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Originally published In Press as doi:10.1074/jbc.M404954200 on August 31, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47125-47131, November 5, 2004
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Syntaxin-1A Inhibits Cardiac KATP Channels by Its Actions on Nucleotide Binding Folds 1 and 2 of Sulfonylurea Receptor 2A*

Youhou Kang{ddagger}§, Yuk-Man Leung{ddagger}§, Jocelyn E. Manning-Fox{ddagger}§||, Fuzhen Xia{ddagger}, Huanli Xie{ddagger}, Laura Sheu{ddagger}, Robert G. Tsushima{ddagger}**, Peter E. Light{ddagger}{ddagger}§§, and Herbert Y. Gaisano{ddagger}¶¶||

From the {ddagger}Departments of Medicine and Physiology, University of Toronto, Toronto M5S 1A8, Canada and the {ddagger}{ddagger}Department of Pharmacology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received for publication, May 4, 2004 , and in revised form, August 20, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234–4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on KATP channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ATP-sensitive potassium (KATP)1 channels couple the metabolic status of the cell to its membrane potential to regulate a number of cellular events, including secretion (neurons and neuroendocrine cells) and muscle contraction (skeletal, cardiac, and vascular smooth muscle) (1–3). The KATP channel is a hetero-octameric complex that comprises two structurally distinct subunits in a 4:4 stoichiometry, including a regulatory sulfonylurea receptor (SUR) and a pore-forming, inwardly rectifying Kir6.X subunit (4–7). The SUR proteins belong to the adenine nucleotide-binding cassette protein superfamily, with each member possessing two cytoplasmic nucleotide binding folds (NBF1 and C-terminal NBF2) (8). Within the SUR family are SUR1 in neurons and neuroendocrine cells (pancreatic islet beta cells), SUR2A in cardiac muscle, and SUR2B in vascular smooth muscle (4, 5, 9–11). Considerable structure-function studies have been done to elucidate the specific actions of ATP and ADP on NBFs of these SUR proteins and their Kir6.X subunits (1–3, 12). However, these studies do not fully explain the distinct regulatory functions of the NBFs of each SUR protein on the KATP channel (1–3, 12).

Syntaxin-1A (Syn-1A), a SNARE protein known to mediate exocytosis (13), also directly binds to voltage-gated K+ and Ca2+ channels to regulate secretion (14, 15). We recently reported that Syn-1A could also bind NBF1 and NBF2 of SUR1 and inhibit pancreatic islet beta cell KATP channels through its actions on NBF1 (16). In fact, Syn-1A was reported to be present in cardiac myocytes (17). These results prompted us to test whether Syn-1A has a similar interaction with cardiac SUR2A. In this study, we found that Syn-1A, present in the plasma membrane of rat cardiac myocyte, inhibited the cardiac KATP channel through its actions on both NBF1 and NBF2 of SUR2A.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs and Recombinant GST Fusion Proteins—The plasmids pECE-SUR2A and pECE-Kir6.2 were generous gifts from S. Seino (Chiba University, Chiba, Japan), and pGEX-4T-1-Syn-1A was from W. Trimble (The Hospital for Sick Children, Toronto, Ontario, Canada). The constructs pGEX-5X-1-SUR2-NBF1 (amino acids 684–872) and -NBF2 (amino acids 1321–1499) were made using the same method as described previously (16). All constructs were verified by DNA sequencing. GST fusion protein expression and purification were performed following manufacturer's instructions (Amersham Biosciences). 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). The purity of each eluted 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.

Cell Culture and Transfection—HEK293 cells were grown at 37 °C in 5% CO2 in minimum Eagle's medium (Invitrogen) containing 1 g/liter glucose and supplemented with 10% fetal bovine serum (Cansera, Rexdale, Ontario, Canada). The HEK293 cells were transiently co-transfected with green fluorescent protein, pECE-Kir6.2, and pECE-SUR2A using LipofectAMINE 2000TM (Invitrogen) according to manufacturer's instructions. One day after transfection, cells were trypsinized and placed in 35-mm culture dishes overnight prior to voltage clamp experiments. Transfected cells were identified by visualization of the fluorescence of the co-expressed green fluorescent protein. For binding assay, the HEK293 cells were transfected with pcDNA3-Syn-1A or pECE-SUR2A and pECE-Kir6.2.

Preparation of Rat Cardiac and Brain Membrane—Rat cardiac membrane was prepared according to the method originally described by Mayanil et al. (18) and modified by Barry et al. (19). To prepare rat brain membranes, we followed the method reported previously by Huttner et al. (20). Finally, both membranes were solubilized in 1x radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 1% Triton X-100, 2 µM pepstatin A, 1 µg/ml leupeptin, and 5 µg/ml aprotinin) on ice for 30 min, and then insoluble material was pelleted by centrifugation at 21,000 x g at 4 °C for 25 min. The supernatant was used for binding assay.

In Vitro Binding Assay—Two days after transfection with pcDNA3-Syn-1A or pECE-SUR2A and pECE-Kir6.2, the HEK293 cells were washed with ice-cold saline-buffered solution (phosphate-buffered saline, pH 7.4) and then harvested in binding buffer (25 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 1% Triton X-100, 20 µM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin A, 1 µg/ml leupeptin, and 10 µg/ml aprotinin). The cells were lysed by sonication, and insoluble materials were removed by centrifugation at 21,000 x g at 4 °C for 30 min. For binding assay, the detergent extract (0.3 ml, 1.2–5.1 µg/µl protein) of HEK293 cells, rat brain synaptic membranes, and rat heart membranes or thrombin-cleaved Syn-1A (650 pmol of protein in 250 µl binding buffer) was mixed with GST (as a negative control, 650 pmol of protein), GST-Syn-1A (650 pmol of protein), GST-SUR2-NBF1, or GST-SUR2-NBF2 (650 pmol of protein in 250 µl of binding buffer or at concentrations indicated in Fig. 2F). (All GST fusion proteins bound to glutathione-agarose beads.) The mixtures were incubated at 4 °C with constant agitation for 2 h. The beads were then washed three times with binding buffer. The samples were separated on 15 or 8% SDS-PAGE, transferred to nitrocellulose membrane (Millipore Corp., Bedford, MA), and identified with mouse anti-Syn-1A monoclonal antibody (1:1000, Sigma) or rabbit anti-SUR2 polyclonal antibody (1:300, catalog no. SC-25684, Santa Cruz Biotechnology, Santa Cruz, CA). The anti-SUR2 antibody was generated against recombinant human SUR2 protein (amino acids 921–1000), which is 97% homologous to rat SUR2A and SUR2B but only 44% homologous to hamster SUR1.



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  		FIG. 2.
Syntaxin-1A binds cardiac SUR2A at its NBF1 and NBF2. GST-Syn-1A, bound to glutathione beads, was able to pull down SUR2 proteins from detergent solubilized rat brain membrane (A) and rat cardiac membrane (B), as well as overexpressed SUR2A in HEK293 cells (C). GST-NBF1 and -NBF2, bound to beads, were capable of pulling down overexpressed Syn-1A from HEK293 cell extract (D) and thrombin-cleaved Syn-1A (E). A–E are representative blots of two or three similar experiments. F shows the dose dependence of GST-NBF1 (i) and -NBF2 (ii) binding to Syn-1A. The upper panel shows a representative blot. The bottom panel shows a summary of quantitative densitometry scanning of the specific bands from three separate experiments. The values are expressed as mean ± S.E. In A–F, molecular mass markers (kDa) are indicated on the left.

 
Isolation of Rat Cardiac Myocytes—Male Sprague-Dawley rats (200–225 g) were injected with heparin (3000 units kg-1 intraperitoneally) and sacrificed under anesthesia with phenobarbitol (Somnotol, 75 mg kg-1 intraperitoneally). Hearts were rapidly excised, immersed in Ca2+-free Tyrode's solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM glucose, 5 mM HEPES, pH 7.4), and gently squeezed to clear the chambers of blood. The aorta was cannulated and perfused in a retrograde fashion on a Langendorff apparatus with Tyrode's solution containing 1 mM CaCl2 for 5 min (10 ml min-1, 37 °C). The heart was perfused for an additional 5 min with Ca2+-free Tyrode's solution followed by a 9-min perfusion period with Ca2+-free Tyrode's solution containing 0.015 mg ml-1 collagenase (500 units mg-1; Yakult Pharmaceutical Ind. Co., Tokyo, Japan) and 0.004 mg ml-1 protease (type XIV, 5.4 units mg-1; Sigma). The hearts were perfused for an additional 5 min with a KB solution containing 100 mM potassium glutamate, 10 mM potassium aspartate, 2.5 mM KCl, 20 mM glucose, 10 mM KH2PO4, 2 mM MgSO4, 20 mM taurine, 5 mM creatine, 0.5 mM EGTA, 5 mM HEPES, pH 7.2. The right free wall of the right ventricle was dissected from the heart, placed in 10 ml of KB medium, and cut into 8–10 pieces. 10 ml of solution was passed through a nylon mesh and stored at room temperature (22–24 °C) until used. Single cardiac myocytes used were calcium-tolerant, quiescent, and had sharp cross-striations without membrane blebs.

Confocal Microscopy—The isolated myocytes were plated on polylysine-coated coverslips, rinsed, fixed in 2% formaldehyde for 0.5 h at room temperature, subjected to blocking with 5% normal goat serum and 0.1% saponin for 0.5 h at room temperature, and then immunolabeled with mouse anti-Syn-1 antibody (1:50, Sigma) overnight at 4 °C. After rinsing with 0.1% saponin/phosphate-buffered saline, the coverslips were incubated with appropriate fluorescein isothiocyanate-labeled secondary antiserum for 1 h at room temperature, mounted on slides in a fading retarder (0.1% p-phenylenediamine in glycerol), and examined using a laser-scanning confocal imaging system (Carl Zeiss, Oberkochen, Germany).

Recording KATP Currents from HEK293 Cells Expressing Kir6.2 and SUR2A—KATP channel current from human embryonic kidney 293 cells was recorded in the whole-cell configuration using an EPC-9 amplifier and Pulse software (HEKA Electronik, Lambrecht, Germany) as we described previously (16). Recording pipettes were pulled from 1.5-mm borosilicate glass capillary tubes (World Precision Instruments, Inc.) using a programmable micropipette puller (Sutter Instrument Co., Novato, CA). Pipettes were then heat-polished with fire, and tip resistances ranged from 1.5–3 mega-ohms when filled with intracellular solution containing 140 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 HEPES, and 0.3 mM MgATP (pH 7.25 adjusted with KOH). Fusion proteins of GST, Syn-1A, and/or SUR2A NBFs were also added to the pipette solution. 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 a pulse of -140 mV was given every 10 s to monitor the current magnitude. When the current reached maximum amplitude, voltage pulses from -160 to 40 mV were given at 20-mV increments to yield the I-V relationship. Steady-state outward currents were determined as the mean current in the final 95–99% of the 500-ms pulse. All experiments were performed at room temperature.

Recording KATP Currents from Cardiac Myocytes—Standard patch clamp techniques were used to record currents in the inside-out configuration so that the internal face of membrane patches could be exposed directly to test solutions using a multi-input perfusion pipette. The time required for solution change at the tip of the recording pipette was <1 s. The pipette/bath solution for excised patch experiments contained 140 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM HEPES-KOH, pH 7.4. Currents were recorded at a holding potential of -60 mV, amplified using Axopatch 200B (Axon Instruments, Inc.), and then digitized and analyzed using Axoscope version 8.0 and pClamp version 8.0 software. Data were sampled at 2.5 kHz and filtered at 1 kHz. Excised patches were initially held in 1 mM ATP to measure base-line current. An application of 50 µM ATP then produced an increase in current to a level defined as "control" for each patch. The current level was then measured following co-application of Syn-1A with or without pre-incubation with SUR2A NBF1 or SUR2A NBF2, and this measured current was expressed as a percentage of the controlled conditions for each patch. All experiments were performed at room temperature.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Syntaxin-1A Is Expressed in the Plasma Membrane of Cardiac Myocytes—We examined the expression and cellular location of Syn-1A in rat cardiac myocytes. Fig. 1A is a Western blot showing that Syn-1A (top band) and Syn-1B (bottom band) were present in rat whole heart cell lysate (100 µg of protein). Both Syn-1 isoforms were recognizable by the rabbit anti-Syn-1A antibody. When the cardiac lysate was purified to a plasma membrane fraction (50 µg of protein), the Syn-1 signals increased with Syn-1A being more abundant than Syn-1B. In Fig. 1B (upper panel), we confirmed the cellular location of Syn-1A to the plasma membrane of cardiac myocyte by confocal microscopy. The bottom panel (Fig. 1B) shows a negative control, wherein the myocytes were not labeled with anti-syntaxin antibody but with fluorescein isothiocyanate-conjugated secondary antibody.



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  		FIG. 1.
Syntaxin-1A is expressed in the plasma membrane of cardiac myocytes. A, Western blot analysis of Syn-1A (top band) and -1B (bottom band) in rat brain lysate (5 µg of protein), cardiac lysate (100 µg of protein), and cardiac membrane (50 µg of protein). B, top panel shows Syn-1A localization in cardiac myocyte by confocal microscopy. The arrowheads point to the plasma membrane location of Syn-1A. Bottom panel shows a negative control, whereby the myocyte was not labeled with anti-syntaxin antibody but with fluorescein isothiocyanate-conjugated secondary antibody.

 
Syntaxin-1A Binds Cardiac SUR2A at Its NBF-1 and NBF-2—We first investigated whether the endogenous SUR2 proteins in rat cardiac muscle and brain are capable of binding the Syn-1A. Although both SUR2 proteins are only found in low abundance in the brain (1, 5, 11, 12), large amounts of purified rat brain membranes could be prepared for the protein binding studies performed in Fig. 2A. Indeed, Fig. 2, A and B, shows that Syn-1A could bind native SUR2 proteins from both rat tissues, as demonstrated by the ability of GST-Syn-1A bound to agarose beads to pull down rat brain membrane and cardiac membrane SUR2-immunoreactive proteins. Because the antibody we have used recognizes a domain (amino acids 921–1000) conserved between SUR2A and SUR2B, the SUR2-immunoreactive proteins likely include both SUR2 proteins, especially the rat brain membranes (11, 12) in Fig. 2A. Furthermore, the molecular mass of SUR2A and SUR2B differs by just 1 kDa (3, 12, 16) and therefore may not be separated on the PAGE. In fact, we observed only a single SUR2 immunoreactive band in Fig. 2, A and B. SUR2A is nonetheless the dominant SUR2 protein in cardiac muscles (1, 3, 11, 12), as shown in Fig. 2B. We have therefore focused on the cardiac SUR2A protein by overexpressing SUR2A/Kir6.2 in HEK293 (Fig. 2C). Here, GST-Syn-1A bound to agarose beads is able to pull down abundant SUR2A proteins from the HEK293 cell lysate extract. This study also demonstrated (Fig. 2C) that Syn-1A binding to SUR2A is direct and not likely via an intermediary ternary protein contained in the brain or heart. We had previously mentioned that Syn-1A did not bind Kir6.2 cytoplasmic N- and C-terminal domains (16).

Our previous study with pancreatic islet beta cells shows that Syn-1A specifically binds to NBF1 and NBF2 domains of SUR1 (16). We therefore examined whether Syn-1A may likewise bind to the NBF1 (amino acids 684–872) and C-terminal NBF2 (amino acids 1321–1499) of SUR2A. Indeed GST-NBF1 and -NBF2 of SUR2A, bound to agarose beads, were both able to pull down overexpressed Syn-1A from HEK293 cell extract (Fig. 2D) and recombinant Syn-1A (thrombin cleaved) (Fig. 2E), but as a negative control, GST did not (Fig. 2, D and E). Finally, we examined the dose dependence of SUR2A-NBF1 (Fig. 2F, i) and NBF2 (Fig. 2F, ii) binding to Syn-1A at saturating concentrations. We found that NBF1 and NBF2 bound Syn-1A with an ED50 of 0.52 ± 0.07 µM (n = 3) and 0.64 ± 0.13 µM (n = 3), respectively.

Syntaxin-1A Inhibits KATP Channels in Cardiac Myocytes through Direct Interactions with NBF1 and NBF2 of SUR2A— To show the functional significance of the interaction between Syn-1A and cardiac SUR2A-NBF-1 and -NBF-2, we examined whether recombinant Syn-1A would directly inhibit KATP currents in rat cardiac myocytes and whether such inhibition is via Syn-1A-NBF(s) interaction. We performed inside-out configuration so that the intracellular face of a cardiac membrane patch containing KATP channels could be exposed to the test solutions. As shown in Fig. 3A,i, the patch had minimum current activities when held in 1 mM ATP but showed bursting activities when ATP was removed, indicative of the opening of multiple KATP channels. Because sustained exposure to ATP-free solution will cause run-down of KATP channels, we applied 50 µM ATP to maintain channel activities, leading to a partial inhibition of KATP currents. The KATP current in the presence of 50 µM ATP is then defined as control for each patch. Application of Syn-1A alone markedly reduced current values to 32.2 ± 6.7% of control values (Fig. 3A, i, and B). The GST control did not affect currents (102.2 ± 20.1%, Fig. 3B). We had previously demonstrated that Syn-1A strongly inhibits pancreatic beta cell KATP currents through binding to the NBF1, but not NBF2, of SUR1 (16). We then investigated whether SUR2A NBF1 or NBF2 would mediate Syn-1A inhibition of cardiac KATP currents. Surprisingly, in contrast to pancreatic beta cells, application of both SUR2A NBF1 and NBF2 abolished Syn-1A effects to 138 ± 17.3% (n = 19) and 129.6 ± 34.5% (n = 9) of control, respectively (Fig. 3A, ii and iii, and B). This result suggests that not only NBF1 but also NBF2 of SUR2A mediates Syn-1A inhibition of cardiac KATP currents. As controls, neither SUR2A NBF1 nor SUR2A NBF2 alone produced any significant effect on current activities, which were 96.2 ± 18.0% and 110.2 ± 11.3% of control, respectively (Fig. 3B).



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  		FIG. 3.
Syn-1A inhibition of cardiac myocyte KATP currents is mediated by both SUR2A NBF1 and NBF2. A, original traces showing small patches of membrane excised from cardiac myocytes to form an inside-out configuration so that the internal face of membrane patches could be exposed directly to test solutions. Excised patches were initially held in 1 mM ATP to measure base-line current. Exposure to zero ATP resulted in a surge of channel activities confirming the ATP sensitivity of the channels. Subsequent application of 50 µM ATP prevented channel run-down and also produced a partial inhibition; the current at this level is defined as control for each patch. i–iii, application of Syn-1A (0.3 µM) with or without pre-incubation with SUR2A NBF1 (1 µM) or SUR2A NBF2 (1 µM). B, quantitative summary of results in A. After the addition of fusion proteins, the currents are expressed as a percentage of the current levels before the addition of fusion proteins. *, significantly different (p < 0.05) from control. Results are mean ± S.E. of 9–19 patches.

 
Both SNARE proteins Syn-1A and SNAP-25 have been shown to interact with Kv1.1 and Kv2.1 channels (15, 21–24). Because cardiac myocytes possess not only Syn-1A (Fig. 1) but also SNAP-25,2 it is possible that SNAP-25 may itself interact with cardiac KATP channel, or it may modulate Syn-1A inhibition of cardiac KATP currents. Therefore we co-expressed Kir6.2 and SUR2A in HEK293 cells (which have no detectable endogenous SNARE proteins) to examine the direct inhibition of cardiac KATP channels by Syn-1A. When Kir6.2/SUR2A-transfected cells were dialyzed with a low ATP concentration (0.3 mM) through the recording pipette, KATP currents gradually developed and reached maximum amplitude in 4–8 min. At this time, I-V relationships were obtained by a family of triggering pulses from -160 to 40 mV in 20-mV increments (Fig. 4, A and E). The I-V relationship is almost linear with the expected reversal potential of about -80 mV. When the cells were dialyzed with a high ATP concentration (5 mM), only very minute currents (0.1–0.2 nA at -140 mV) were elicited, showing the ATP sensitivity of the currents. Dialyzing Syn-1A into the cell caused significant inhibition at both hyperpolarized and depolarized potentials (Fig. 4, B and E). Consistent with the myocyte results, co-dialysis of Syn-1A with either SUR2A-NBF1 or -NBF2 prevented Syn-1A inhibition (Fig. 4, C–E). To investigate whether SUR2A NBF1 and NBF2 had any effect on the currents on their own, we dialyzed either SUR2A-NBF1 or -NBF2 into Kir6.2/SUR2A-transfected cells. Neither of these NBFs had a significant effect on KATP currents (Fig. 4F). Therefore, our results indicate that Syn-1A inhibits the cardiac KATP channel by binding to both NBFs of SUR2A.



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  		FIG. 4.
Syn-1A inhibition of KATP currents from HEK293 cells expressing Kir6.2/SUR2A is mediated by both SUR2A NBF1 and NBF2. A–D, original traces showing effects of Syn-1A (0.3 µM) and SUR2A NBFs (1 µM each) on KATP currents from HEK293 cells expressing Kir6.2/SUR2A. After KATP currents reached maximum amplitude, voltage pulses from -160 to 40-mV were given at 20 mV increments to yield the I-V relationship (see "Experimental Procedures"). E, steady-state currents were determined as the mean current in the final 95–99% of the 500-ms pulse and then normalized with cell membrane capacitance to give current densities. The normalized values are plotted against membrane potential to obtain the I-V curves. *, Syn-1A alone group is significantly different (p < 0.05) from other groups. Results are mean ± S.E. of 6–18 cells. F, I-V curves showing that SUR2A-NBF1 or -NBF2 alone (1 µM each) did not significantly affect KATP currents. pF, picofarad.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, we demonstrated that wild-type Syn-1A acted on the SUR1 subunit to close the KATP channel in cultured insulinoma cells and freshly isolated rat islet beta cells (16). Here, we show that Syn-1A can also act on cardiac myocyte SUR2A protein to close the KATP channel, even under low cytosolic ATP concentrations, indicating that Syn-1A could close the cardiac KATP channel independently of ATP. This suggests that Syn-1A may provide an inhibitory "braking" mechanism on the cardiac KATP channel isoform (SUR2A/Kir6.2). These findings therefore describe a novel regulatory pathway of KATP channel function, which has important implications in both physiological and pathophysiological conditions and which could be influenced by the dynamic changes of ATP and ADP concentrations occurring under these conditions. In support, our previous report showed that ADP could reduce the efficacy of Syn-1A inhibition of islet beta cell SUR1/KATP channels (16). In contrast, additional cellular factors, including phosphatidylinositol 4,5-bisphosphate (PIP2) (25) and acyl-CoA (26), act directly on the Kir6.2 subunit to activate KATP channels, providing a further level of fidelity to fine-tune channel activity and hence cardiac electrical excitability.

These actions of Syn-1A on the cardiac KATP channels were directly on the plasma membrane SUR2A protein because Syn-1A was able to bind the full-length SUR2A protein in SUR2A/Kir6.2-expressing HEK293 cells, which resulted in inhibition of KATP currents. Also, SUR2A is the dominant SUR2 protein in the heart plasma membrane (1, 3, 11, 12, 17) where we found Syn-1A to be also most abundant. We had previously shown that the Kir6.2 cytoplasmic N or C terminus did not bind Syn-1A or modify Syn-1A inhibition of KATP currents (16). In our previous report (16), we found that only recombinant NBF1 blocks the inhibitory action of Syn-1A on pancreatic beta cell KATP channels, whereas NBF2 does not, even though NBF2 binds with similar affinity to Syn-1A as NBF1. In contrast, in the present study, we used inside-out cardiac patches and found that both NBF1 and NBF2 of SUR2A were similarly effective in completely blocking Syn-1A inhibition of the plasma membrane cardiac KATP channels. These results are surprising because NBF2 is highly conserved between the SUR isoforms, whereas more heterogeneity exists at the NBF1 domains, N-terminal transmembrane, and distal C-terminal domain (3, 12). In fact, the distal 42-amino acid C-terminal portion of the SUR1 is distinct and has been shown to confer higher ATP sensitivity of SUR1 (4-fold higher) than SUR2A and SUR2B (27); it also conferred the distinct MgADP actions (28). It therefore seems that the Syn-1A binding domain of NBF2 of SUR2A and SUR1 is likely at the N-terminal portion of both NBFs and transduces the Syn-1A binding into channel modulation, whereas the distinct 42-amino acid C-terminal domain in SUR1-NBF2 seems to reduce the ability of Syn-1A in transducing this inhibitory action on KATP opening. Consistent with this thinking, the structural determinants within the NBF2s which confer adenine nucleotide binding were found to be distinct from those which transduce nucleotide binding into KATP channel activation (29). Previous studies have shown that NBF1 and NBF2 exist as heterodimers in the intact cells (30). Our findings demonstrate that Syn-1A binds both NBF1s and NBF2s of SUR2A and SUR1 (16) with similar affinity, therefore suggesting that an important action of Syn-1A might be to stabilize this NBF1/NBF2 complex and finely modulate its interactions. It should be noted that NBF2 is functionally distinct between the SUR1 and SUR2A isoforms (1, 2, 3, 12). Although NBF1 has a similar role in regulating gating kinetics of the channel complex, NBF2 in SUR1 renders the channel susceptible to the antagonistic stimulatory action of ADP. The ADP sensitivity in the SUR2A/Kir6.2 channel is markedly lower. The differing sensitivities between SUR1- and SUR2A-NBF2 to Syn-1A may contribute to these differences in nucleotide sensitivity.

Syn-1A is known to regulate exocytosis and several membrane ion channels involved in secretion. Less work has been done in examining the role of SNARE proteins in non-secretory cells. Here, we show that Syn-1A plays an important role in modulating membrane excitability of a muscle cell, the cardiac myocyte, through its action on the KATP channel. Consistently, we had previously shown that SNAP-25, another SNARE protein, could modulate voltage-gated K+ channels in gastrointestinal smooth muscles (23). Because NBF-1 and NBF-2 domains are highly homologous between SUR2A and SUR2B (1, 3, 12, 16), Syn-1A would be expected to interact similarly with the NBF-1 and NBF-2 of SUR2B, which regulate vascular smooth muscle KATP channels. In fact, the SUR2 immunoreactive proteins in the brain (this study) pulled down by Syn-1A are likely to contain more SUR2B than SUR2A, which in part are contributed by the vascular beds in the brain (11, 12). More work will have to be done to examine the specific actions of Syn-1A on vascular KATP (SUR2B) channel activity. Nonetheless, Syn-1A binding to both SUR1 (16) and SUR2 (this study) proteins indicate the importance of Syn-1A regulation of these KATP channels. Taken together, these studies also indicate that SNARE proteins, particularly Syn-1A, play an important role in coordinating the ion channel events in both secretory and non-secretory cells.

Syn-1A interactions with KATP channels are potentially important in the health and disease of the heart, pancreatic islet beta cell, vascular smooth muscle, and neurons (1). In insulin-secreting beta cells, we recently demonstrated that Syn-1A and other SNARE proteins are sequestered within plasma membrane lipid raft domains along with Ca2+ and Kv2.1 channel proteins, whereas SUR1/Kir6.2 KATP channel proteins are in non-lipid raft domains (31). Interestingly, cholesterol depletion shifted Syn-1A out of the lipid rafts into non-lipid raft domains, which had a profound effect on channel regulation and insulin exocytosis. It therefore appears that membrane compartmentalization of ion channels and SNARE proteins is critical for the temporal and spatial coordination of insulin release and also presumably for cardiac membrane excitability and contractility, which could be profoundly altered by lipid metabolism occurring in health and diabetes.

KATP channel regulation plays a central role in protecting the heart from ischemia/reperfusion injury (32) and calcium homeostasis during exercise (33). ATP levels decrease during exercise and ischemia, leading to opening of KATP channels, thereby shortening the action potential duration to reduce Ca2+ influx and myocardial contraction. This leads to energy conservation and an optimization of the contractile response (33). Increased local concentrations of Syn-1A may inhibit cardiac KATP channels and potentially offset pro-arrhythmic activity via excessive action potential shortening. Syn-1A-mediated inhibition may therefore act as a brake to oppose the potentially deleterious effects of sustained KATP channel activity during periods of metabolic compromise. However, this remains to be tested experimentally.


   FOOTNOTES
 
* This work was supported by grants from the Juvenile Diabetes Research Foundation Grant 1-2001-521 (to H. Y. G.), Canadian Institutes for Health Research (CIHR) Grants MOP-69083 and PPP-52103 (to H. Y. G.), Canadian Association of Gastroenterology Gastroenterology/Abbott Laboratories/CIHR Grant DOP-68570 (to H. Y. G.), and the Canadian Diabetes Association (to P. E. L.). 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. Back

§ These authors contributed equally to this work. Back

Supported by a fellowship from the Canadian Diabetes Association. Back

|| Supported by a fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). Back

** Supported by a New Investigator Award from the Heart and Stroke Foundation of Canada. Back

§§ AHFMR scholar and CIHR new investigator. Back

¶¶ To whom correspondence should be addressed: Rm. 7226, Medical Science Bldg., University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1526; Fax: 416-978-8765; E-mail: Herbert.gaisano{at}utoronto.ca.

1 The abbreviations used are: KATP channel, ATP-sensitive potassium channel; SUR, sulfonylurea receptor; NBF, nucleotide binding fold; Syn-1A, syntaxin-1A; GST, glutathione S-transferase; ADP, adenosine 5'-diphosphate; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; HEK, human embryonic kidney; SNAP-25, synaptic protein of 25 kDa. Back

2 R. G. Tsushima and H. Y. Gaisano, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Bob Norman for critical review of the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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