Huanli Xie

University of Toronto, Toronto, Ontario, Canada

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Publications (16)77.12 Total impact

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    ABSTRACT: 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.
    Journal of Molecular and Cellular Cardiology 07/2014; · 5.15 Impact Factor
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    ABSTRACT: In β-cells, Syntaxin (Syn)-1A interacts with SUR1 to inhibit KATP channels. PIP2 binds Kir6.2 subunit to open KATP channels. PIP2 also modifies Syn-1A clustering in plasma membrane (PM) which may alter Syn-1A actions on PM proteins like SUR1. Here, we assessed whether PIP2's actions on activating KATP channels is contributed by sequestering Syn-1A from binding SUR1. In vitro binding showed PIP2 dose-dependently disrupted Syn-1A-SUR1 complexes, corroborated by 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 β-cells, INS-1 and SUR1/Kir6.2-expressing HEK293 cells showed PIP2 dose-dependent activation of KATP currents were 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 physiologically low PIP2 concentration that did not disrupt Syn-1A-SUR1 complex, compared to 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 β-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.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
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    ABSTRACT: RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane 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 channels. RalA knockdown in INS-1 cells and primary rat β-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Ca(v) 1.2 and Ca(v) 1.3) and R-type (Ca(v) 2.3) Ca(2+) channels. Restoration of RalA expression in RalA knockdown cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Ca(v) α(2) δ-1 auxiliary subunit known to bind the three Ca(v) s. Moreover, the functional molecular interactions between Ca(v) α(2) δ-1 and RalA on the plasma membrane shown by TIRF microscopy/FRET analysis could be induced by glucose stimulation. Knockdown of RalA inhibited trafficking of α(2) δ-1 to insulin granules without affecting the localization of the other Ca(v) subunits. Furthermore, we confirmed that RalA and α(2) δ-1 functionally interact since RalA knockdown-induced inhibition of Ca(v) currents could not be recovered by RalA when α(2) δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α(2) δ-1 on insulin granules to tether these granules to plasma membrane 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.
    Traffic 01/2013; · 4.65 Impact Factor
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    ABSTRACT: 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.
    Journal of Molecular and Cellular Cardiology 08/2011; 51(5):790-802. · 5.15 Impact Factor
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    ABSTRACT: The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-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 β-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.
    Journal of Biological Chemistry 05/2011; 286(26):23308-18. · 4.65 Impact Factor
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    ABSTRACT: 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 β-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 β-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 β-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 β-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.
    Journal of Biological Chemistry 02/2011; 286(7):5876-83. · 4.65 Impact Factor
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    ABSTRACT: 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. 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. 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.
    Cardiovascular Research 09/2008; 80(3):365-74. · 5.81 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Pancreas 02/2008; 36(1):10-7. · 2.95 Impact Factor
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    ABSTRACT: 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.
    Diabetes 09/2007; 56(8):2124-34. · 7.90 Impact Factor
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    ABSTRACT: SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier K(+) channels, K(v)1.1 and K(v)2.1, in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/E166A) inhibits K(v)2.1 channels more optimally than wild-type Syn-1A. In this report we examined whether Syn-1A in its wild-type and open conformations would exhibit similar differential actions on the gating of K(v)1.2, a major delayed rectifier K(+) channel in nonsecretory smooth muscle cells and some neuronal tissues. In coexpression and acute dialysis studies, wild-type Syn-1A inhibited K(v)1.2 current magnitude. Of interest, wild-type Syn-1A caused a right shift in the activation curves of K(v)1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on K(v)2.1 (steady-state availability reduction without changes in voltage dependence of activation). Also, although both wild-type and open-form Syn-1A bound equally well to K(v)1.2 in an expression system, open-form Syn-1A failed to reduce K(v)1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open-form Syn-1A on K(v)2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in nonsecretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different K(v) channels by Syn-1A and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and nonsecretory (smooth muscle) cell types.
    AJP Gastrointestinal and Liver Physiology 06/2007; 292(5):G1233-42. · 3.65 Impact Factor
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    ABSTRACT: During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of K(ATP) channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125-47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac K(ATP) channels. K(ATP) channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-deltaC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn-1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the K(ATP) channels, which may serve as a "brake" to temper the fluctuation of low pH-induced K(ATP) channel opening that could induce fatal reentrant arrhythmias.
    Journal of Biological Chemistry 07/2006; 281(28):19019-28. · 4.65 Impact Factor
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    ABSTRACT: 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.
    Biochemical Journal 06/2006; 396(2):363-9. · 4.65 Impact Factor
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    ABSTRACT: 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.
    Biochemical Journal 05/2005; 387(Pt 1):195-202. · 4.65 Impact Factor
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    ABSTRACT: The ATP-sensitive potassium (K(ATP)) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the K(ATP) channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K(+) and Ca(2+) channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K(ATP) channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell K(ATP) channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the K(ATP) channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the K(ATP) current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis.
    Journal of Biological Chemistry 01/2005; 279(51):53259-65. · 4.65 Impact Factor
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    ABSTRACT: 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 K(ATP) 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.
    Journal of Biological Chemistry 12/2004; 279(45):47125-31. · 4.65 Impact Factor
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    ABSTRACT: Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.
    Journal of Biological Chemistry 06/2003; 278(19):17532-8. · 4.65 Impact Factor