1 N terminus.
A, after dialysis of Syn-1A (n = 9), GST
(n = 8), Syn-1A + C1 (n = 5), Syn-1A + C2 (n = 10), Syn-1A + C1 + C2 (n = 4),
or Syn-1A + N terminus (n = 5) (all at 1 µM) into the
-cell for 8 min, the current-voltage
relationship of
-cell outward K+ currents was studied
using the protocol in which the cell was held at
70 mV and was then
given depolarizing pulses (250 ms) at 10 mV increments. Currents were
normalized by cell capacitance to yield current densities. B
and C, for clarity, the current densities for each treatment
group are presented as bar graphs at two voltages, +10 and
+60 mV. *, p < 0.05 when compared with the GST
control.
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Fig. 2.
Binding of Syn-1A to Kv2.1 C- and N-terminal
fragments. Binding assays were performed as described under
"Materials and Methods." Top panel, using a specific
anti-Syn-1A antibody, Syn-1A bound strongly with Kv2.1 C1 and C2. Kv2.1
N terminus bound only weakly with Syn-1A while GST did not bind Syn-1A
at all. Bottom panel, Ponseau S staining of the blot to
demonstrate the amount of GST protein loaded.
-cell Kv2.1 channels, C2 was more effective than C1 in blocking the
effects of the dialyzed GST-Syn-1A. However, Kv2.1 N terminus was
completely ineffective in preventing such reduction. These data
indicate that Syn-1A inhibited Kv2.1 currents by a direct interaction
with the cytoplasmic C terminus.
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Fig. 3.
Reduction of HEK293 cell Kv2.1 current by
dialysis of Syn-1A fusion protein was abolished by Kv2.1 C-terminal
fragments but not affected by Kv2.1 N terminus. The effects of
dialysis of Syn-1A (n = 15), GST (n = 12), Syn-1A + C1 (n = 9), Syn-1A + C2
(n = 4) or Syn-1A + N terminus (n = 10)
(all at 10 nM) on Kv2.1 current magnitude were tested by
giving a +70 mV pulse (250 ms) from a holding potential of 70 mV
every 40 s after membrane break-in. Currents are normalized to the
initial current magnitude immediately after membrane rupture. *,
p < 0.05 when compared with GST alone.
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Fig. 4.
Syntaxin 1A reduces Kv2.1 current density by
inhibiting the channel surfacing to the plasma membrane.
A, Syn-1A reduced Kv2.1 current density. Outward
K+ currents triggered by a +70 mV pulse (250 ms) from a
holding potential of 70 mV in HEK293 cells expressing Kv2.1 alone
(n = 13), Kv2.1 with Syn-1A (n = 22) or
Kv2.1 with Syn-2 (n = 7) were normalized by cell
capacitance to yield current density. *, p < 0.05 when
compared with Kv2.1 alone. B, confocal microscopy of Syn-1A
inhibition of Kv2.1 surface expression. HEK293 cells were transfected
with Kv2.1 in the absence or presence of Syn-1A or -2. Expression of
Kv2.1 alone was visualized as a bright and clear FITC fluorescence at
the cell periphery (left panel). Kv2.1 fluorescence was
faint and diffuse with Syn-1A co-expression. Inset shows
expression of Syn-1A, as visualized by Texas Red fluorescence
(middle panel). Kv2.1 fluorescence was bright and clear at
the cell periphery with Syn-2 co-expression. Inset shows
expression of Syn-2, as visualized by Texas Red fluorescence
(right panel). C, plasma membrane Kv2.1 protein
expression. Transfected HEK293 cells were biotinylated and solubilized
as described under "Materials and Methods." Biotinylated proteins
(plasma membrane fraction) were isolated using the streptavidin resin.
The proteins eluted from the resin (upper panel) and whole
cell lysates obtained prior to the streptavidin precipitation
(lower panel) were then separated by PAGE, and the Kv2.1
protein identified by a specific antibody by Western blotting. The
whole cell lysate panel is a gross assessment of the effects on Kv2.1
synthesis.
of 5.6 ± 0.6 ms (Fig.
5A). The overexpressed Syn-1A
significantly (p < 0.05) slowed down the activation
rate (
= 8.5 ± 0.7 ms) while Syn-2 had no effect. Kv2.1
exhibited a very slow inactivation rate (Fig. 5B), which was
neither affected by Syn-1A nor Syn-2 co-expression.
View larger version (19K):
[in a new window]
Fig. 5.
Syn-1A slowed Kv2.1 current activation but
did not affect current inactivation. A, outward
K+ currents were triggered by a +70 mV pulse (250 ms) from
a holding potential of 70 mV in HEK293 cells expressing Kv2.1 with
(n = 14) or without Syn-1A (n = 21).
Currents shown are normalized to its peak magnitude and overlapped for
comparison. Activation time constants were obtained by an exponential
fit to the rising phase of the current and were summarized in the graph
shown. Syn-1A, but not Syn-2 (n = 7), significantly
slowed down activation. *, p < 0.05 when compared with
Kv2.1 alone. B, to show inactivation, outward K+
currents were triggered by a prolonged +70 mV pulse (10 s) from a
holding potential of
70 mV in HEK293 cells expressing Kv2.1 with
(n = 5) or without Syn-1A (n = 5).
Currents shown are normalized to its peak magnitude and overlapped for
comparison. Inactivation time constants were obtained by an exponential
fit to the decaying phase of the current, and are summarized in the
graph shown. Syn-1A and Syn-2 (n = 7) did not affect
the rate of inactivation.
50 to +70 mV in 10 mV increments
were followed by a
40 mV step to trigger tail currents. Normalized
peak tail currents are then plotted against the various voltage steps
and fit by the Boltzmann equation (Fig.
6A). Syn-1A did not
significantly alter the voltage dependence of activation of Kv2.1.
View larger version (14K):
[in a new window]
Fig. 6.
Syn-1A did not affect voltage dependence of
Kv2.1 current activation but increased the voltage sensitivity of
steady-state inactivation in HEK293 cells. A, instantaneous
activation curves. Voltage steps from 50 to +70 mV (250 ms) delivered
in 10 mV increments from a holding potential of
70 mV were followed
by a
40 mV step to trigger tail currents. Normalized peak tail
currents are then plotted against the various voltage steps. The curves
are best fit by the Boltzmann equation. There is no significant
difference between Kv2.1 with (n = 8) and without
Syn-1A (n = 10). B, steady-state
inactivation experiments. A dual-pulse protocol was used in which a
test pulse step of +70 mV (largest conductance, see above) was preceded
by a long pre-pulse (12 s) of different potentials. The test pulse
currents are normalized to the largest test pulse current and plotted
against the pre-pulse voltages. The curves are best fit by the
Boltzmann equation. There is no significant difference in
V1/2 between Kv2.1 with (n = 5) and
without Syn-1A (n = 7), but Syn-1A significantly
decreased the slope factor (see "Results").
29.9 ± 2.5 mV. The left shift of the inactivation curve
(V1/2 value of
34.7 ± 1.3 mV) caused by
Syn-1A was slight and statistically insignificant. However, Syn-1A
significantly decreased the slope factor from 6.2 ± 0.63 to
4.4 ± 0.25 (p < 0.05), indicating that Syn-1A
increased the sensitivity of voltage-dependent inactivation within the physiological range (
40 to
10 mV). Syn-2 did not significantly alter V1/2 values and slope factor of
inactivation curves (
28.7 ± 2.2 mV and 6.3 ± 0.22, respectively, n = 7).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell, wherein a more optimal insulin
secretion would be able to achieve euglycemia in the treatment of
diabetes. Toward the latter, we have recently shown that SNAP-25 binds
the N terminus of rat islet
-cell Kv2.1 channels (17). In this work,
we have found that Syn-1A also regulates the rat islet
-cell Kv2.1 channel.
-cell
Kv2.1 channels by binding to a novel domain, the cytoplasmic C
terminus. First, dialysis of GST-Syn-1A inhibited the outward currents
in rat islet
-cell, which could be blocked by the cytoplasmic C
terminus domain proteins. Second, dialysis of Syn-1A-GST fusion protein
into Kv2.1-transfected HEK293 cells caused a similar reduction in
current magnitude, which could also be blocked by the Kv2.1 C terminus
domain proteins. Third, our GST-Syn-1A was able to specifically pull
down the recombinant Kv2.1 cytoplasmic C terminus domain proteins, C1
(412-633) and C2 (634-853), but not GST, and to a much lesser extent,
the cytoplasmic N terminus. Peculiarly, despite the slight binding to
the Kv2.1 N terminus, dialysis of this peptide fragment had no effect
on the inhibitory effect of Syn-1A on Kv2.1 channel activity, in
contrast to Kv1.1 channel activity (19). Whereas Syn-1A seems to bind
C1 better than C2, C2 was more effective in blocking Syn-1A inhibition
of Kv2.1 activity. Our previous reports (16, 17, 19), together with the
present study, therefore demonstrate the heterogeneity in the binding of these SNARE proteins with distinct domains (C and N terminus) within
the different Kv channel families. This is in contrast to the
interactions of these SNARE proteins to a common cytoplasmic domain
linking repeats II and III (II-III linker, or the so-called synprint
site) with the different families of Ca2+ channels (5, 6).
We therefore further explored and compared these distinct features of
Syn-1A interactions with Kv1.1 and Kv2.1 channels by examining the
activation and inactivation kinetics and voltage dependence of
activation and steady-state inactivation.
subunit resulted in a fast
inactivation component, and we reported before that Syn-1A could
increase the extent of such fast inactivation. In this work, we did not
investigate such an effect of Syn-1A, as it has been reported that
Kv2.1 does not interact with Kv
subunits (23).
-cells are suppressed
by overexpression of Syn-1A, not Syn-2 (7). We here show that Kv2.1
channels also interact specifically with Syn-1A, but not Syn-2. Syn-1A inhibition of Kv2.1 currents in
-cells implicate the potential physiological significance of such Syn-1A-Kv2.1 interaction in regulating insulin secretion. The interactions between Syn-1A with
distinct domains within the different Kv channel families further
suggest distinct interacting sites may provide opportunities for Kv
channel-specific drug design (15). Kv2.1 is also present in vascular
smooth muscle cells and cardiac myocytes (25, 26). Interestingly, we
have shown by confocal microscopy that Syn-1A and SNAP-25 are also
present in cardiac myocytes and could modify the outward
currents.2 Since myocytes are
not actively secreting cells, this suggests the importance of SNARE
protein regulation of Kv channels independent of exocytosis. In further
support, we had also reported that SNAP-25 modulated Kv and
Ca2+-dependent outward K+ currents
in feline esophageal smooth muscle cells (27).
-cell depolarization, but
may be modulated or attenuated by SNAP-25 (and perhaps synaptobrevin) during core complex formation. More experiments have to be done to
elucidate the dynamics and role of the SNARE protein-Kv channel complex
during exocytosis. Our very recent report (19) that G-protein
subunits enhanced Syn-1A binding to Kv1.1/Kv
and augmented the
inactivating effect of Syn-1A on Kv1.1/Kv
current adds complexity to
the regulation of the SNARE protein-Kv1.1 channel complex. Hence, the
possibility that additional factors may modulate the functions of the
SNARE protein-Kv2.1 channel complex awaits exploration.
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FOOTNOTES |
---|
* This work was supported by Grant DK55160 from the National Institutes of Health, the Juvenile Diabetes Research Foundation and the Canadian Diabetes Association (to H. Y. G.) and by Grant MOP 39498 from the Canadian Institutes of Health Research, J. H. Cummings Foundation, and J. P. Bickell Foundation (to R. G. T.), and the Heart and Stroke Foundation of Ontario (NA 5012, to R. G. T. and H. Y. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Fellowship Awards from the Heart and Stroke Foundation of Canada and the Dept. of Medicine (University of Toronto).
Supported by a New Investigator Award from the Heart and
Stroke Foundation of Canada. To whom correspondence may be addressed: Medical Sciences Bldg., Rm. 7308, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-8899; Fax:
416-978-8765; E-mail: r.tsushima@utoronto.ca.
** To whom correspondence may be addressed: Medical Sciences Bldg., Rm. 7226, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1526; Fax: 416-978-8765; E-mail: herbert.gaisano@utoronto.ca.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M213088200
2 H. Y. Gaisano and R. G. Tsushima, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: VDCC, voltage-dependent Ca2+ channels; Syn-1A, syntaxin 1A; SNAP-25, synaptosome-associated protein of 25 kDa; Kv, voltage-dependent K+ channels; GST, glutathione S-transferase; GFP, green fluorescent protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; FITC, fluorescein isothiocyanate; HEK, human embryonic kidney.
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