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Am J Physiol Gastrointest Liver Physiol 292: G1233-G1242, 2007. First published January 18, 2007; doi:10.1152/ajpgi.00473.2006
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NEUROREGULATION AND MOTILITY

Distinct modulation of Kv1.2 channel gating by wild type, but not open form, of syntaxin-1A

Leila Neshatian,1,2 Yuk M. Leung,1 Youhou Kang,1 Xiaodong Gao,1 Huanli Xie,1 Robert G. Tsushima,1 Herbert Y. Gaisano,1,* and Nicholas E. Diamant1,2,*

1Departments of Medicine and Physiology, and 2Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Canada

Submitted 12 October 2006 ; accepted in final form 16 January 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier K+ channels, Kv1.1 and Kv2.1, in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/E166A) inhibits Kv2.1 channels more optimally than wild-type Syn-1A. In this report we examined whether Syn-1A in its wild-type and open conformations would exhibit similar differential actions on the gating of Kv1.2, a major delayed rectifier K+ channel in nonsecretory smooth muscle cells and some neuronal tissues. In coexpression and acute dialysis studies, wild-type Syn-1A inhibited Kv1.2 current magnitude. Of interest, wild-type Syn-1A caused a right shift in the activation curves of Kv1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on Kv2.1 (steady-state availability reduction without changes in voltage dependence of activation). Also, although both wild-type and open-form Syn-1A bound equally well to Kv1.2 in an expression system, open-form Syn-1A failed to reduce Kv1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open-form Syn-1A on Kv2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in nonsecretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different Kv channels by Syn-1A and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and nonsecretory (smooth muscle) cell types.

ion channels; electrophysiology; SNARE protein

IN NEUROTRANSMITTER RELEASE and endocrine secretion, Ca2+-triggered vesicle fusion is preceded by a priming process whereby a vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin assembles with two plasma membrane target-SNARE proteins SNAP-25 and syntaxin 1A (Syn-1A) to form a complex (6, 11, 22, 31, 32). This complex pulls the vesicle and the plasma membrane into close proximity. For this complex to form, the conformation of syntaxin changes from a closed state to an open state, and Munc18-1 and Munc13 appear to be instrumental in initiating the priming process by promoting this conformational change (5, 9, 32, 36). When Syn-1A is in the closed state, the NH2-terminal HABC domain folds back onto the COOH-terminal H3 domain (SNARE motif), preventing the latter from interacting with SNAP-25 and synaptobrevin; opening up of Syn-1A therefore allows SNARE complex formation (Fig. 1). After exocytosis, the SNARE complex disassembles and Syn-1A resumes its closed form in complex with Munc18-1 (5, 9, 32, 36).


Figure 1
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  		Fig. 1. Models of the open and closed conformations of syntaxin-1A (Syn-1A). A: model for the closed conformation of syntaxin-1A. The three helices of the NH2-terminal domain (N) or the HABC domain and the helix predicted for H3 domain are shaded in dark and light gray, respectively. B: on the right, the open conformation of Syn-1A is shown. The transmembrane domain of Syn-1A is shown preceding the COOH terminus (C) of the protein [adapted and modified from Dulubova et al. (9)].

 
SNARE proteins also play an important role in the secretory process through their regulation and modulation of ion channels. For example, the opening of voltage-dependent Ca2+ channels (VDCC) provides a high local Ca2+ concentration that is sensed by synaptotagmin, which by still unclear mechanisms, interacts with the SNARE complex, leading to membrane fusion (6, 22) and exocytosis (2, 7). SNARE proteins have been found to directly modulate VDCC gating (2, 7, 17, 18). VDCCs open upon membrane depolarization [caused by voltage-gated Na+ channel opening or ATP-sensitive K+ (KATP) channel closure] and close upon repolarization as a result of voltage-gated K+ (Kv) channel opening (12, 29, 37). Therefore regulation of K+ channels is also important in the secretory process.

SNARE proteins also interact with Kv channels to alter their activity. For example, Kv1.1 and Kv2.1 are the dominant delayed rectifying Kv channels in secretory cells, particularly neurons (10, 28) and neuroendocrine cells (24). Using coimmunoprecipitation and electrophysiological experiments, we have shown that SNARE proteins interact with Kv1.1 and Kv2.1 in HEK293 cell and Xenopus oocyte expression systems (10, 16, 20, 25–27). We also showed that wild-type Syn-1A binds Kv2.1 to inhibit Kv2.1 gating (slowing of activation and decreasing steady-state availability) (20). A constitutively open form of Syn-1A generated by the induction of two mutations at the linker region (L165A, E166A) (9) was found to be more potent than the wild-type (largely closed-form) Syn-1A in inhibiting Kv2.1 gating (21). This finding suggests that the Syn-1A conformational change to its open form through its interaction with Munc13-1 and Munc18-1 could induce a stronger inhibition of Kv2.1 during exocytosis (21). This effect would serve to limit K+ efflux and delay repolarization, thus optimizing Ca2+ influx via the VDCCs and exocytosis. These reports led us to postulate that SNARE modulation of both VDCC and Kv channels acts to orchestrate cell excitability to fine tune secretion.

Intriguingly, SNARE proteins are present in the plasma membrane of not only secretory cells but also in some nonsecretory cells. For example, Syn-1A is found in cardiac myocytes and gastrointestinal smooth muscle cells (14, 19), suggesting that Syn-1A must have additional functions in addition to promotion of secretion. Using immunofluorescence confocal microscopy of circular muscle tissue sections and single freshly isolated muscle cells, we have demonstrated the presence of SNARE proteins on the plasma membrane of esophageal smooth muscle (ESM) in the feline body circular and longitudinal layers and lower esophageal sphincter (14). Furthermore, we showed that SNAP-25 has a dose-dependent modulatory effect on the outward potassium currents by inhibiting both Ca2+-activated channel currents (KCa) and delayed rectifier K+ channel currents in ESM (15). Notably, an increase in the outward currents after the cleavage of endogenous SNAP-25 by botulinum neurotoxin A indicates that SNAP-25 inhibition of ESM K+ channels is physiological (15).

Kv1.2 is an important delayed rectifier K+ channel in a number of nonsecretory cells, including vascular (1) and ESM cells (35) and cardiac myocytes (4) and is also present in certain neuronal tissues as well (34). Kv1.2 displays gating properties distinct from those of Kv2.1, including a hyperpolarized shift in V1/2 (voltage for half-activation) and more rapid activation kinetics (33). The objectives of the present work were directed to assessing the interaction of Syn-1A with Kv1.2, an important Kv channel in nonsecretory cells, by examining 1) whether Syn-1A inhibits nonsecretory Kv1.2; 2) whether a conformational change of Syn-1A is required to more effectively modulate Kv1.2 gating; and 3) whether Munc 13 and Munc18-1, normally important for the conformational change to occur, are present in the nonsecretory cell, ESM.


   EXPERIMENTAL METHODS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Constructs and recombinant GST-fusion proteins. The plasmid pGEX-4T-1-Syn-1A was a gift from W. Trimble (The Hospital for Sick Children, Toronto, Ontario, Canada), pCMV-Syn-1A (wild type) from R. Scheller (Genentech, South San Francisco, CA), pcDNA3-Kv1.2 from D. McKinnon (State University of New York at Stony Brook, NY), and pCMV-Syn-1A-L165A/E166A and pGEX-4T-1-Syn-1AL165A/E166A from S. Sugita (Toronto Western Hospital Research Institute, Toronto, Ontario, Canada). Constructs pGEX-4T-1-Syn-1A-HABC (corresponding to amino acids 1–160) and pGEX-4T-1-Syn-1A-H3 (amino acids 191–256) were generated as described previously (8). All constructs were verified by DNA sequencing. Glutathione S-transferase (GST)-fusion protein expression and purification were performed according to the manufacturer's (Amersham Biosciences, Baie d'Urfé, Québec) instructions. Before elution of the GST-fusion protein from glutathione-agarose beads, Syn-1A protein was obtained by cleavage of GST-Syn-1A with thrombin (Sigma, St. Louis, MO). The purity of each eluted or thrombin-cleaved recombinant GST protein was confirmed by PAGE as a strong single band, which was identified by Coomassie blue staining as the molecular weight of the desired protein.

Tissue harvesting and preparations. Smooth muscle cells were harvested from circular smooth muscle of feline esophagus following a previously described protocol with minor modification (30). Briefly, fasted adult cats of either sex were anesthetized with ketamine hydrochloride (Bimeda-MTC, Cambridge, Ontario, Canada) (0.15 ml/kg im). After 5–10 min when adequately anesthetized, the cats were killed by intravenous injection of an overdose of pentobarbital sodium (Bimeda-MTC, Cambridge, Ontario, Canada) (100 mg/kg) following a protocol approved by the University Health Network Animal Care Committee. The lower part of the esophagus including the gastroesophageal junction was exposed by a midline incision from upper abdomen extended to the chest and quickly excised and placed in cold (4°C) Krebs solution oxygenated with 5% CO2 and 95% O2 and containing the following composition (in mM): 115.0 NaCl, 4.6 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 22.0 NaHCO3, 2.5 CaCl2, and 11.0 glucose. All the connective tissue and surrounding fascia were removed. The esophagus was stretched to its in situ length and then opened along the greater gastric curvature. The circular fibers of the smooth muscle, visible after the mucosa was stripped off by sharp dissection, were dissected out and stored at –80°C.

Cell culture and transfection. HEK293 and TSA cells were grown at 37°C in 5% CO2 in DMEM supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin (100 units/ml, 100 µg/ml) (Invitrogen). The cells were transiently transfected with pEGFP (enhanced green fluorescent protein) (Clontech, Palo Alto, CA) and pcDNA3-Kv1.2 with or without pCMV-Syn-1A (wild type) or pCMV-Syn-1A-L165A/E166/A (open-form Syn-1A) by using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. At 24 h after transfection, the HEK293 cells were trypsinized and placed in 35-mm dishes for voltage-clamp experiments, the experiments were performed 24 h later. Since HEK293 cells are known to pick multiple plasmids, the coexpressed GFP served as an in vivo cell marker to identify the cells expressing the SNARE and/or Kv1.2 proteins.

The efficacy of the transfection based on the number of GFP expressing cells was ~40%. More than 80% of the GFP-positive cells had an outward current greater than 3 nA in all Kv1.2-expressing cells with or without SNARE expression. This is almost 10 times bigger than the endogenous HEK293 outward K+ currents and, therefore, is considered to be attributed to exogenous Kv1.2.

In vitro binding assay. Two days after transfection, the TSA cells were washed with ice-cold saline buffered solution (PBS, pH 7.4) and then harvested in binding buffer [25 mM HEPES (pH 7.4), 100 mM KCl, 2 mM EDTA, 2% Triton X-100, 20 µM NaF, and protease inhibitors]. The cells were homogenized by sonication on ice using a Vibra Cell sonicator (Sonics Materials, Danbury, CT). The samples were then subjected to centrifugation at 35,000 g at 4°C for 30 min, and the resulting supernatants containing the solubilized membrane proteins were collected.

To prepare smooth muscle solubilized plasma membrane protein extracts, muscle tissues, isolated as described above, were minced into small pieces and homogenized with three volumes of homogenization buffer per gram frozen tissue. Homogenization buffer consisted of 25 mM HEPES (pH 7.5), 100 mM KCl, 1.5% Triton X-100, 5 mM EDTA, 4 mM EGTA, and 5 mM NaN3, supplemented with a cocktail of protease inhibitors (Sigma). Homogenization was performed in three steps, first homogenized on ice using an electrohomogenizer (Ultra-Turrax T25 Basic; IKA Labor Technical, Staufen, Germany) at 16,000 rpm for 1 min x 5, second by hand in a Pyrex 7727 glass homogenizer on ice, and then with sonication using a Vibra Cell sonicator (Sonics Materials, Danbury, CT) at output control 40, 30 s/ml x 3 on ice. For a better yield of solubilized membrane protein extraction, samples were kept in homogenization buffer for another 30 min on ice and then centrifuged at 35,000 g at 4°C for 30 min. Supernatants containing the solubilized membrane proteins were collected, and remaining pellets were subjected to the membrane protein extraction protocol one more time. All supernatants were pooled and stored at –20°C.

For binding assay, the smooth muscle or the TSA cell extracts (0.3 ml, 1.2–1.8 µg protein/µl) were incubated with GST (as a negative control), GST-Syn-1A-wild type, GST-Syn-1A-open form, GST-Syn-1A-H3 or GST-Syn-1A-HABC (immobilized on glutathione-agarose beads from Sigma, 650 pmol protein each) at 4°C with constant agitation for 2 h. The beads were then spun down and washed three times with binding buffer without the protease inhibitors.

Western immunoblotting. After being washed three times, the precipitated proteins immobilized on glutathione-agarose beads were boiled for 5 min in SDS sample buffer. Moreover, in a separate set of experiment to determine the Munc proteins' presence in smooth muscle, 10-µg proteins of smooth muscle solubilized plasma membrane protein extracts were boiled for 5 min in SDS sample buffer. The samples were then separated on 10% SDS-PAGE and transferred to nitrocellulose membranes (Sigma). Nonspecific binding sites were blocked by incubating membranes in 5% nonfat dry milk in PBS containing 0.5% Tween 20 overnight at 4°C before incubation with primary antibody. The next day, the membranes were then incubated for 1.5 h at room temperature with primary antibodies, including mouse anti-Syn-1A antibody (1:2,000, Sigma), rabbit anti-Kv1.2 COOH-terminal antibody (1:2,000, Alomone Labs, Jerusalem, Israel), mouse anti-Munc 18-a (1:1,000, Transduction Laboratories, Lexington, KY), rabbit anti-Munc 18-b and anti-Munc 18-c (both 1:1,000, gifts from V. Olkkonen, Helsinki, Finland) and mouse anti-Munc 13-1 antibody (1:1,000, Transduction Laboratories), followed by three washes with PBS containing 0.5% Tween 20. The secondary antibody, a peroxidase-conjugated goat affinity pure anti-mouse or anti-rabbit antibody (Jackson Immunoresearch Laboratories, West Grove, PA), was applied using a dilution of 1:40,000 in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20 for 1 h. Detection was achieved with an enhanced chemiluminescence agent (BioLynx, Brockville, Ontario, Canada) and by exposing the membranes to autoradiographic film. Molecular mass of the blotted proteins was estimated by comparison of sample bands with a protein ladder (Fermentas, Burlington, Ontario, Canada). Each experiment was carried out at least three times, and results representative of the overall trend are displayed in the figures.

Electrophysiology. Electrophysiological experiments were performed as previously reported (22). HEK293 cells transiently expressing pcDNA3-Kv1.2 channel with or without pcDNA3-WT Syn-1A or pcDNA3-Syn-1A-L165A/E166/A were voltage clamped in the whole-cell configuration. Thin-walled borosilicate glass tubes (OD 1.5 mm, ID 1.10 mm, Sutter Instrument, Novato, CA) were pulled with a micropipette puller (SP-97, Sutter Instrument) and then heat polished by a microforge (World Precision Instruments, Sarasota, FL). The typical pipette resistance filled with intracellular solution, containing (in mM) 140 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, and 5 MgATP (pH 7.25 adjusted with KOH) was 2–4 M{Omega}.

Indicated recombinant proteins were added to the pipette solution for dialysis into the cells. The bath solution contained (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES (pH 7.3 adjusted with NaOH). The currents were recorded using an EPC-10 amplifier with Pulse 8.60 acquisition software and analyzed by Pulsefit 8.60 software (HEKA Electronik, Lambrecht, Germany). Data were filtered at 2 KHz and sampled at 10 KHz. All experiments were performed at room temperature (~22°C). Data for voltage dependence of activation and steady-state inactivation were fit by the Boltzmann equation: I/Imax = 1/{1+exp[(V1/2 – V)/k]} (for fitting voltage dependence of activation), or I/Imax = 1/{1+exp[(V – V1/2)/k]} (for fitting steady-state inactivation), where I is current, Imax is current peak magnitude, and V is voltage. 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 is the slope factor.

Cell-surface biotinylation. Two days after transfection, HEK293 cells were washed and harvested in PBS. After further washing of cells with borate buffer (154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl2 and 10 mM boric acid, pH 9.0), they were incubated in 5 ml of sulfo-NHS-SS-biotin (0.5 mg/ml; Pierce Biotechnology, Rockford, IL) in borate buffer at 4°C for 30 min. After being washed three times with ice-cold quenching buffer (192 mM glycine and 25 mM Tris, pH 8.3), cells were solubilized on ice in 500 ml of immunoprecipitation buffer (1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 10 mM Tris·HCl, pH 7.5) containing a cocktail of protease inhibitors (Roche Diagnostics, Mannheim, Germany). The cell lysate was centrifuged for 20 min at 16,000 g, and the supernatant was retained. Immobilized streptavidin resin (50 ml; Pierce; 50% slurry in PBS 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 immunoprecipitation buffer. The protein was eluted from the resin by the addition of SDS/PAGE sample buffer containing 10% (wt/vol) 2-mercaptoethanol and incubation at 65°C for 5 min. The protein concentration of the samples was first determined by the Lowry method using bovine serum albumin as a standard (23). The samples were analyzed for Kv1.2 expression by Western blotting using anti-Kv1.2 (1:2,000, Alomone Labs). A commercial software (Scion Image Beta 4.02; Scion, Frederick, MA) was used to determine integrated density of the bands.

Statistical analysis. Data are presented as means ± SE. ANOVA was used to compare data among three or more groups followed by Tukey's test using SigmaStat Software. A value of P < 0.05 was considered to have significant difference.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Wild type, but not open form, of Syn-1A reduced Kv1.2 current density. We first examined whether Syn-1A would inhibit Kv1.2 and whether such inhibition depends on the conformational status of Syn-1A. Kv1.2 currents were markedly attenuated by wild-type Syn-1A, but surprisingly not by open-form Syn-1A (Fig. 2A). The results are summarized in the current-voltage relationship shown in Fig. 2B. Seventy-four percent of the cells expressing wild-type Syn-1A and Kv1.2 had lower current density than the control mean. Control current density (measured at +70 mV) was 384.07 ± 69 pA/pF and was significantly reduced with wild-type Syn-1A coexpression to 239.12 ± 25 pA/pF. In contrast, open-form Syn-1A coexpression did not cause a significant reduction of current density (359.53 ± 68 pA/pF). When we compared the raw data using a dot presentation (Fig. 2C), we saw in both wild-type and open-form Syn-1A groups cells with low current density, but in the wild-type Syn-1A expressing cells the entire group of cells was clustered toward the low current density (mean of 239.12 ± 25 pA/pF), whereas the open-form group was spread out over a range of 18–1,096 pA/pF, which is quite similar to the control group (101–1,225 pA/pF). This clustering toward low currents suggests that wild-type Syn-1A may be more effective in inhibiting cells with higher currents. The latter analysis indeed reaffirms that only the wild type but not the open form of Syn-1A could significantly inhibit Kv1.2.


Figure 2
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  		Fig. 2. Effects of Syn-1A on Kv1.2 current density. A: representative traces showing outward K+ currents triggered by a series of depolarizing pulses. HEK293 cells expressing Kv1.2 alone, Kv1.2 with wild-type (WT) Syn-1A, or Kv1.2 with the open form of Syn-1A were held at –70 mV and given depolarizing pulses (250 ms) from –70 to 70 mV at 10-mV increments. B: quantitative summary of A. Currents were normalized by cell capacitance to yield current densities. Wild-type Syn-1A (n = 31) decreased the Kv1.2 outward currents significantly by 36% (P < 0.05). Open-form Syn-1A (n = 23) did not reduce the Kv1.2 current density significantly. Results are means ± SE. *Significantly (P < 0.05) lower than control. C: dot presentation showing the distribution of the current density measured at +70 mV in cells coexpressing WT or open Syn-1A.

 
The reduction of Kv1.2 current density with wild-type Syn-1A coexpression could be due to inhibition of Kv1.2 channel trafficking to the plasma membrane, as we had observed with Kv2.1 coexpressed with Syn-1A (20, 21). We therefore investigated whether Syn-1A would affect Kv1.2 plasma membrane surface expression by the technique of cell-surface biotinylation. Figure 3A shows that the level of plasma membrane Kv1.2 protein pulled down by the streptavidin resin was comparable between the control and open-form Syn-1A coexpression (100 and 92%, respectively) but was paradoxically increased by 72% with wild-type Syn-1A coexpression. This suggests that the reduction of Kv1.2 current density by wild-type Syn-1A coexpression must be a direct effect of Syn-1A on the channel and not due to inhibition of channel trafficking. It is noteworthy that the Kv1.2 protein content in the total lysates determined just before the treatment with streptavidin resin was not affected by coexpression of either Syn-1A, suggesting Syn-1A did not affect total synthesis of Kv1.2 (Fig. 3B).


Figure 3
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  		Fig. 3. Effect of Syn-1A on Kv1.2 channel trafficking to the plasma membrane. Transfected HEK293 cells were biotinylated and solubilized, whole cell lysates were obtained, and plasma-membrane fractions were isolated as described in EXPERIMENTAL METHODS. Kv1.2 was identified by Western blotting in plasma membrane fractions (A) and whole cell lysates (B) of cells transfected with or without WT or open-form Syn-1A. The whole cell lysates are assessments of total cellular synthesis of Kv1.2. WT Syn-1A, but not the open form, increased Kv1.2 channel trafficking to the plasma membrane. Results are means of 2 separate experiments.

 
The greater effect of the wild-type Syn-1A than open Syn-1A in decreasing Kv1.2 current density could potentially be due to a higher expression of wild-type Syn-1A at the plasma membrane. However, this is unlikely given that we have already shown in our previous report that membrane expression and total synthesis of wild-type and open-form Syn-1A were similar (21).

Acute inhibition of Kv1.2 current by wild-type Syn-1A. To confirm the direct inhibition of the Kv1.2 channel by Syn-1A, we dialyzed GST-fusion proteins or GST-Syn-1A fusion proteins (1 µM) into the Kv1.2-expressing HEK293 cells via patch pipette. Whereas dialysis of the control GST protein did not significantly affect the current magnitude, dialysis of GST-Syn-1A-WT into the cell for 7 min caused an 18 ± 3% decrease in the magnitude of current (P < 0.05; Fig. 4). The decrease in current magnitude by dialysis of the open-form Syn-1A was small (8 ± 2%) and insignificant compared with GST.


Figure 4
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  		Fig. 4. Direct inhibition of Kv1.2 currents by glutathione S-transferase (GST)-Syn-1A fusion proteins of different conformations and the H3 domain. The effects of dialysis of GST, GST-WT Syn-1A, open-form GST-Syn-1A or GST-Syn-1A-H3 domain (all at 1 µM) on Kv1.2 current magnitude were tested by giving +70 mV pulses (250 ms) from a holding potential of –70 mV after membrane break-in. Currents were measured 7 min after break-in and normalized to the initial current magnitude immediately after membrane rupture. Results are means ± SE from 6–14 cells. *Significantly lower (P < 0.05) than the GST control.

 
The H3 domain has been shown to be the biologically active domain of Syn-1A, particularly when Syn-1A assumes the open conformation during exocytosis (9, 36). Furthermore, the H3 domain is also the putative Syn-1A domain that inhibits Kv2.1 (21). We therefore examined whether the H3 domain would inhibit Kv1.2 currents. However, when dialyzed into Kv1.2-expressing HEK293 cells, the H3 domain was unable to inhibit the currents significantly (Fig. 4). Taken together, these results suggest that inhibition of Kv1.2 currents requires the full-length Syn-1A protein molecule in its closed form.

Both wild-type and open-form Syn-1A bound to Kv1.2 in an expression system. The distinct actions of the Syn-1A conformations may be due to their different abilities to bind the Kv1.2 channel protein. We therefore performed protein-binding assays to examine whether recombinant (GST-proteins) wild-type and open-form Syn-1A are able to bind expressed Kv1.2 (in HEK cells). Both open-form and wild-type GST-Syn-1A were able to bind to Kv1.2 (Fig. 5A). The GST-Syn-1A-H3 domain bound only weakly to Kv1.2 whereas GST-Syn-1A-HABC did not bind at all to Kv1.2 (Fig. 5A). The results indicate that the H3 domain of Syn-1A is the domain that bound Kv1.2, but optimal binding requires the full-length Syn-1A protein. That is, the H3 domain or full-length open-form Syn-1A bound to Kv1.2, but these proteins did not result in Kv1.2 inhibition. Therefore, it appears that significant inhibition of Kv1.2 requires the binding of the full-length wild-type Syn-1A, presumably in its closed form, to Kv1.2.


Figure 5
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  		Fig. 5. Binding of Syn-1A proteins or domains to Kv1.2 in an expression system and in esophageal smooth muscle (ESM). HEK293 cells were transfected with Kv1.2. The cell extracts were incubated with equal concentrations of GST, GST-WT-Syn-1A, GST-open-Syn-1A (Syn-1A-L165A/E166A), GST-Syn-1A-HABC or -H3 (all bound to glutathione Sepharose beads) as described in EXPERIMENTAL METHODS. The Kv1.2 protein pulled down by these Syn-1A domain proteins was separated on SDS-polyacrylamide gel after washing, the samples were probed with anti-Kv1.2 antibody. A: representative results from 3 separate experiments. B: ESM cell extracts were incubated with equal concentrations of GST, GST-Syn-1A-WT, GST-Syn-1A-HABC or -H3 (all bound to glutathione Sepharose beads). The Kv1.2 protein pulled down by these Syn-1A domain proteins was probed and identified with anti-Kv1.2 antibody. The cellular Kv1.2 was found to bind to the Syn-1A-WT protein and to the Syn-1A-H3 but not the HABC domain.

 
Syn-1A bound to Kv1.2 in ESM. Next, we examined whether the endogenous Kv1.2 protein in cat esophageal circular smooth muscle is capable of binding the Syn-1A. Indeed, Fig. 5B shows that wild-type Syn-1A can bind native Kv1.2 proteins from the cat ESM tissue, as demonstrated by the ability of GST-Syn-1A-WT bound to Sepharose beads to pull down cat esophageal circular smooth muscle Kv1.2-immunoreactive proteins. GST alone did not bind Kv1.2 in ESM (Fig. 5B).

The wild-type Syn-1A binding to ESM Kv1.2 prompted us to examine which Syn1A domain(s) might be mediating this physical interaction. We used GST-Syn-1A-H3 and GST-Syn-1A-HABC (all bound to glutathione Sepharose beads) to pull down Kv1.2 from the ESM cell extract (Fig. 5B). The GST-Syn-1A-HABC did not bind to the ESM Kv1.2. In contrast, the GST-Syn-1A-H3 domain bound to Kv1.2, but the binding was less than that of the GST-Syn-1A-WT (Fig. 5B).

Wild type, but not open form, of Syn-1A modulates Kv1.2 channel gating. Wild-type Syn-1A has been shown to modulate Kv1.1 and Kv2.1 channel gating properties (10, 20, 21, 26, 27), and the open-form Syn-1A inhibits Kv2.1 gating more than wild-type Syn-1A (21). Therefore, we explored whether coexpression of wild-type Syn-1A or open-form Syn-1A would exhibit distinct effects on Kv1.2 channel gating properties. Since HEK293 cells express endogenous outward K+ currents as high as 0.3 nA, only cells expressing currents greater than 3 nA were used for gating analysis.

Kv1.2 channel activated with a fairly rapid rate, with an activation time constant ({tau}) of 3.6 ms (Fig. 6A). Overexpression with wild-type Syn-1A, but not open-form Syn-1A, significantly (P < 0.05) slowed down activation ({tau} = 5.3 ms). Next we examined whether Kv1.2 inactivation rate is affected by Syn-1A. Outward K+ currents were triggered by a prolonged +70-mV pulse (10 s) from a holding potential of –70 mV in HEK293 cells expressing Kv1.2 with or without Syn-1A. Inactivation time constants were obtained by an exponential fit to the decaying phase of the current. Neither wild-type nor open-form Syn-1A modified the Kv1.2 inactivation rate (Fig. 6B).


Figure 6
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  		Fig. 6. Effects of Syn-1A on Kv1.2 activation and inactivation kinetics. A: outward K+ currents were triggered by a +70 mV pulse (250 ms) from a holding potential of –70 mV in HEK293 cells expressing Kv1.2 with or without WT and open-form Syn-1A. Each current (I) is normalized to its peak magnitude (Imax). B: activation time constants were obtained by an exponential fit to the rising phase of the currents. WT Syn-1A but not open form significantly increased the activation time constant (P < 0.05). C: outward K+ currents were triggered by a prolonged +70 mV pulse (10 s) from a holding potential of –70 mV in HEK293 cells expressing Kv1.2 with or without Syn-1A. Each current is normalized to its peak magnitude. D: inactivation time constants were obtained by an exponential fit to the decaying phase of the current. Neither WT nor open form of Syn-1A modified the Kv1.2 inactivation time constant.

 
We then examined whether wild-type and/or open-form Syn-1A would affect the voltage dependence of activation of Kv1.2. Activation curves were derived from the protocol in which voltage steps delivered in 10-mV increments from a holding potential of –70 mV were followed by a –40 mV step to trigger tail currents. The peak tail currents are normalized with the maximum tail current and plotted against the various voltage steps. The curves are best fit by the Boltzmann equation (Fig. 7A). Wild-type Syn-1A coexpression caused a right-shift in activation curve, with V1/2 of –4.3 mV in control group shifting to +6.4 mV in the presence of wild-type Syn-1A (P < 0.05). The slope of the curve did not change significantly. By contrast, open-form Syn-1A did not significantly alter the voltage dependence of activation of Kv1.2.


Figure 7
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  		Fig. 7. Effects of Syn-1A on voltage dependence of Kv1.2 activation and steady-state inactivation. A: voltage dependence of activation. Voltage steps 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. WT Syn-1A shifted the half-maximal activation potential from –4.3 mV in control group (n = 20) to +6.4 mV (n = 18). The slope of the curve did not change significantly. Open-form Syn-1A (n = 14) did not have any effect on the voltage dependence of channel activation. B: steady-state inactivation: a dual-pulse protocol was used in which a test pulse step of +70 mV was preceded by a long prepulse (12 s) of different potentials. The test pulse currents are normalized to the largest test pulse current and plotted against the prepulse voltages. The curves are best fit by the Boltzmann equation. Neither WT (n = 6) nor the open form of (n = 12) Syn-1A changed the steady-state inactivation curves compared with the control (n = 11).

 
We next performed steady-state inactivation experiments to determine channel availability 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 prepulse (12 s) of different potentials. The test pulse currents are normalized to the largest test pulse current and plotted against the prepulse voltages. The curves are best fit by the Boltzmann equation (Fig. 7B). Neither wild-type nor the open-form Syn-1A significantly altered V1/2 values and slope factors of the inactivation curves.

Munc13 and Munc18 proteins are not present in ESM. In nonsecretory cells such as smooth muscle cells, the functional role of open-form Syn-1A is unclear. Furthermore, Munc18a and/or Munc-13-1, which are required to convert Syn-1A from closed to open configuration (5, 9, 32, 36), may not be present in smooth muscle cells. To investigate this, we performed Western blot analysis of ESM cell homogenates with specific antibodies against the three isoforms of the Munc18 family and compared their expression with crude rat brain homogenates (Fig. 8A). Incubation of rat brain homogenates with antibodies against mammalian Munc18a, b, and c revealed strong bands. However, in subcellular fractions of smooth muscle cells, there were no corresponding Munc18 protein bands in either the cell cytosol or membrane fractions.


Figure 8
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  		Fig. 8. Expression of Munc proteins in ESM. A: Western blot analysis of Munc18a, b, and c in rat brain lysate (5 µg of protein) and ESM lysate (100 µg of protein). B and C: rat brain or ESM cell extracts were incubated with equal concentrations of GST, GST-Syn-1A-WT, or GST alone, all bound to glutathione Sepharose beads. The Munc proteins pulled down by WT Syn-1A was probed and identified with anti-Munc18a (B) or b (C) antibodies. Shown are representative blots of 2 separate similar experiments. SMC, smooth muscle cell. D: GST and GST-Syn-1A-WT bound to glutathione agarose beads were incubated with ESM cell extracts paired with rat brain incubation with GST-Syn-1A-open or GST-Syn-1A-WT glutathione agarose beads as described in EXPERIMENTAL METHODS. The Munc13 protein pulled down by this Syn-1A protein was then identified by Western blotting. Each panel shown is a representative blot of 2 separate experiments.

 
To confirm that Munc18 proteins are not expressed in the ESM, we also performed pull-down assays with GST fusion proteins, followed by Western blot analysis. By this method even a minute amount of Munc18 proteins from ESM would be identified. We used GST-Syn-1A-WT, bound to glutathione agarose beads, to pull down Munc18 proteins from ESM and rat brain cell extracts (Fig. 8, B and C). GST-Syn-1A-WT pulled down substantial amounts of Munc18a and b from rat brain. However, none of these Munc18 proteins were pulled down from ESM extracts by GST-Syn-1A-WT. GST alone did not bind Munc18 proteins at all.

Munc13-1 binds with high affinity to the NH2-terminal domain of Syn-1A, which would displace the Munc18a, a process that facilitates the transition of Syn-1A to an open conformation (5, 9, 32, 36). Therefore, we further looked for Munc13-1 in the ESM using both wild-type and open-form GST-Syn-1A. GST-Syn-1A-WT and GST-Syn-1A-Open have a great affinity for Munc13 in rat brain. Nevertheless, GST-Syn-1A-WT was unable to pull down any Munc13-1 from ESM cell lysate, substantiating the absence of this protein in the smooth muscle (Fig. 8D).


   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL METHODS
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We have previously reported that Syn-1A binds to the Kv2.1 COOH terminus to directly inhibit channel gating and trafficking to the plasma membrane (20). Recently, we reported that the Syn-1A-Kv2.1 interaction changes with the conformation of Syn-1A (21). A mutant open form of Syn-1A inhibits Kv2.1 more strongly than the closed form, suggesting that open-form Syn-1A limits K+ efflux and thus delays membrane repolarization during exocytosis. This may ultimately lead to optimization of secretion. These findings led us in the present study to investigate first whether Syn-1A inhibited Kv1.2, a major delayed rectifier, in nonsecretory smooth muscles (4, 35) and in some neuronal tissues and second whether a similar distinct inhibition is induced by the different Syn-1A conformations.

Wild-type Syn-1A reduced the magnitude of Kv1.2 current, which was particularly observed in coexpression experiments. Wild-type Syn-1A coexpression did not result in inhibition of surfacing of Kv1.2 but paradoxically enhanced the trafficking of Kv1.2 to the cell surface. This suggests that Syn-1A strongly inhibited Kv1.2 despite the increased population of Kv1.2 channels in the plasma membrane, and the ~40% inhibition in the presence of wild-type Syn-1A was apparently an underestimation of the actual inhibition. It is unknown how wild-type Syn-1A coexpression enhanced Kv1.2 surface expression. Tyrosine phosphorylation of Kv1.2 by GTPases has been implicated in the endocytosis of Kv1.2, which would lead to decreased channel expression (13). It is plausible that Syn-1A coexpression could enhance Kv1.2 surface expression by preventing such phosphorylation.

To confirm that Syn-1A directly inhibited Kv1.2, we dialyzed wild-type Syn-1A fusion proteins (without the transmembrane domain) into Kv1.2-expressing HEK cells and observed a direct inhibition (17%). The inhibition was not as substantial as in the coexpression experiment (≥40%), which utilized the full-length Syn-1A that includes the COOH-terminal transmembrane domain. The Syn-1A transmembrane domain would better "anchor" Syn-1A to the plasma membrane and this domain may be required to stabilize Kv1.2 channel-Syn-1A interactions. The recombinant GST-WT Syn-1A dialyzed into the cytosol may not anchor properly to the plasma membrane or to Kv1.2.

Wild-type Syn-1A modulation of Kv1.2 gating shares some similar but also distinct features from Kv2.1 (20, 21). As with Kv2.1, wild-type Syn-1A slowed Kv1.2 activation without affecting Kv1.2 inactivation rate. The effects of wild-type Syn-1A on voltage dependence of activation and steady-state availability of Kv1.2 are opposite to its effects on Kv2.1 (20, 21). Specifically, wild-type Syn-1A right shifted the activation curves without affecting steady-state availability of Kv1.2. In its interaction with Kv2.1, wild-type Syn-1A decreased steady-state channel availability while having no effect on voltage dependence of activation (20, 21). These results suggest that although Syn-1A inhibits both Kv1.2 and Kv2.1 current magnitude, it does have subtly different effects on gating of different Kv channels.

Perhaps the most interesting observation in this work is that open-form Syn-1A completely lost its ability to modulate Kv1.2. This was the case with either Syn-1A coexpression or acute dialysis studies into Kv1.2-expressing HEK cells. This is in contrast to secretory Kv2.1 in which open-form Syn-1A caused a stronger inhibition than wild-type Syn-1A (21). In fact, open-form Syn-1A also did not affect Kv1.2 trafficking. The failure to directly inhibit Kv1.2 or modulate Kv1.2 gating by open-form Syn-1A was not due to its inability to bind Kv1.2. Indeed, open-form and wild-type Syn-1A bound equally well to Kv1.2 in an expression system. The H3 domain, and not the HABC, is the putative domain that binds Kv1.2, albeit weakly; however, the H3 domain did not inhibit Kv1.2 current. These results suggest that optimal inhibition of Kv1.2 by Syn-1A requires Syn-1A to be in its closed conformation and perhaps conferred by the H3 domain (or subdomains) in the closed conformation.

Syn-1A inhibition of rat brain Kv1.1/Kvbeta1.1 channel is mediated by binding to its cytoplasmic NH2 terminus (26), which increased the extent of fast inactivation of this channel (10). On the basis of the high homology of the NH2 termini of Kv1.1 and Kv1.2 (86%), wild-type Syn-1A likely binds to the NH2 terminus of Kv1.2 to inhibit the channel. The Syn-1A effects on Kv1.1 inactivation was reversed by disrupting the Syn-1A-channel interaction by injecting the synprint peptide that competes efficiently with the binding of Syn-1A to the Kvbeta1.1 subunit of the channel, thereby demonstrating the significance of the endogenous protein–protein interactions (10). In Kv1.1, Syn-1A also affected the current amplitudes in a concentration-dependent biphasic manner: at low concentration it augments current without affecting surface channel expression, whereas at high concentration it decreases current amplitude by reducing surface channel expression (10). However, our results showing that Syn-1A paradoxically enhanced the trafficking of Kv1.2 to the cell surface suggest that Syn-1A actions on these highly homologous isoforms of the Kv1 family may exhibit further differences, which would require future studies to elucidate.

In contrast to Kv1.1, Syn-1A binds to both cytoplasmic NH2 and COOH termini of Kv2.1, channel inhibition is transduced via the Kv2.1 COOH terminus (20). A most intriguing but speculative explanation for the distinct actions of these Syn-1A conformations on Kv1.2 and Kv2.1 might perhaps be due to possibly different effects of the Syn-1A conformations on assembly of these Kv channels in the plasma membrane. This is suggested by the results that wild-type Syn-1A but not open-form Syn-1A increased Kv1.2 surface expression, whereas both Syn-1A conformations reduced Kv2.1 surface expression (21). The higher potency of open-form Syn-1A than closed-form Syn-1A in inhibiting Kv2.1 may serve to slow down membrane repolarization during exocytosis in cells where Kv2.1 predominates, such as pancreatic islet beta-cells (24). This might have the effect of prolonging depolarization to optimize VDCC channel opening and Ca2+ influx, which in turn would optimize insulin release. This appears to be physiologically relevant because insulin release is relatively slow and sustained. In secretory cells (some neurons), the inability of the open form of Syn-1A to inhibit Kv1.2 may imply that during exocytosis Kv1.2 can potentially eventually open in a full-fledged manner to speed up repolarization and thus terminate fusion. Perhaps in Kv1.2-bearing neurons such a mechanism is necessary to ensure the high turnover rate of the exocytotic cycle of neurotransmitter release. These distinct effects of Syn-1A conformations on different Kv demonstrate the plasticity of Syn-1A modulation of Kv channels during the exocytotic cycle.

Both Syn-1A and Kv1.2 are present not only in certain neuronal tissues, but also in a variety of nonsecretory cells such as smooth muscle cells and cardiac myocytes (1, 3, 14, 19, 35). In cardiac myocytes, Syn-1A inhibits KATP channels through interaction with SUR2A nucleotide binding fold (NBF)-1 and NBF-2 (19). Syn-1A cognate partner, SNAP-25, inhibited Kv and KCa currents in ESM (15). Kv1.2 is an important delayed rectifier K+ channel in ESM and is involved in setting the resting membrane potential. In smooth muscle cells, Syn-1A might cause a constitutive inhibition of Kv1.2, through binding to the channel, and potentially serve as modulator of membrane potential and cell excitability. Since open-form Syn-1A loses its inhibitory effects on Kv1.2, it seems that in smooth muscle cells there is no need for dynamic regulation of Kv1.2 by Syn-1A in a conformation-dependent manner.

In secretory cells, the regulatory proteins Munc18a and Munc13-1 facilitate the rearrangement in Syn-1A structure. Munc18a stabilizes a closed Syn-1A conformation by binding to the multiple regions along the length of the Syn-1A protein, whereas Munc13-1 binding to the NH2-terminal domain of Syn-1A displaces the Munc18, serving to facilitate the transition of Syn-1A to an open conformation (5, 9, 36). Our finding in ESM of the absence of Munc18a and Munc13-1 is consistent with the concept that Syn-1A regulation of ion channels in nonsecretory cells may be different than in secretory cells. That is, Syn-1A would not exist in the open form in smooth muscle cells, because these nonsecretory cells do not contain the synaptic proteins (i.e., Munc18a, Munc13-1) required to induce such a conformational change in Syn-1A. Thus, whereas Syn-1A needs to be "activated" to its open conformation in secretory cells, the "active form" of Syn-1A in nonsecretory cells (smooth muscles) is the closed form.

In summary, our data demonstrate that the specific effects of Syn-1A on gating of various Kv channels can vary from one cell type to another. This report demonstrates a Syn-1A regulation of Kv1.2 gating very distinct from that of Kv2.1 previously described, suggesting the plasticity of Syn-1A modulation, one mechanism of modulation designed for secretion and another for excitability, depending on the different cell types.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work is supported by grants from Canadian Institutes for Health Research (MOP-36499 to N. E. Diamant and H. Y. Gaisano; MOP-69083 to H. Y. Gaisano, MOP-69083 to H. Y. Gaisano and R. G. Tsushima) and Canadian Association of Gastroenterology/Abbott Laboratories/CIHR (CIHR DOP 68570 to H. Y. Gaisano). L. Neshatian was supported by the Margaret J. Santalo Fellowship Award (University of Toronto). Y. M. Leung would like to thank Canadian Diabetes Association for a Fellowship Award and China Medical University, Taiwan for providing a start-up fund (CMU95-049).


   ACKNOWLEDGMENTS
 
Present address for Y. M. Leung: Department of Physiology, School of Medicine, China Medical University, Taichung, Taiwan, R.O.C.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Diamant or Y. M. Leung, Rm. 12-419 McLaughlin Pavilion, The Toronto Western Hospital, 399 Bathurst St., Toronto, Ontario, Canada M5T 2S8 (e-mail: ndiamant{at}sympatico.ca)

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.

* H.Y. Gaisano and N. E. Diamant contributed equally as senior authors. Back


   REFERENCES
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 REFERENCES
 

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