RESUMEN
The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic ß cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the ß-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Qa-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Canales de Potasio Shab/metabolismo , Animales , Membrana Celular/metabolismo , Exocitosis/fisiología , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Microscopía Fluorescente , Unión Proteica , Dominios Proteicos , Proteínas Qa-SNARE/química , Ratas , Ratas Wistar , Proteínas SNARE/metabolismo , Canales de Potasio Shab/fisiologíaRESUMEN
Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/-) and human pancreas (lenti-Munc18c-shRNA-treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/-) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/- mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18c's role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.
Asunto(s)
Ceruletida/efectos adversos , Proteínas Munc18/metabolismo , Pancreatitis/metabolismo , Anciano , Animales , Ceruletida/metabolismo , Colecistoquinina/efectos adversos , Colecistoquinina/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Exocitosis , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas Munc18/genética , Páncreas/metabolismo , Pancreatitis/genética , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
BACKGROUND & AIMS: Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. METHODS: We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-µm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. RESULTS: Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. CONCLUSIONS: In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.
Asunto(s)
Autofagia/genética , Exocitosis/genética , Páncreas/citología , Pancreatitis/genética , Sintaxina 1/metabolismo , Células Acinares/metabolismo , Animales , Membrana Celular/metabolismo , Ceruletida , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirugía , Pancreatitis/inducido químicamente , Vesículas Secretoras/fisiología , Tripsinógeno/metabolismoRESUMEN
RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo R/metabolismo , Insulina/metabolismo , Subunidades de Proteína/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo R/genética , Membrana Celular/metabolismo , Exocitosis , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Células Secretoras de Insulina/metabolismo , Unión Proteica , Subunidades de Proteína/genética , Transporte de Proteínas , ARN Interferente Pequeño , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS/HYPOTHESIS: Of the four exocytotic syntaxins (Syns), much is now known about the role of Syn-1A (pre-docked secretory granules [SGs]) and Syn-3 (newcomer SGs) in insulin exocytosis. Some work was reported on Syn-4's role in biphasic glucose-stimulated insulin secretion (GSIS), but its precise role in insulin SG exocytosis remains unclear. In this paper we examine this role in human beta cells. METHODS: Endogenous function of Syn-4 in human islets was assessed by knocking down its expression with lentiviral single hairpin RNA (lenti-shRNA)-RFP. Biphasic GSIS was determined by islet perifusion assay. Single-cell analysis of exocytosis of red fluorescent protein (RFP)-positive beta cells (exhibiting near-total depletion of Syn-4) was by patch clamp capacitance measurements (Cm) and total internal reflection fluorescence microscopy (TIRFM), the latter to further assess single SG behaviour. Co-immunoprecipitations were conducted on INS-1 cells to assess exocytotic complexes. RESULTS: Syn-4 knockdown (KD) of 77% in human islets caused a concomitant reduction in cognate Munc18c expression (46%) without affecting expression of other exocytotic proteins; this resulted in reduction of GSIS in the first phase (by 42%) and the second phase (by 40%). Cm of RFP-tagged Syn-4-KD beta cells showed severe inhibition in the readily releasable pool (by 71%) and mobilisation from reserve pools (by 63%). TIRFM showed that Syn-4-KD-induced inhibition of first-phase GSIS was attributed to reduction in exocytosis of both pre-docked and newcomer SGs (which undergo minimal residence or docking time at the plasma membrane before fusion). Second-phase inhibition was attributed to reduction in newcomer SGs. Stx-4 co-immunoprecipitated Munc18c, VAMP2 and VAMP8, suggesting that these exocytotic complexes may be involved in exocytosis of pre-docked and newcomer SGs. CONCLUSIONS/INTERPRETATION: Syn-4 is involved in distinct molecular machineries that influence exocytosis of both pre-docked and newcomer SGs in a manner functionally redundant to Syn-1A and Syn-3, respectively; this underlies Syn-4's role in mediating portions of first-phase and second-phase GSIS.
Asunto(s)
Insulinas Bifásicas/sangre , Exocitosis , Células Secretoras de Insulina/citología , Insulina/metabolismo , Proteínas Qa-SNARE/metabolismo , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Humanos , Secreción de Insulina , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Proteínas Munc18/metabolismo , Técnicas de Placa-Clamp , Proteínas R-SNARE/metabolismo , ARN Interferente Pequeño/metabolismo , Análisis de la Célula Individual , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Proteína Fluorescente RojaRESUMEN
In ß-cells, syntaxin (Syn)-1A interacts with SUR1 to inhibit ATP-sensitive potassium channels (KATP channels). PIP2 binds the Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) that may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether the actions of PIP2 on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed that PIP2 dose-dependently disrupted Syn-1A·SUR1 complexes, corroborated by an in vivo Forster resonance energy transfer assay showing disruption of SUR1(-EGFP)/Syn-1A(-mCherry) interaction along with increased Syn-1A cluster formation. Electrophysiological studies of rat ß-cells, INS-1, and SUR1/Kir6.2-expressing HEK293 cells showed that PIP2 dose-dependent activation of KATP currents was uniformly reduced by Syn-1A. To unequivocally distinguish between PIP2 actions on Syn-1A and Kir6.2, we employed several strategies. First, we showed that PIP2-insensitive Syn-1A-5RK/A mutant complex with SUR1 could not be disrupted by PIP2, consequently reducing PIP2 activation of KATP channels. Next, Syn-1A·SUR1 complex modulation of KATP channels could be observed at a physiologically low PIP2 concentration that did not disrupt the Syn-1A·SUR1 complex, compared with higher PIP2 concentrations acting directly on Kir6.2. These effects were specific to PIP2 and not observed with physiologic concentrations of other phospholipids. Finally, depleting endogenous PIP2 with polyphosphoinositide phosphatase synaptojanin-1, known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR1, causing inhibition of KATP channels that could no longer be further inhibited by exogenous Syn-1A. These results taken together indicate that PIP2 affects islet ß-cell KATP channels not only by its actions on Kir6.2 but also by sequestering Syn-1A to modulate Syn-1A availability and its interactions with SUR1 on PM.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Sulfonilureas/metabolismo , Sintaxina 1/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Células Secretoras de Insulina/citología , Masculino , Mutación , Fosfatidilinositol 4,5-Difosfato/genética , Canales de Potasio de Rectificación Interna/genética , Ratas , Ratas Wistar , Receptores de Sulfonilureas/genética , Sintaxina 1/genéticaRESUMEN
Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cells showed that increasing endogenous or exogenous PIP2 diminished the inhibitory effect of Syn-1A on KATP currents. The physiological relevance of these findings was confirmed by ability of exogenous PIP2 to block exogenous Syn-1A inhibition of cardiac KATP currents in inside-out patches of mouse ventricular myocytes. The effect of PIP2 on physical and functional interactions between Syn-1A and KATP channels is specific and not observed with physiologic concentrations of other phospholipids. To unequivocally demonstrate the specificity of PIP2 interaction with Syn-1A and its impact on KATP channel modulation by Syn-1A, we employed a PIP2-insensitive Syn-1A-5RK/A mutant. The Syn-1A-5RK/A mutant retains the ability to interact with SUR2A in both in vitro binding and in vivo FRET assays, although as expected the interaction is no longer disrupted by PIP2. Interestingly, at physiological PIP2 concentrations, Syn-1A-5RK/A inhibited KATP currents to a greater extent than Syn-1A-WT, indicating that the inhibitory effect of Syn-1A on KATP channels is not due to direct competition between Syn-1A and Kir6.2 for PIP2 binding. At high-dose PIP2, however, inhibition of KATP currents by Syn-1A-5RK/A was greatly reduced, likely overridden by the direct activating effect of PIP2 on KATP channels. Finally, depleting endogenous PIP2 with polyphosphoinositide phosphatase synaptojanin-1 known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR2A, causing optimal inhibition of KATP channels. These results taken together led us to conclude that PIP2 affects cardiac KATP channels not only by its actions on the channel directly but also by multi-modal effects of dynamically modulating Syn-1A mobility from Syn-1A clusters and thereby the availability of Syn-1A to inhibit KATP channels via interaction with SUR2A on the plasma membrane.
Asunto(s)
Canales KATP/metabolismo , Miocardio/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacología , Receptores de Sulfonilureas/metabolismo , Sintaxina 1/metabolismo , Animales , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Ratas , Sintaxina 1/químicaRESUMEN
The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic ß-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat ß-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Células Secretoras de Insulina/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Pliegue de Proteína , Receptores de Droga/metabolismo , Sintaxina 1/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células HEK293 , Humanos , Células Secretoras de Insulina/citología , Masculino , Canales de Potasio de Rectificación Interna/genética , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Receptores de Droga/genética , Receptores de Sulfonilureas , Sintaxina 1/genéticaRESUMEN
ATP-sensitive potassium (K(ATP)) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg(2+), phospholipids, and pH). We previously reported that K(ATP) channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K(ATP) channel activity in pancreatic islet ß-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg(2+) causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic ß-cells demonstrated that reduced ATP concentrations increased K(ATP) channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates K(ATP) channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit ß-cell K(ATP) channel gating, they could also influence the sensitivity of K(ATP) channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic ß-cell K(ATP) channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Células Secretoras de Insulina/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Sintaxina 1/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/genética , Animales , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Ratones , Canales de Potasio de Rectificación Interna/genética , Unión Proteica , Ratas , Ratas Sprague-Dawley , Receptores de Droga/genética , Receptores de Sulfonilureas , Sintaxina 1/genéticaRESUMEN
Enhancing pancreatic ß-cell secretion is a primary therapeutic target for type-2 diabetes (T2D). Syntaxin-2 (Stx2) has just been identified to be an inhibitory SNARE for insulin granule exocytosis, holding potential as a treatment for T2D, yet its molecular underpinnings remain unclear. We show that excessive Stx2 recruitment to raft-like granule docking sites at higher binding affinity than pro-fusion syntaxin-1A effectively competes for and inhibits fusogenic SNARE machineries. Depletion of Stx2 in human ß-cells improves insulin secretion by enhancing trans-SNARE complex assembly and cis-SNARE disassembly. Using a genetically-encoded reporter, glucose stimulation is shown to induce Stx2 flipping across the plasma membrane, which relieves its suppression of cytoplasmic fusogenic SNARE complexes to promote insulin secretion. Targeting the flipping efficiency of Stx2 profoundly modulates secretion, which could restore the impaired insulin secretion in diabetes. Here, we show that Stx2 acts to assist this precise tuning of insulin secretion in ß-cells, including in diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Insulina , Humanos , Sintaxina 1/genética , Sintaxina 1/metabolismo , Insulina/metabolismo , Exocitosis/fisiología , Proteínas SNARE/metabolismo , Membrana Celular/metabolismoRESUMEN
We previously demonstrated that syntaxin (Syn)-1A is present in the sarcolemma of rat cardiomyocytes and binds sulfonylurea receptor (SUR) 2A nucleotide binding folds (NBFs) to inhibit ATP-sensitive potassium (K(ATP)) channel. Here, we examined for the precise domains within the NBFs of SUR2A that may interact with Syn-1A. Specifically, we tested truncated NBF protein segments encompassing the conserved motifs Walker A (W(A)), signature/Linker (L), and Walker B (W(B)). In vitro binding results indicate that the domains encompassing W(A) and L of NBF-1 and all three conserved motifs of NBF-2 bound Syn-1A. Electrophysiological studies, employing inside-out patch-clamp recordings from SUR2A/Kir6.2 expressing HEK cells and mouse cardiomyocytes, show that W(B) and L of NBF-1 and all three NBF-2 truncated protein segments reduced Syn-1A inhibition of SUR2A/K(ATP) channels. Remarkably, these same NBF-1 and -2 truncated proteins could independently disrupt the intimate FRET interactions of full length SUR2A (-mCherry) and Syn-1A (-EGFP). These results taken together indicate that Syn-1A possibly maintains inhibition of cardiac ventricular K(ATP) channels by binding to large regions of NBF-1 and NBF-2 to stabilize the NBF-1-NBF-2 heterodimer formation and prevent ATP-binding and ATP hydrolysis. Since K(ATP) channels are closely coupled to metabolic states, we postulate that these very intimate Syn-1A-SUR2A interactions are critically important for myocardial protection during stress, in which profound changes in metabolic factors (pH, ATP) could modulate these Syn-1A-SUR2A interactions.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Canales KATP/metabolismo , Miocitos Cardíacos/metabolismo , Oligopéptidos/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Droga/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Sintaxina 1/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/biosíntesis , Secuencias de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/metabolismo , Secuencia Conservada , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Canales KATP/química , Canales KATP/genética , Masculino , Ratones , Microscopía Fluorescente , Miocitos Cardíacos/citología , Oligopéptidos/química , Oligopéptidos/genética , Técnicas de Placa-Clamp , Plásmidos , Potasio/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Droga/química , Receptores de Droga/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Receptores de Sulfonilureas , Sintaxina 1/química , Sintaxina 1/genéticaRESUMEN
Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKß-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKß inhibitors, and confirmed by the inability of IKBKB/IKKß phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKß's involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.Abbreviations: AL: autolysosome; AP: acute pancreatitis; AV: autophagic vacuole; CCK: cholecystokinin; IKBKB/IKKß: inhibitor of nuclear factor kappa B kinase subunit beta; SNAP23: synaptosome associated protein 23; SNARE: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; STX: syntaxin; TAP: trypsinogen activation peptide; VAMP: vesicle associated membrane protein; ZG: zymogen granule.
Asunto(s)
Pancreatitis , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Enfermedad Aguda , Animales , Autofagia , Exocitosis , Humanos , Lisosomas , Páncreas , Pancreatitis/genética , Pancreatitis/prevención & control , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Ratas , Tripsina/farmacología , Proteínas de Transporte VesicularRESUMEN
Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.
Asunto(s)
Activación del Canal Iónico , Estructura Terciaria de Proteína , Canales de Potasio Shab/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Tetraetilamonio/farmacología , Regulación Alostérica , Animales , Antidiscinéticos/metabolismo , Antidiscinéticos/farmacología , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/farmacología , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Péptidos/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sensibilidad y Especificidad , Canales de Potasio Shab/genética , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/farmacología , Tetraetilamonio/metabolismo , TransfecciónRESUMEN
Of the four syntaxins specialized for exocytosis, syntaxin (Syn)-2 is the least understood. In this study, we used Syn-2/epimorphin knockout mice to examine the role of Syn-2 in insulin secretory granule (SG) exocytosis. Unexpectedly, Syn-2 knockout mice exhibited paradoxical superior glucose homeostasis resulting from an enhanced insulin secretion. This was confirmed in vitro by pancreatic islet perifusion showing an amplified biphasic glucose-stimulated insulin secretion arising from an increase in size of the readily releasable pool of insulin SGs and enhanced SG pool refilling. The increase in insulin exocytosis was attributed mainly to an enhanced recruitment of the larger pool of newcomer SGs that undergoes no residence time on plasma membrane before fusion and, to a lesser extent, also the predocked SGs. Consistently, Syn-2 depletion resulted in a stimulation-induced increase in abundance of exocytotic complexes we previously demonstrated as mediating the fusion of newcomer SGs (Syn-3/VAMP8/SNAP25/Munc18b) and predocked SGs (Syn-1A/VAMP2/SNAP25/Muncn18a). This work is the first to show in mammals that Syn-2 could function as an inhibitory SNARE protein that, when relieved, could promote exocytosis in pancreatic islet ß-cells. Thus, Syn-2 may serve as a potential target to treat diabetes.
Asunto(s)
Exocitosis/genética , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas SNARE/metabolismo , Sintaxina 1/genética , Animales , Western Blotting , Inmunoprecipitación , Secreción de Insulina , Ratones , Ratones Noqueados , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Fracciones Subcelulares , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.
Asunto(s)
Antígenos de Superficie/química , Antígenos de Superficie/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Antígenos de Superficie/biosíntesis , Canales de Potasio de Tipo Rectificador Tardío , Humanos , Activación del Canal Iónico/fisiología , Riñón/química , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Técnicas de Placa-Clamp/métodos , Canales de Potasio con Entrada de Voltaje/biosíntesis , Canales de Potasio con Entrada de Voltaje/genética , Estructura Cuaternaria de Proteína/fisiología , Canales de Potasio Shab , Sintaxina 1 , Transfección/métodosRESUMEN
Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Cav). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Cav opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet ß-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Cavα1 pore-forming subunits of R-type Cav2.3 and to lesser extent L-type Cavs, while confirming the preferential interactions between Syn-1A with L-type (Cav1.2, Cav1.3) Cavs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Cav2.3, whereas Syn-1A prefers L-type Cavs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Cav channels. Syn-3 KD enhanced Ca2+ currents by 46% attributed mostly to R- and L-type Cavs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Cav activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Cav activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Cavs, but preferring Cav2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo R/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Qa-SNARE/metabolismo , Línea Celular , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Sintaxina 1/química , Sintaxina 1/metabolismoRESUMEN
Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic ß-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in ß-cells remain unknown. We show that Syt-7 is abundant in human ß-cells. Adenovirus-short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca(2+)-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human ß-cells than its previously purported role in exocytotic fusion per se.
Asunto(s)
Exocitosis/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretoras/metabolismo , Sinaptotagminas/metabolismo , Adulto , Anciano , Animales , Calcio/metabolismo , Exocitosis/efectos de los fármacos , Femenino , Péptido 1 Similar al Glucagón/farmacología , Glucosa/farmacología , Humanos , Imidazoles/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proteínas Qa-SNARE/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas SNARE/metabolismoRESUMEN
AIMS: Syntaxin (Syn)-1A binds sulfonylurea receptor (SUR) nucleotide binding folds of cardiac myocyte (SUR2A) and islet beta-cells (SUR1) to inhibit ATP-sensitive potassium (K(ATP)) channels. We further reported that Syn-1A reduced the potency and efficacy of beta-cell-specific K(ATP) channel openers (KCOs). Here, we examined whether Syn-1A would influence non-specific (diazoxide) and SUR2-specific KCOs [N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P-1075) and cromakalim] on cardiac myocyte K(ATP) channels activation. METHODS AND RESULTS: Confocal microscopy and Western blotting verified the presence of both Syn-1A and -1B expressions on rodent cardiac ventricular myocytes. Inside-out patch-clamp electrophysiology was utilized to examine the effects of these syntaxins on K(ATP) macroscopic currents activated by various KCOs from a stable cell line expressing the potassium inward rectifier 6.2 (Kir6.2)/SUR2A and from C57BL/6 male mouse ventricular myocytes. Syn-1A inhibited the current amplitude activated by P-1075, cromakalim and diazoxide via its H3 but not Habc domain. Syn-1B exhibited similar inhibitory effects on P-1075 activation of K(ATP) currents. In examining for direct effects of Syn-1A on the KCO binding to cardiac SUR2 receptors, we found that Syn-1A did not directly affect [(3)H]-P-1075 binding to rat cardiac membrane SUR2A at maximum binding capacity, but was able to mildly reduce the affinity of cold P-1075 and cromakalim to displace [(3)H]-P-1075 binding. CONCLUSION: In conclusion, Syn-1A (and Syn-1B) could inhibit K(ATP) currents activated by SUR2A-acting KCOs. Potential fluctuations in the levels of these syntaxins in the myocardium may affect the therapeutic effectiveness of cardiac KCOs.
Asunto(s)
Cromakalim/antagonistas & inhibidores , Diazóxido/antagonistas & inhibidores , Guanidinas/antagonistas & inhibidores , Canales KATP/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Piridinas/antagonistas & inhibidores , Sintaxina 1/farmacología , Vasodilatadores/antagonistas & inhibidores , Animales , Línea Celular , Células Cultivadas , Cromakalim/farmacología , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Guanidinas/farmacología , Humanos , Canales KATP/metabolismo , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Canales de Potasio de Rectificación Interna/metabolismo , Piridinas/farmacología , Sintaxina 1/metabolismo , Vasodilatadores/farmacologíaRESUMEN
OBJECTIVES: Synaptosome-associated protein of 25 kd (SNAP-25) regulates pancreatic islet beta-cell-delayed rectifier K channels (Kv2.1) in addition to insulin exocytosis. Botulinum neurotoxin A (BoNT/A) and E (BoNT/E) cleavage and presumed deletion of SNAP-25 have been used to examine SNAP-25 function. We hypothesized that proteolytic products of SNAP-25 (206 amino acids) resulting from BoNT/A and BoNT/E cleavage, SNAP-25(1-197) and SNAP-25(1-180), have independent actions on beta-cell Kv gating. METHODS: We examined by confocal microscopy and immunoblotting BoNT/A and BoNT/E cleavage of SNAP-25 to these N-terminal fragments, and the consequent effects of these BoNTs and SNAP-25 fragments on Kv currents in rat beta cells and MIN6 cells by patch clamp electrophysiology. RESULTS: Confocal microscopy and immunoblotting showed that MIN6 cells transfected with BoNT/A or BoNT/E generated SNAP-25(1-197) and SNAP-25(1-180) fragments that were retained in the cytosol. Both BoNTs caused increased rate of channel activation and slowed channel inactivation, mimicked by these SNAP-25 fragments, but not full-length SNAP-25. These SNAP-25 fragments potentiated tetraethylammonium block of beta-cell Kv currents. CONCLUSIONS: BoNT/A or BoNT/E treatment of beta cells generates N-terminal SNAP-25 fragments that are retained in beta cells to directly influence Kv channel gating in a manner distinct from full-length SNAP-25, contributing to overall actions of these BoNTs on insulin secretion.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas/metabolismo , Células Secretoras de Insulina/química , Fragmentos de Péptidos/farmacología , Canales de Potasio Shab/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismo , Animales , Western Blotting , Línea Celular , Glutatión Transferasa/genética , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/fisiología , Activación del Canal Iónico/efectos de los fármacos , Microscopía Confocal , Técnicas de Placa-Clamp , Fragmentos de Péptidos/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Canales de Potasio Shab/fisiología , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/farmacología , TransfecciónRESUMEN
Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) 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 beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) 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 beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) 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 K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) 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 K(ATP) channel openers.