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Syntaxin-1A Inhibits Cardiac KATP Channels by Its Actions on Nucleotide Binding Folds 1 and 2 of Sulfonylurea Receptor 2A

2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês

10.1074/jbc.m404954200

1083-351X

Youhou Kang, Yuk‐Man Leung, Jocelyn Manning-Fox, Fuzhen Xia, Huanli Xie, Laura Sheu, Robert G. Tsushima, Peter E. Light, Herbert Y. Gaisano,

Ion channel regulation and function

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. 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. ATP-sensitive potassium (KATP) 1The 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.1The 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. 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) (1Seino S. J. Diabetes Complicat. 2003; 17: 2-5Crossref PubMed Scopus (29) Google Scholar, 2Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. 1998; 274: C25-C37Crossref PubMed Google Scholar). 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 (4Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1607) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar, 6Clement J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 7Shyng S. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (418) Google Scholar). 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) (8Higgins C.F. Cell. 1995; 82: 693-696Abstract Full Text PDF PubMed Scopus (340) Google Scholar). 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 (4Inagaki N. Gonoi T. Clement J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1607) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar, 9Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1278) Google Scholar, 10Sakura H. Ammala C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Crossref PubMed Scopus (402) Google Scholar, 11Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). 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 (1Seino S. J. Diabetes Complicat. 2003; 17: 2-5Crossref PubMed Scopus (29) Google Scholar, 2Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. 1998; 274: C25-C37Crossref PubMed Google Scholar, 12Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (453) Google Scholar). However, these studies do not fully explain the distinct regulatory functions of the NBFs of each SUR protein on the KATP channel (1Seino S. J. Diabetes Complicat. 2003; 17: 2-5Crossref PubMed Scopus (29) Google Scholar, 2Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. 1998; 274: C25-C37Crossref PubMed Google Scholar, 12Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (453) Google Scholar).Syntaxin-1A (Syn-1A), a SNARE protein known to mediate exocytosis (13Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1760) Google Scholar), also directly binds to voltage-gated K+ and Ca2+ channels to regulate secretion (14Leung Y.M. Kang Y. Gao X. Xia F. Xie H. Sheu L. Tsuk S. Lotan I. Tsushima R.G. Gaisano H.Y. J. Biol. Chem. 2003; 278: 17532-17538Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 15Wiser O. Bennett M.K. Atlas D. EMBO J. 1996; 15: 4100-4110Crossref PubMed Scopus (232) Google Scholar). 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 (16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In fact, Syn-1A was reported to be present in cardiac myocytes (17Sevilla L. Tomas E. Munoz P. Guma A. Fischer Y. Thomas J. Ruiz-Montasell B. Testar X. Palacin M. Blasi J. Zorzano A. Endocrinology. 1997; 138: 3006-3015Crossref PubMed Google Scholar). 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 PROCEDURESConstructs 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 (16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 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 2000™ (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. (18Mayanil C.S. Richardson R.M. Hosey M.M. Mol. Pharmacol. 1991; 40: 900-907PubMed Google Scholar) and modified by Barry et al. (19Barry D.M. Trimmer J.S. Merlie J.P. Nerbonne J.M. Circ. Res. 1995; 77: 361-369Crossref PubMed Google Scholar). To prepare rat brain membranes, we followed the method reported previously by Huttner et al. (20Huttner W.B. Schiebler W. Greengard P. De Camilli P. J. Cell Biol. 1983; 96: 1374-1388Crossref PubMed Scopus (884) Google Scholar). Finally, both membranes were solubilized in 1× 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 × 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 × 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.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 (16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 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.RESULTSSyntaxin-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.Fig. 1Syntaxin-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.View Large Image Figure ViewerDownload (PPT)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 (1Seino S. J. Diabetes Complicat. 2003; 17: 2-5Crossref PubMed Scopus (29) Google Scholar, 5Inagaki N. Gonoi T. Clement J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar, 11Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 12Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (453) Google Scholar), 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 (11Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 12Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (453) Google Scholar) in Fig. 2A. Furthermore, the molecular mass of SUR2A and SUR2B differs by just 1 kDa (3Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. 1998; 274: C25-C37Crossref PubMed Google Scholar, 12Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (453) Google Scholar, 16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) 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 (1Seino S. J. Diabetes Complicat. 2003; 17: 2-5Crossref PubMed Scopus (29) Google Scholar, 3Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. 1998; 274: C25-C37Crossref PubMed Google Scholar, 11Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 12Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (453) Google Scholar), 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 (16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Our previous study with pancreatic islet beta cells shows that Syn-1A specifically binds to NBF1 and NBF2 domains of SUR1 (16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 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 (16Pasyk E.A. Kang Y. Huang X. Cui N. Sheu L. Gaisano H.Y. J. Biol. Chem. 2004; 279: 4234-4240Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). 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).Fig. 3Syn-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.View Large Image Figure ViewerDownload (PPT)Both SNARE proteins Syn-1A and SNAP-25 have been shown to interact with Kv1.1 and Kv2.1 channels (15Wiser O. Bennett M.K. Atlas D. EMBO J. 1996; 15: 4100-4110Crossref PubMed Scopus (232) Google Scholar, 21MacDonald P.E. Wang G. Tsuk S. Dodo C. Kang Y. Tang L. Wheeler M.B. Cattral M.S. Lakey J.R. Salapatek A.M. Lotan I. Gaisano H.Y. Mol. Endocrinol. 2002; 16: 2452-2461Crossref PubMed Scopus (70) Google Scholar, 22Ji J. Tsuk S. Salapatek A.M. Huang X. Chikvashvili D. Pasyk E.A. Kang Y. Sheu L. Tsushima R. Diamant N. Trimble W.S. Lotan I. Gaisano H.Y. J. Biol. Chem. 2002; 277: 20195-20204Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 23Ji J. Salapatek A.M. Lau H. Wang G. Gaisano H.Y. Diamant N.E. Gastroenterology. 2002; 122: 994-1006Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 24Fili O. Michaelevski I. Bledi Y. Chikvashvili D. Singer-Lahat D. Boshwitz H. Linial M. Lotan I. J. Neurosci. 2001; 21: 1964-1974Crossref PubMed Google Scholar). Because cardiac myocytes possess not only Syn-1A (Fig. 1) but also SNAP-25, 2R. G. Tsushima and H. Y. Gaisano, unpublished observation. 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 r