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Published online May 11, 2007
Diabetes 56:2124-2134, 2007
DOI: 10.2337/db07-0030
© 2007 by the American Diabetes Association
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The Actions of a Novel Potent Islet ß-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Betty Ng1, Youhou Kang1, Chadwick L. Elias1, Yan He1, Huanli Xie1, John B. Hansen2, Philip Wahl2, and Herbert Y. Gaisano1

1 Departments of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada
2 Novo Nordisk, Malov, Denmark

Address correspondence and reprint requests to Herbert Y. Gaisano, Room 7226 Medical Science Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: herbert.gaisano{at}utoronto.ca

Abbreviations: EC50, half-maximal effective concentration; KATP, ATP-sensitive K+ channel; GFP, green fluorescence protein; GST, glutathione S-transferase; IKATP, ATP-sensitive K+ current; NNC55-0462, 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide; SNARE, soluble N-ethylmaleimide–sensitive factor attachment protein receptor; SUR, sulfonylurea receptor


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Islet ß-cell–specific ATP-sensitive K+ (KATP) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in ß-cells to inhibit KATP channels. As a strategy to elucidate the molecular mechanism of action of these KATP channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A–SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A–SUR1 interactions. Dialysis of GST–syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet ß-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST–syntaxin-1A and its H3 domain inhibited KATP channels previously activated by NNC55-0462. This action on KATP channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST–syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A–SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the KATP channel openers NNC55-0462 and diazoxide on KATP channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of KATP channel openers.

Chronic exposure to hyperglycemia in diabetes can impair insulin secretory function (1) and accelerate apoptosis (2) in islet ß-cells. Expression levels of soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins, the critical machinery for insulin granule exocytosis (3), particularly in first-phase secretion (4), are severely reduced in islets of rodent models and human patients with type 2 diabetes (5–8). Hyperglycemia further reduced SNARE protein levels in Goto-Kakizaki (GK) rats, but levels could be partially restored by normoglycemia (6). Restoration of SNARE protein levels partially normalized insulin secretion in these animal models (6,7). In addition to direct glucotoxic effects, chronic hyperglycemia can lead to ß-cell overstimulation (9). Prolonged exposure to high glucose and other secretagogues induces desensitization of islet ß-cells to subsequent stimuli, with Ca2+ influx–induced toxicity (10) and insulin stores depletion (11) suggested as possible mechanisms. In diabetes, chronic hyperglycemia and insulin resistance increase insulin secretory demand, and failure of ß-cells to sustain this compensatory increase in workload may contribute to disease progression (12).

A novel therapeutic approach, therefore, is to pharmacologically induce "ß-cell rest" by ATP-sensitive K+ (KATP) channel openers to preserve residual secretory function and insulin stores (13). Pancreatic KATP channels are octamers of four sulfonylurea receptor 1 (SUR1) subunits in complex with four pore-forming Kir6.2 subunits, which act to modulate cell membrane potential, linking glucose-mediated changes in the ATP-to-ADP ratio to insulin secretion (14,15). The opening of KATP channels leads to membrane hyperpolarization sufficient to induce ß-cell rest. The nonselective KATP channel opener diazoxide can protect ß-cells of humans and rodents against hyperglycemia-induced desensitization in vitro (16,17), restoring first-phase and pulsatile insulin release (18), and can protect against streptozotocin-induced injury (18,19). Remarkably, diazoxide treatment also preserves residual insulin secretion in patients with type 1 (20,21) and type 2 (22) diabetes. However, clinical use of diazoxide is limited because of its nonselective opening of extrapancreatic KATP channels, causing cardiovascular side effects (23). Recently, a class of islet ß-cell–specific KATP channel openers (thiadiazine dioxides) being developed (24–27) have been shown to be capable of inducing ß-cell rest in rodent (10,28,29) and human islets (30,31), improving insulin stores and secretory responses, and protecting against autoimmune injury in an animal model of type 1 diabetes (32). These ß-cell–specific KATP channel openers interact with cytoplasmic and transmembrane domains of the SUR1 subunit and displace 3H-glibenclamide binding to HEK cells stably expressing human Kir6.2/SUR1 (BA8 cells) (24,27). Their precise molecular mechanism of action, however, remains unknown (13).

We recently reported that the plasma membrane–bound SNARE protein syntaxin-1A directly binds the cytoplasmic nucleotide binding folds of SUR proteins of ß-cells (SUR1) (33,34) and cardiac myocytes (SUR2A) (35) to modulate KATP channels. We further demonstrated that the COOH-terminal H3 domain of syntaxin-1A is the putative domain mediating KATP channel inhibition (33). In the current study, we explored the molecular basis by which a novel islet ß-cell (SUR1)-specific KATP channel opener, 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) (Fig. 1), modulates KATP channel opening. NNC55-0462 is very potent in inhibiting insulin release from rat islets, being >100 times more potent than its parent compound diazoxide (27). Using rat islet ß-cells and BA8 cells, we now examined the possibility that NNC55-0462 actions on pancreatic KATP channels could modulate or be modulated by syntaxin-1A–SUR1 interactions.


Figure 1
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  		FIG. 1. Structure of NNC55-0462 (A) compared with diazoxide (B).

 

   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The constructs pcDNA3–syntaxin-2 and pcDNA3–syntaxin-1A were from Richard Scheller (Genentech, San Francisco, CA), pGEX-4T-1–syntaxin-1A from W. Trimble (Hospital for Sick Children, Toronto, Canada), and pcDNA3–syntaxin-1B from G. Zamponi (University of Calgary, Calgary, Canada). The coding sequences corresponding to syntaxin-1A (amino acid 1–266, without transmembrane domain), syntaxin-1A–Habc domain (amino acid 1–160), and syntaxin-1A–H3 domain (amino acid 191–256) were amplified by PCR and cloned into pGEX-4T-1 expression vector for generation of glutathione S-transferase (GST) fusion proteins. GST fusion protein expression, purification, and thrombin (Sigma) cleavage were performed according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ).

Cell culture and transfection.
HEK-293 cells stably expressing human Kir6.2/SUR1 (BA8 cells) have been previously characterized (24,27), and cell culture and transfection were performed as previously described (33–35). For a detailed description, refer to the supplementary appendix, available online at http://dx.doi.org/10.2337/db07-0030.

Preparation of rat islet ß-cells.
Pancreatic islets were isolated from male Sprague-Dawley rats by collagenase digestion and dispersed into single cells by treatment with 0.05% trypsin in Ca2+- and Mg2+-free PBS as previously described (33,34). Islet cells were plated on glass coverslips in 35-mm tissue culture dishes and cultured overnight in RPMI-1640 medium (Invitrogen), containing 2.8 mmol/l glucose and supplemented with 7.5% FBS, before electrophysiological recordings.

Electrophysiology.
Current recordings from single-islet ß-cells and BA8 cells were performed using standard patch-clamp techniques in the whole-cell and inside-out configurations as previously described (33–35). Refer to the supplementary appendix for a detailed description.

In vitro binding assay and Western blotting analysis.
BA8 cells were washed with 1x PBS (pH 7.4) and harvested in binding buffer (25 mmol/l HEPES, pH 7.4, 100 mmol/l KCl, 1.5% Triton X-100, 2 µmol/l pepstatin A, 1 µg/ml leupeptin, and 10 µg/ml aprotinin). The cells were lysed by sonication, and insoluble materials were removed by centrifugation (55,000g, 4°C, 30 min). The detergent extract (0.3 ml, 1.2 µg/µl protein) of BA8 cells was incubated for 2 h at 4°C with GST (as negative control) or GST–syntaxin-1A (500 pmol protein bound to glutathione agarose beads) in the absence or presence of indicated concentrations of NNC55-0462 or untagged syntaxin-1A and/or ATP. The beads were washed three times with binding buffer, the samples were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane, and the proteins were identified with mouse anti-SUR1 antibody (1:750; a gift from J. Ferrer, University of Barcelona, Barcelona, Spain). For Western blotting analysis of syntaxin-1A, -1B, and -2 expression, whole-cell lysates were prepared from rat brain (as positive control), rat islet, syntaxin-1A–or syntaxin-1B–expressing HEK293 cells (as negative or positive control), and rat pancreatic acinar cells (as positive control) by sonication or with a homogenizer. Samples were separated by 12% SDS-PAGE, and proteins of interest were identified with specific antibodies (syntaxin-1A and -1B from Syntaxinaptic Systems [Goettingen, Germany] and syntaxin-2 from V. Olkkonen [Helsinki, Finland]).

Data analysis.
Data analysis and curve fitting were performed using Origin 6.0 (Microcal Software, Northampton, MA). Data are means ± SE. Differences in means were compared using either one-way ANOVA, followed by Dunnett's post hoc test, or paired/unpaired Student's t test, with P < 0.05 considered as statistically significant. Concentration-response curves were fitted to the equation: y = {(A1-A2)/[1+(X/X0){rho}]} + A2, where y is the KATP current at different concentrations of NNC55-0462, X is the NNC55-0462 concentration applied externally to the cells, X0 is the NNC55-0462 concentration that produced half-maximal KATP channel activation, A1 is the KATP current before NNC55-0462 was applied, A2 is the maximal KATP current level activated by NNC55-0462, and {rho} is the slope of the curve.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Syntaxin-1A decreases the KATP opening action of NNC55-0462 in BA8 cells.
Previously, we had reported that syntaxin-1A inhibited Kir6.2/SUR1 currents by its actions on nucleotide binding fold-1 and -2 of SUR1 (33,34). Here, we examined the possibility that NNC55-0462, belonging to a class of thiadiazine dioxide KATP channel openers (Fig. 1) and acting on distinct domains in SUR1 (23,27), might influence the actions of syntaxin-1A on the nucleotide binding folds, or vice versa. To examine the direct functional effects of syntaxin-1A on KATP channels, we again first used BA8 cells, a HEK cell line stably expressing human Kir6.2/SUR1 (24,27) that does not contain endogenous syntaxin-1A (33), and dialyzed into the cytoplasm of these cells soluble GST–syntaxin-1A lacking its transmembrane domain. Here, external perfusion of NNC55-0462 dose-dependently activated Kir6.2/SUR1 KATP channels, effective starting at 0.1 µmol/l and achieving maximum effect at 1 µmol/l (Fig. 2A). A higher concentration of NNC55-0462 (3 µmol/l) did not elicit a further increase in channel amplitude; instead, it induced channel rundown. GST–syntaxin-1A (100 nmol/l) (Fig. 2B), when compared with GST control (Fig. 2A), reduced the ability of NNC55-0462 to open KATP channels. We analyzed the data in several ways to show that syntaxin-1A (10 and 100 nmol/l) reduced both drug potency (Fig. 2C) and efficacy (Fig. 2D).


Figure 2
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  		FIG. 2. Syntaxin-1A reduces potency and efficacy of NNC55-0462 (NNC) on BA8 KATP channels. Shown in A and B are representative recordings of whole-cell K+ currents from BA8 cells subjected to extracellular perfusion of increasing NNC55-0462 concentrations with 100 nmol/l GST (A) or 100 nmol/l GST–syntaxin-1A (B) dialysis. Currents were evoked by 500-ms hyperpolarizations to –120 mV in 10-s intervals from a holding potential of –70 mV. Intracellular solution contains 1 mmol/l MgATP. C: Summary of results with each curve (100 nmol/l GST-control, 10 and 100 nmol/l GST–syntaxin-1A) normalized to its own maximum current level (Imax) to illustrate shifts in potency. D: Summary of results with each curve normalized to the maximum current level of GST-control group (Imax-GST) to illustrate changes in efficacy. E: Bar diagram summary showing GST–syntaxin-1A dialysis increased EC50 ({square}, left axis) and reduced efficacy ({blacksquare}, right axis) of NNC55-0462 on BA8 KATP channel currents. Results are the means ± SE of 5–7 cells in each group. One-way ANOVA followed by Dunnett's post hoc test was used for statistical analysis. *P < 0.05 compared with GST control group. Syn, syntaxin.

 
Figure 2C examines drug potency, where dose-response curves were normalized to each group's maximal channel activation at 1 µmol/l NNC55-0462, which demonstrated a rightward shift in potency caused by GST–syntaxin-1A dialysis. This is further illustrated in Fig. 2E, showing the half-maximal effective concentration (EC50) of NNC55-0462 in GST control group was 0.11 ± 0.02 µmol/l (n = 7), whereas GST–syntaxin-1A dialysis at 10 and 100 nmol/l increased it to 0.15 ± 0.05 µmol/l (n = 5, not significant) and 0.21 ± 0.04 µmol/l (n = 7, P < 0.05), respectively. Fig. 2D examines drug efficacy, where dose-response curves were normalized to the maximum channel activation observed with the GST-dialyzed control group, which demonstrated a downward shift in efficacy caused by GST–syntaxin-1A dialysis. This is further illustrated in Fig. 2E, where maximum current density activated by NNC55-0462 was 283 ± 14 pA/pF (n = 7) with GST dialysis, whereas syntaxin-1A dialysis at 10 and 100 nmol/l decreased it to 256 ± 28 pA/pF (n = 5, not significant) and 194 ± 12 pA/pF (n = 7, P < 0.01), respectively, with the latter being a significant reduction of 31 ± 4%.

To reiterate, these modulatory effects of syntaxin-1A on the effectiveness of NNC55-0462 on BA8 KATP channels are caused by direct interactions with the SUR1 protein because BA8 cells are devoid of endogenous syntaxin-1A and syntaxin-1A–interacting proteins (33). Note that the concentrations of GST–syntaxin-1A required likely represent an overestimation because of suboptimal targeting of soluble GST–syntaxin-1A to the sites of KATP channels on the plasma membrane.

Syntaxin-1A decreases the KATP opening action of NNC55-0462 in rat islet ß-cells.
To demonstrate the physiological implication of the BA8 study, we performed similar studies on rat islet ß-cells. Shown in Fig. 3A and B, NNC55-0462 dose-dependently activated KATP channel activity in rat islet ß-cells, effective starting at 0.03 µmol/l, with maximal effects reached at 1 µmol/l. Like BA8 cells, ß-cell KATP channels displayed rundown on further perfusion with a supramaximal concentration of NNC55-0462 at 3 µmol/l. The ß-cell currents were sensitive to tolbutamide inhibition applied in the presence of 3 µmol/l NNC55-0462, confirming the identity of the activated currents to be KATP currents (IKATP). KATP channel openers have negative allosteric effects on glibenclamide binding to SUR (36,37). In fact, such SUR1-specific KATP channel openers could displace 3H-glibenclamide binding (24), which, taken along with the high affinity of NNC55-0462 for SUR1, led us to use a higher concentration of tolbutamide to inhibit the channels.


Figure 3
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  		FIG. 3. Syntaxin-1A reduces potency and efficacy of NNC55-0462 (NNC) on rat islet ß-cell KATP channels. Rat islet ß-cells were subjected to the same experimental protocol as the BA8 cells in Fig. 2. Tolbutamide (Tolb.) was applied at the end of the experimental protocol in the presence of the final dose of NNC55-0462. A: Representative trace showing the effect of GST (1 µmol/l) dialysis into the cell cytoplasm. B: Representative trace showing the effect of GST–syntaxin-1A (1 µmol/l) dialysis into the cell cytoplasm. C: Summary of results with each curve (GST and GST–syntaxin-1A dialysis) normalized to its own maximum current level (Imax) to illustrate a shift in potency. D: Summary of results with each curve normalized to the maximum current level of GST-control group (Imax-GST) to illustrate changes in efficacy. E: Bar diagram summary showing GST–syntaxin-1A dialysis increased EC50 ({square}, left axis) and reduced efficacy ({blacksquare}, right axis) of NNC55-0462 on ß-cell KATP channel currents. Results are the means ± SE from 7–10 cells. Unpaired Student's t test was used for statistical analysis. *P < 0.05. Syn, syntaxin.

 
Consistent with the BA8 results (Fig. 2), both potency (Fig. 3C) and efficacy (Fig. 3D) of NNC55-0462 in opening islet ß-cell KATP channels were decreased by dialysis of GST–syntaxin-1A into the ß-cell cytoplasm. Here, GST–syntaxin-1A (1 µmol/l) increased the EC50 of NNC55-0462 from 0.050 ± 0.009 µmol/l (n = 10) to 0.11 ± 0.03 µmol/l (n = 7, P < 0.05) (Fig. 3E). With respect to efficacy, the maximum current density activated by NNC55-0462 was suppressed by GST–syntaxin-1A dialysis from 157 ± 8 pA/pF (n = 10) to 110 ± 4 pA/pF (n = 7, P < 0.01), which is a 30 ± 3% reduction (Fig. 3E). It is possible that soluble GST–syntaxin-1A lacking the transmembrane domain could bind a number of syntaxin-1A binding proteins in ß-cells that might have independent effects on KATP channels or, alternatively, sequester syntaxin-1A (i.e., endogenous v-SNAREs on insulin granules) to reduce the amount of GST–syntaxin-1A available for KATP channel interaction. Although we could not rule out these possibilities, their contributions are not likely to be significant in our study because the results of dialyzed syntaxin-1A on ß-cell KATP channel opener potency and efficacy were remarkably similar to those of BA8 cells, albeit the KATP channel density is much higher in the KATP-overexpressing BA8 cell line.

Syntaxin-1A, but not syntaxin-2, reduces the efficacy of NNC55-0462 in BA8 cells.
It is possible that other syntaxins in the ß-cell (38) might have actions similar to that of syntaxin-1A on KATP channels. Syntaxin-1B and -2 exhibit the highest homology to syntaxin-1A (>90 and >70%, respectively) (39). Using Syntaxin isoform–specific antibodies and overexpressed proteins as controls, Fig. 4A shows that syntaxin-2 is abundant in rat islets, but not syntaxin-1B, the latter confirming a previous report (40). We therefore proceeded to explore the specificity of action of syntaxin-1A on NNC55-0462–induced ß-cell KATP channel opening by comparing the effects of syntaxin-1A versus syntaxin-2.


Figure 4
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  		FIG. 4. Syntaxin-1A, but not syntaxin-2, reduces efficacy of NNC55-0462 (NNC) in BA8 cells. A: Syntaxin-1A (left upper panel) and syntaxin-2 (right panel), but not syntaxin-1B (left lower panel), are expressed in rat pancreatic islets (30 µg protein). Lysates were separated by 12% SDS-PAGE and probed with Syntaxin isoform–specific antibodies as indicated. Positive controls used included rat brain (2 µg), HEK293 cells (15 µg) overexpressing syntaxin-1A or -1B, and pancreatic acinar cells (15 µg, for syntaxin-2). Shown are representative blots of three similar experiments. Because syntaxin-1A and -2 are present in rat islets, which could modulate ß-cell KATP channels, we next performed whole-cell recordings in BA8 cells transiently transfected with either GFP alone or cotransfected with syntaxin-1A or -2 (B–D). B: Representative current-voltage (I-V) relationships, stepped from –140 to –20 mV at 20-mV intervals every 2 s from a holding potential of –70 mV, were recorded when maximum channel activation had occurred at 1 µmol/l perfusion of NNC55-0462. C: Representative current-voltage traces showing that currents activated were sensitive to tolbutamide inhibition (3 mmol/l) applied in the presence of 1 µmol/l NNC55-0462 after maximum channel activation in the same experiment. D: Summary of results showing that syntaxin-1A transfection, but not syntaxin-2, reduced the maximum current density activated by NNC55-0462 recorded at –120 mV (I-120 mV). Results are the means ± SE from 9–11 cells. One-way ANOVA followed by Dunnett's post hoc test was used for statistical analysis. *P < 0.05 compared with GST control group. Syn, syntaxin.

 
BA8 cells were transiently transfected with green fluorescence protein (GFP) alone as control, or cotransfected with full-length (including transmembrane domain) syntaxin-1A or -2 to localize the expressed syntaxin to the plasma membrane, where KATP channels are located. Fig. 4B shows representative current-voltage relationships demonstrating that maximum current density activated by 1 µmol/l NNC55-0462 was specifically reduced in the syntaxin-1A–but not the syntaxin-2–transfected group. Activated currents were confirmed to be of IKATP by their sensitivity to tolbutamide inhibition (Fig. 4C), applied in the presence of NNC55-0462 after maximal channel activation had occurred. Fig. 4D summarizes these results, showing that the maximum current density activated by NNC55-0462 (at –120 mV) was reduced by GFP/syntaxin-1A transfection to 206 ± 14 pA/pF (n = 10, P < 0.01) from 323 ± 14 pA/pF in the GFP control group (n = 9), which is a ~36% reduction. The lack of effect of GFP/syntaxin-2 transfection on KATP channel opening (349 ± 17 pA/pF, n = 11, not significant), despite its high homology to syntaxin-1A, indicates that syntaxin-1A effects on KATP channels are isoform specific within the syntaxin family in ß-cells.

Syntaxin-1A reverses the KATP opening action of NNC55-0462 in inside-out membrane patches.
We used the inside-out patch configuration on BA8 cells (Fig. 5) for patch-clamp recording to examine: 1) if NNC55-0462 can still open KATP channels when applied intracellularly, 2) if syntaxin-1A could influence these NNC55-0462 actions, and 3) which domain within syntaxin-1A (H3 versus Habc) effects these actions. Membrane patches were first exposed to 0 and 3 mmol/l Mg-ATP to confirm the ATP sensitivity of currents recorded. Patches were then held at 0.3 mmol/l Mg-ATP to produce partial channel inhibition and to prevent rapid channel rundown. NNC55-0462 (0.3 µmol/l) was applied in a 0.3 mmol/l Mg-ATP environment to induce channel opening, after which GST, GST–syntaxin-1A, GST–syntaxin-1A–Habc, or GST–syntaxin-1A–H3 was applied to the solution. NNC55-0462 was able to induce KATP channel opening when applied on the intracellular side of the membrane, achieving a remarkable >80% of maximum channel opening at 0.3 µmol/l (Fig. 5A–D). Application of GST (Fig. 5A) or GST–syntaxin-1A–Habc (300 nmol/l) (Fig. 5C) had no effect on NNC55-0462–mediated channel opening, whereas GST–syntaxin-1A (Fig. 5B) and GST–syntaxin-1A–H3 (300 nmol/l) (Fig. 5D) significantly suppressed the current activity activated by NNC55-0462. Figure 5E summarizes the data showing that the percentage of maximal current activated by NNC55-0462 in the presence of GST (92 ± 13%, n = 9) was not significantly different from NNC55-0462 alone (84 ± 11%), whereas NNC55-0462 plus GST–syntaxin-1A showed a current of 39 ± 6% (n = 9), which was a significant reduction of ~52% (P < 0.01) from NNC55-0462 alone (91 ± 10%). Although the percentage of current activation by NNC55-0462 plus GST–syntaxin-1A–Habc (88 ± 10%, n = 6) was not significantly different from NNC55-0462 alone (93 ± 10%), NNC55-0462 plus GST–syntaxin-1A–H3 caused a similar (to GST–syntaxin-1A), ~54% reduction of current (38 ± 7%, n = 6, P < 0.01) compared with NNC55-0462 alone (92 ± 6%). These results indicate that 1) NNC55-0462 can access its site of action on KATP channels when pplied intracellularly and that 2) the H3 domain of syntaxin-1A is the putative syntaxin-1A domain inhibiting KATP channels previously activated by this KATP channel opener.


Figure 5
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  		FIG. 5. Syntaxin-1A inhibits NNC55-0462 (NNC)-activated KATP channels in inside-out membrane patches of BA8 cells. Macroscopic currents were recorded from inside-out membrane patches of BA8 cells held at –100 mV in symmetrical 140 mmol/l K+ conditions. A–D: Representative traces showing the effect of GST (A), GST–syntaxin-1A (B), GST–syntaxin-1A–Habc (C), and GST–syntaxin-1A–H3 (D) (300 nmol/l each) on 0.3 µmol/l NNC55-0462–activated KATP currents. E: Summary of results are the means ± SE with n = 6–9 in each group. Paired Student's t test was used for statistical analyses. *P < 0.05. Syn, syntaxin.

 
Syntaxin-1A decreases the KATP opening action of diazoxide in rat islet ß-cells.
We next examined whether the effect of syntaxin-1A is specific to this particular KATP channel opener compound. We chose to study the parent compound, diazoxide, which shares the same backbone structure as NNC55-0462 (Fig. 1) but interacts with SUR1 at slightly different domains. We repeated the experiment performed with NNC55-0462 on rat islet ß-cells, but instead we perfused increasing concentrations of diazoxide (3–300 µmol/l). Similar to the NNC55-0462 study (Fig. 3), GST–syntaxin-1A (Fig. 6B) dialyzed into the cytoplasm of rat islet ß-cells significantly reduced the potency and efficacy of diazoxide, compared with GST dialysis (Fig. 6A). Analysis of drug potency (Fig. 6C and E) shows the EC50 of diazoxide was increased from 21 ± 5 µmol/l (n = 10) to 42 ± 7 µmol/l (n = 7, P < 0.05) by GST–syntaxin-1A dialysis. Analysis of drug efficacy (Fig. 6D and E) shows the maximum current density activated by diazoxide was reduced by GST–syntaxin-1A from 136 ± 4 pA/pF (n = 10) to 92 ± 5 pA/pF (n = 7, P < 0.01), which is a ~32% inhibition. These results together suggest that the effect of syntaxin-1A on the opening of KATP channels by KATP channel openers is not specific to NNC55-0462 alone, but rather is shared among different dioxide compounds.


Figure 6
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  		FIG. 6. Syntaxin-1A reduces potency and efficacy of diazoxide (DZX) on rat islet ß-cell KATP channels. Rat islet ß-cells were subjected to the same experimental protocol as the BA8 cells in Fig. 2. Tolbutamide (Tolb.) was applied at the end of the experimental protocol in the presence of the final dose of DZX. A: Representative trace showing the effect of GST (1 µmol/l) dialysis into the cell cytoplasm. B: Representative trace showing the effect of GST–syntaxin-1A (1 µmol/l) dialysis into the cell cytoplasm. C: Summary of results with each curve (GST and GST–syntaxin-1A dialysis) normalized to its own maximum current level (Imax) to illustrate a shift in potency. D: Summary of results with each curve normalized to the maximum current level of GST-control group (Imax-GST) to illustrate changes in efficacy. E: Bar diagram summary showing GST–syntaxin-1A dialysis increased EC50 ({square}, left axis) and reduced efficacy ({blacksquare}, right axis) of diazoxide on ß-cell KATP channel currents. Results are the means ± SE from 7–10 cells. Unpaired Student's t test was used for statistical analysis. *P < 0.05. Syn, syntaxin.

 
NNC55-0462 does not affect syntaxin-1A–SUR1 binding interactions.
Because we showed that NNC55-0462 was effective when applied not only extracellularly but also intracellularly, the latter suggests the possibility that this KATP channel opener might share common binding domains with syntaxin-1A on SUR1 nucleotide binding folds to influence KATP channel opening. We therefore examined whether this KATP channel opener could affect syntaxin-1A–SUR1 binding interactions. GST–syntaxin-1A bound to glutathione agarose beads was able to pull down the same amount of SUR1 protein from BA8 cell lysate extract with increasing NNC55-0462 concentrations (up to 10 µmol/l) both in the absence (Fig. 7A) or presence of 0.3 mmol/l ATP (Fig. 7B) or 5 mmol/l ATP (Fig. 7C). ATP at 5 mmol/l appeared to reduce the amount of SUR1 pulled down by GST–syntaxin-1A at all concentrations of NNC55-0462 (Fig. 7C). A positive control with increasing concentrations of thrombin-cleaved syntaxin-1A (instead of NNC55-0462) showed that the assay used is indeed capable of detecting competitive binding inhibition (Fig. 7D). A negative control experiment showed that GST bound to beads could not pull down SUR1, representing nonspecific binding (Fig. 7E); however, glutathione agarose beads alone did bind a small amount of SUR1 (~20% of that bound by GST–syntaxin-1A). This inability of NNC55-0462 to modulate syntaxin-1A binding to SUR1 suggests that this KATP channel opener does not likely bind the same domains that syntaxin-1A binds to in SUR1 at its nucleotide binding folds (33,34). The ability of syntaxin-1A to block this KATP channel opener action on KATP channels suggests that syntaxin-1A could modulate NNC55-0462 binding to the SUR1 protein. However, because of the lack of a radiolabeled NNC55-0462, we were unable to investigate this possibility.


Figure 7
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  		FIG. 7. NNC55-0462 (NNC) does not influence syntaxin-1A binding to SUR1 protein. GST–syntaxin-1A (2 µmol/l), bound to glutathione agarose beads, was incubated with BA8 cell lysate extract in the presence of increasing concentration of NNC55-0462 at 0 (A), 0.3 (B), or 5 mmol/l ATP (C). Panels D and E were performed as positive and negative control experiments, respectively. D: Increasing concentrations of thrombin-cleaved syntaxin-1A prevent syntaxin-1A bound to beads from pulling down SUR1. E: GST bound to beads does not pull down SUR1 compared with the strong binding of SUR1 to syntaxin-1A bound to beads. In these experiments, the proteins bound to beads were eluted, separated on SDS-PAGE, and probed with anti-SUR1 antibody. Shown are representative blots (top panels) and summaries of 3–4 experiments (lower panels) (means ± SE) expressed as a percentage of the control binding value determined at 0 ATP in the absence of NNC55-0462 (A–C) or thrombin-cleaved syntaxin-1A (D) and, in panel E, as a percentage of GST–syntaxin-1A bound to glutathione agarose beads. Molecular mass markers (kD) are indicated on the left. C, control lane; Syn, syntaxin.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current study is the first to demonstrate an effect of the SNARE protein syntaxin-1A on the actions of NNC55-0462, a member of a novel class of thiadiazine dioxide KATP channel openers, and its parent compound diazoxide. Our results showed that syntaxin-1A reduces the potency and, more significantly, the efficacy of NNC55-0462 and diazoxide. In inside-out membrane patches, syntaxin-1A can also inhibit channels previously activated by NNC55-0462. Within the syntaxin family present in ß-cells, these actions were specific to syntaxin-1A because syntaxin-2, which shares >70% homology with syntaxin-1A (39), had no effect on KATP channels. We further determined that the H3 domain, and not the Habc domain, is the putative syntaxin-1A domain that modulated the KATP channel opener's actions on the KATP channel. Because syntaxin-1A also influenced the KATP channel–opening actions of diazoxide, a nonspecific KATP channel opener, this would suggest that syntaxin-1A might also influence the other KATP channel openers’ actions on SUR2 proteins, which will require further study.

The isoform specificity of syntaxin-1A's inhibition on NNC55-0462–mediated KATP channel opening is in line with previous studies examining other syntaxin-1A–ion channel interactions. Studies with Lc- and N-type calcium channels showed similar results, with the basis of such specificity mapped out to two critical cysteine residues found in syntaxin-1A but not in syntaxin-2 (41). However, these cysteine residues on the transmembrane domain of syntaxin-1A are unlikely candidates for the basis of its actions toward KATP channel modulation because: 1) a soluble GST–syntaxin-1A fusion protein lacking the transmembrane domain inhibited NNC55-0462–mediated KATP channel opening as efficaciously as expression of the full-length syntaxin-1A, and 2) the soluble GST–syntaxin-1A–H3 domain induced a similar inhibition as syntaxin-1A, consistent with our previous study, which mapped out the nucleotide binding fold–interacting domain to be the H3 domain (33). Nonetheless, that report (41) does point out that differences as small as one to two amino acid residues are enough to produce isoform-specific action. Moreover, a study of syntaxin-1A versus syntaxin-1B, which shares 93% homology, shows that only syntaxin-1A is capable of mediating tonic voltage-dependent inhibition on N-type calcium channels (42). With regard to other K+ channels, we previously reported that syntaxin-1A, but not syntaxin-2, reduced Kv2.1 current density by direct actions on specific Kv2.1 cytoplasmic domains and by inhibiting channel trafficking (43).

Although the mechanisms of action of syntaxin-1A on calcium channel regulation have been well characterized, those involved in KATP channel modulation are less clear. We postulated three possibilities: 1) syntaxin-1A may influence these KATP channel openers’ interaction with SUR1, 2) conversely, these KATP channel openers may influence syntaxin-1A interactions with SUR1, or 3) syntaxin-1A may influence the mechanism of action of KATP channel openers by interfering with adenosine nucleotide binding and/or hydrolysis.

For the first possibility, syntaxin-1A interacting with the nucleotide binding folds may induce conformational changes to SUR1 that affect the affinity of NNC55-0462 to its binding sites. Unfortunately, NNC55-0462 could not be labeled to directly test this possibility. Nonetheless, direct competitive inhibition is unlikely because of the inability of increasing concentrations of NNC55-0462 to overcome the reduced efficacy caused by syntaxin-1A. The SUR1 domains critical to the actions of a closely related compound, NNC55–9216, were mapped out to transmembrane domains 8–11 and both nucleotide binding folds (24). Because syntaxin-1A can bind both nucleotide binding folds of SUR1 (33,34), such interactions may induce negative allosteric modulation onto the KATP channel opener binding sites of SUR1, thereby decreasing the KATP channel opener binding affinity. In addition, previous studies have determined the sites of action of diazoxide to be transmembrane domains 6–11 and nucleotide binding fold-1 (44). Therefore, it is not surprising that syntaxin-1A is capable of influencing the effects of both KATP channel openers because of potential overlapping binding or signal transduction sites. Further mutational studies will be required to elucidate these sites.

For the second possibility, NNC55-0462 may act to relieve syntaxin-1A inhibition on KATP channels, and thus increased levels of syntaxin-1A would reduce the effectiveness of this KATP channel opener. Our in vitro binding studies showed that increasing concentrations of NNC55-0462 did not affect syntaxin-1A binding to SUR1 protein, thus eliminating this possibility.

For the third possibility, the binding and activity of a wide range of KATP channel openers are dependent on the presence of Mg2+ and hydrolyzable ATP (45–47), and a postulated mechanism of action of these compounds is the acceleration of nucleotide hydrolysis by nucleotide binding folds (48). Syntaxin-1A may affect KATP channel openers by interfering with this mechanism, either via slowing the rate of nucleotide hydrolysis or by affecting the binding of Mg-ATP or Mg-ADP to their respective nucleotide binding folds. In support of this, NNC55-9216 required both nucleotide binding folds (24), whereas diazoxide required nucleotide binding fold-1 to exert their KATP channel opening action (44). Our in vitro binding study revealed reduced syntaxin-1A binding to SUR1 when the Mg-ATP concentration was increased to 5 mmol/l, which suggests competitive binding displacement at the nucleotide binding folds. Should syntaxin-1A influence the efficacy of these KATP channel openers via this postulated mechanism, further modulating effects may be observed with combinations of adenosine nucleotides and Mg2+. These complex interactions are not within the scope of the current study and will require much further investigation.

Our previous report on syntaxin-1A–overexpressing mice showed that a moderate increase of ~30% of islet syntaxin-1A levels was sufficient to cause hyperglycemia from reduction in ß-cell insulin exocytosis and inhibition of L-type Ca2+ current density (49). However, there was no effect on KATP or Kv2.1 channels, suggesting that these distinct components of the insulin secretory process exhibit differing sensitivity to syntaxin-1A modulation. This also suggests that moderate increases in syntaxin-1A levels beyond normal islet levels (which do not occur physiologically) are not likely to influence KATP channel opener actions.

The converse, however, may not be true—that is, when islet syntaxin-1A levels become reduced. Alemzadeh et al. (50) reported that 6 weeks of treatment with NN414, a SUR1-specific KATP channel opener of the same family as NNC55-0462, led to improvements in fasting and post–intraperitoneal glucose tolerance test glucose levels in Zucker obese diabetic rats but not in lean control rats, which was postulated to be attributable to lower insulin levels in lean animals during basal conditions and after NN414 treatment. We propose an alternative explanation. Islet syntaxin-1A levels are severely reduced in type 2 diabetic rodent models and human patients (percent of normal controls: ~35% for obese Zucker rats, ~40% for GK rats, and ~20% for humans) (5–8). This may at least partly explain the increased effectiveness of KATP channel opener therapy in the Zucker rats (50) by enhancing KATP channel opener potency and efficacy on ß-cells, whereas normal islet levels of syntaxin-1A would dampen the KATP channel opener actions. Because brain levels of syntaxin-1A are high and do not fluctuate with diabetes (6), the KATP channel opener would not affect neuronal SUR1 KATP channels, thereby increasing the drug's specificity. Normoglycemic control partially restored islet syntaxin-1A levels in GK rats (6), which could dampen the KATP channel opener effects that may be misconstrued as loss of drug effectiveness. These dynamics of islet syntaxin-1A expression occurring during the course of therapy may profoundly modulate KATP channel opener drug potency and efficacy on ß-cells, rendering the drug more effective at stages of severe diabetes while dampening its actions as normoglycemic control is gradually attained. These exciting possibilities will require further experimental verification in diabetic rodent models.


   ACKNOWLEDGMENTS
 
H.Y.G. has received support from the Canadian Institutes for Health Research (CIHR MOP-69083) and the Heart and Stroke Foundation of Ontario (T-6064). B.N is supported by studentships from the Ontario Graduate Studentship and the Banting and Best Diabetes Center (University of Toronto). Y.H. is supported by a postdoctoral award from the Janssen-Ortho/Canadian Association of Gastroenterology.

We thank X. Gao for technical assistance in preparing the rat pancreatic islets and R. Tsushima and Y.-M. Leung for active discussion.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 11 May 2007. DOI: 10.2337/db07-0030.

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0030.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication January 9, 2007 and accepted in revised form May 8, 2007


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