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TRPC3-Calcineurin Microdomains Govern Orai1 Signaling in Mast Cells
610a
Wednesday, March 2, 2016
flexible architecture of this porin not only successfully binds protein targets, but also provides unique detection. Our engineered OmpG sensor is sensitive enough to provide, for the first time, the ability to resolve homologous protein analytes. The ability of our biosensor to discriminate between homologous proteins reveals the important need in better understanding the underlying mechanism of OmpG gating. This understanding will allow us to further engineer OmpG and tailor it for sensing an array of protein analytes. In addition, this knowledge will aid in a deeper understanding of the biology of this membrane protein itself. In this work, we highlight the pH-dependent gating mechanism of OmpG by investigating repulsive forces of OmpG’s extracellular loops. 3011-Pos Board B388 Characterization of Transmembrane Synthetic Chloride Ion Transporters Ahmed Fuwad. Inha University, Incheon, Korea, Republic of. Homeostasis of cells is essential for sustainable biological processes like proliferation, signaling, and differentiation. Among the most abundant anions in a biological system, chloride plays a vital role in maintaining homeostasis via chloride ion channels. Malfunctioning of the ion transporters may leads to diseases like cystic fibrosis, barrter syndrome, and osteoporosis. Thus the development of synthetic ion channels for replacing the malfunctioning ion channel may be one potential remedy for diseases. In this work we synthesized anion transporters by introducing different functional groups to make them Cl selective. Chloride efflux across the membrane that bears the synthetic ion channels was measured by a model membrane in a form of vesicles under controlled composition of intra and extravesicular solutions. We further verify ion transports across the membrane using Axopatch amplifier and concluded the antiporter ability of the synthetic ion channels. Toxicity of these compounds was also measured by using live/dead cell assay found no evidence of Toxicity. Considering the aforementioned characteristics and efflux potency they can be employed for treatment of different biological diseases together with membrane based sensors and separation technologies. Our future approach includes highly selective channels for further applications.
TRP Channels II 3012-Pos Board B389 Store-Operated Ca2D Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat b-Cells Jessica Sabourin1, Loı¨c Le Gal2, Jacques-Antoine Haefliger3, Eric Raddatz4, Florent Allagnat3. 1 Faculty of Pharmacy, Inserm UMR-S1180, Chatenay-Malabry, France, 2 Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 3Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 4Department of Physiology, University of Lausanne, Lausanne, Switzerland. Store-Operated Ca2þ Channels (SOCs) are voltage-independent Ca2þ channels activated upon depletion of the endoplasmic reticulum (ER) Ca2þ stores. Early studies suggest the contribution of such channels to Ca2þ homeostasis in insulin-secreting pancreatic b-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remain unclear. In this study, ER Ca2þ depletion triggered by acetylcholine (ACh) or thapsigargin (Tg) stimulated the formation of a ternary complex composed of Orai1, TRPC1 and STIM1, the key proteins involved in the formation of SOCs. Ca2þ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to Tg or ACh. Pharmacological SOCs inhibition or dominant-negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D or TRPC1F562A impaired GSIS in rat b-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant-negative blockade of TRPC3 had no effect on extracellular Ca2þ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1 and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process which may be impaired in type 2 diabetes. 3013-Pos Board B390 TRPC3-Calcineurin Microdomains Govern Orai1 Signaling in Mast Cells Michael Poteser1, Bernadett Bacsa1, Oleksandra Tiapko1, Michaela Lichtenegger1, Irene Frischauf2, Christoph Romanin2, Klaus Groschner1. 1 Institute of Biophysics, Medical University of Graz, Graz, Austria, 2Institute of Biophysics, University of Linz, Linz, Austria.
Molecular communication between TRPC and Orai channel proteins has been proposed and is still a matter of debate. We investigated functional crosstalk of TRPC3 and Orai1 in RBL mast cells in terms Orai-mediated NFAT signalng. Overexpression or shRNA knock-down of TRPC3 failed to affect NFAT activation in response to Orai1-mediated Ca2þ entry. Nonetheless, expression of a calcineurin (CaN) binding-deficient TRPC3 mutant (TRPC3P704Q) abolished NFAT translocation initiated by Ca2þ entry into store-depleted RBL cells without significant reduction of store-operated Ca2þ currents. TIRF-FRET experiments revealed a subpopulation of resting Orai1 complexes that are physically coupled to CaN and colocalized with TRPC3 proteins. Overexpressed TRPC3 proteins interfered with CaN recruitment into resting Orai-complexes in that Orai-CaN association was enhanced by TRPC3 and eliminated by TRPC3P704Q expression. Impaired Orai-CaN association in TRPC3P704Q expressing RBL cells was accompanied by reduced targeting of Orai into preformed PM-ER junctional connections. We conclude that TRPC3 governs Orai-mediated activation of NFAT by promoting resting-state Orai-CaN interactions and recruitment of resting-state Orai channels into PM-ER junctions. 3014-Pos Board B391 Modulation of Neuronal Activity by Synthetic Activators of Lipid-Gated TRPC Channels Oleksandra Tiapko1, Toma Glasnov2, Gemma Guedes de la Cruz2, Michael Poteser1, Michaela Lichtenegger1, Klaus Groschner1. 1 Institute of Biophysics, Medical University of Graz, Graz, Austria, 2Institute of Chemistry, University of Graz, Graz, Austria. Transient receptor potential canonical (TRPC) cation channels are highly expressed in the central nervous system. Neuner et al. showed an inverse correlation between TRPC3 expression/activity and firing of hippocampal neurons. These results lead to the concept of TRPC3 being crucially involved in regulation of hippocampal excitability and constitution of fear memory. A small molecule TRPC3/6 agonist GSK1702934A has recently been introduced as a tool to activate lipid-sensitive TRPC channels directly, bypassing phospholipase C signaling. In an attempt to test the potential and pharmacological value of TRPC3 as a direct pharmacological target for interference with hippocampal functions, we characterized the effects of a series of novel synthetic TRPC3 activators, including molecules that are suitable for optical control of agonist activity. All compounds that proved effective in eliciting TRPC3 conductances in TRPC3 overexpressing, but not in wild type HEK293 cells, significantly reduced firing frequency in hippocampal neurons. Experiments with selective TRPC channel inhibitors as well as TRPC knock down strategies confirmed direct modulation of lipid-sensitive TRPC channels by small molecule activators as a suitable basis for pharmacological and/or optopharmacological interventions to govern neuronal functions. Supported by FWF, DK-MCD. 3015-Pos Board B392 Mechanosensitivity of TRPC6 Ion Channel Reconstituted in the Liposomes Yury A. Nikolaev1,2, Paul R. Rohde1, Derek R. Laver2, Boris Martinac1,3. 1 Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Sydney, Australia, 2University of Newcastle, Newcastle, Australia, 3 Univ New South Wales, Sydney, Australia. The transient receptor potential ion channel family is a diverse group of channels gated by various physical and chemical stimuli. One of the members, TRPC6, is a calcium permeable cation channel located in the ventricular cardiomyocytes. TRPC6 has been reported to be activated by mechanical force, however it is still unclear if TRPC6 is activated directly by membrane tension from the lipid bilayer or whether activation requires additional membrane components including cytoskeleton and/or extracellular matrix for its activation. Thus, it is important to elucidate the gating mechanism of TRPC6. In this study, we purified TRPC6 protein and investigated the mechanosensitivity of TRPC6 reconstituted into the liposomes using the patch clamp technique. We have transfected C-GFP TRPC6 construct into HEK293 cells and demonstrated that the channel is localised in the plasma membrane. Purification were performed using immobilized metal-affinity chromatography. To assess the integrity of the TRPC6 protein, the elution fraction was analysed by fluorescence sized exclusion chromatography (FSEC) and SDS/PAGE gel. GFP fluorescence peak using FSEC showed that TRPC6 channel is found mostly in tetramer state. Fluorescent scan of the gels demonstrated a single fluorescent band at the correct TRPC6 - GFP size. However, Coomassie staining of the same gel revealed additional bands in the gel showing that purification needs optimisation. Using isolated protein, liposome reconstitution was carried out using DR method. We recorded TRPC6 single channel activity in liposomes by patch clamping. TRPC6 ion channel activity demonstrated outward rectification and calculated conductance was ~44 pS. By applying negative pressure to the patch pipette, we did observe multiple channel activity. TRPC6 was
Wednesday, March 2, 2016
flexible architecture of this porin not only successfully binds protein targets, but also provides unique detection. Our engineered OmpG sensor is sensitive enough to provide, for the first time, the ability to resolve homologous protein analytes. The ability of our biosensor to discriminate between homologous proteins reveals the important need in better understanding the underlying mechanism of OmpG gating. This understanding will allow us to further engineer OmpG and tailor it for sensing an array of protein analytes. In addition, this knowledge will aid in a deeper understanding of the biology of this membrane protein itself. In this work, we highlight the pH-dependent gating mechanism of OmpG by investigating repulsive forces of OmpG’s extracellular loops. 3011-Pos Board B388 Characterization of Transmembrane Synthetic Chloride Ion Transporters Ahmed Fuwad. Inha University, Incheon, Korea, Republic of. Homeostasis of cells is essential for sustainable biological processes like proliferation, signaling, and differentiation. Among the most abundant anions in a biological system, chloride plays a vital role in maintaining homeostasis via chloride ion channels. Malfunctioning of the ion transporters may leads to diseases like cystic fibrosis, barrter syndrome, and osteoporosis. Thus the development of synthetic ion channels for replacing the malfunctioning ion channel may be one potential remedy for diseases. In this work we synthesized anion transporters by introducing different functional groups to make them Cl selective. Chloride efflux across the membrane that bears the synthetic ion channels was measured by a model membrane in a form of vesicles under controlled composition of intra and extravesicular solutions. We further verify ion transports across the membrane using Axopatch amplifier and concluded the antiporter ability of the synthetic ion channels. Toxicity of these compounds was also measured by using live/dead cell assay found no evidence of Toxicity. Considering the aforementioned characteristics and efflux potency they can be employed for treatment of different biological diseases together with membrane based sensors and separation technologies. Our future approach includes highly selective channels for further applications.
TRP Channels II 3012-Pos Board B389 Store-Operated Ca2D Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat b-Cells Jessica Sabourin1, Loı¨c Le Gal2, Jacques-Antoine Haefliger3, Eric Raddatz4, Florent Allagnat3. 1 Faculty of Pharmacy, Inserm UMR-S1180, Chatenay-Malabry, France, 2 Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 3Department of Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, 4Department of Physiology, University of Lausanne, Lausanne, Switzerland. Store-Operated Ca2þ Channels (SOCs) are voltage-independent Ca2þ channels activated upon depletion of the endoplasmic reticulum (ER) Ca2þ stores. Early studies suggest the contribution of such channels to Ca2þ homeostasis in insulin-secreting pancreatic b-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remain unclear. In this study, ER Ca2þ depletion triggered by acetylcholine (ACh) or thapsigargin (Tg) stimulated the formation of a ternary complex composed of Orai1, TRPC1 and STIM1, the key proteins involved in the formation of SOCs. Ca2þ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to Tg or ACh. Pharmacological SOCs inhibition or dominant-negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D or TRPC1F562A impaired GSIS in rat b-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant-negative blockade of TRPC3 had no effect on extracellular Ca2þ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1 and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process which may be impaired in type 2 diabetes. 3013-Pos Board B390 TRPC3-Calcineurin Microdomains Govern Orai1 Signaling in Mast Cells Michael Poteser1, Bernadett Bacsa1, Oleksandra Tiapko1, Michaela Lichtenegger1, Irene Frischauf2, Christoph Romanin2, Klaus Groschner1. 1 Institute of Biophysics, Medical University of Graz, Graz, Austria, 2Institute of Biophysics, University of Linz, Linz, Austria.
Molecular communication between TRPC and Orai channel proteins has been proposed and is still a matter of debate. We investigated functional crosstalk of TRPC3 and Orai1 in RBL mast cells in terms Orai-mediated NFAT signalng. Overexpression or shRNA knock-down of TRPC3 failed to affect NFAT activation in response to Orai1-mediated Ca2þ entry. Nonetheless, expression of a calcineurin (CaN) binding-deficient TRPC3 mutant (TRPC3P704Q) abolished NFAT translocation initiated by Ca2þ entry into store-depleted RBL cells without significant reduction of store-operated Ca2þ currents. TIRF-FRET experiments revealed a subpopulation of resting Orai1 complexes that are physically coupled to CaN and colocalized with TRPC3 proteins. Overexpressed TRPC3 proteins interfered with CaN recruitment into resting Orai-complexes in that Orai-CaN association was enhanced by TRPC3 and eliminated by TRPC3P704Q expression. Impaired Orai-CaN association in TRPC3P704Q expressing RBL cells was accompanied by reduced targeting of Orai into preformed PM-ER junctional connections. We conclude that TRPC3 governs Orai-mediated activation of NFAT by promoting resting-state Orai-CaN interactions and recruitment of resting-state Orai channels into PM-ER junctions. 3014-Pos Board B391 Modulation of Neuronal Activity by Synthetic Activators of Lipid-Gated TRPC Channels Oleksandra Tiapko1, Toma Glasnov2, Gemma Guedes de la Cruz2, Michael Poteser1, Michaela Lichtenegger1, Klaus Groschner1. 1 Institute of Biophysics, Medical University of Graz, Graz, Austria, 2Institute of Chemistry, University of Graz, Graz, Austria. Transient receptor potential canonical (TRPC) cation channels are highly expressed in the central nervous system. Neuner et al. showed an inverse correlation between TRPC3 expression/activity and firing of hippocampal neurons. These results lead to the concept of TRPC3 being crucially involved in regulation of hippocampal excitability and constitution of fear memory. A small molecule TRPC3/6 agonist GSK1702934A has recently been introduced as a tool to activate lipid-sensitive TRPC channels directly, bypassing phospholipase C signaling. In an attempt to test the potential and pharmacological value of TRPC3 as a direct pharmacological target for interference with hippocampal functions, we characterized the effects of a series of novel synthetic TRPC3 activators, including molecules that are suitable for optical control of agonist activity. All compounds that proved effective in eliciting TRPC3 conductances in TRPC3 overexpressing, but not in wild type HEK293 cells, significantly reduced firing frequency in hippocampal neurons. Experiments with selective TRPC channel inhibitors as well as TRPC knock down strategies confirmed direct modulation of lipid-sensitive TRPC channels by small molecule activators as a suitable basis for pharmacological and/or optopharmacological interventions to govern neuronal functions. Supported by FWF, DK-MCD. 3015-Pos Board B392 Mechanosensitivity of TRPC6 Ion Channel Reconstituted in the Liposomes Yury A. Nikolaev1,2, Paul R. Rohde1, Derek R. Laver2, Boris Martinac1,3. 1 Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Sydney, Australia, 2University of Newcastle, Newcastle, Australia, 3 Univ New South Wales, Sydney, Australia. The transient receptor potential ion channel family is a diverse group of channels gated by various physical and chemical stimuli. One of the members, TRPC6, is a calcium permeable cation channel located in the ventricular cardiomyocytes. TRPC6 has been reported to be activated by mechanical force, however it is still unclear if TRPC6 is activated directly by membrane tension from the lipid bilayer or whether activation requires additional membrane components including cytoskeleton and/or extracellular matrix for its activation. Thus, it is important to elucidate the gating mechanism of TRPC6. In this study, we purified TRPC6 protein and investigated the mechanosensitivity of TRPC6 reconstituted into the liposomes using the patch clamp technique. We have transfected C-GFP TRPC6 construct into HEK293 cells and demonstrated that the channel is localised in the plasma membrane. Purification were performed using immobilized metal-affinity chromatography. To assess the integrity of the TRPC6 protein, the elution fraction was analysed by fluorescence sized exclusion chromatography (FSEC) and SDS/PAGE gel. GFP fluorescence peak using FSEC showed that TRPC6 channel is found mostly in tetramer state. Fluorescent scan of the gels demonstrated a single fluorescent band at the correct TRPC6 - GFP size. However, Coomassie staining of the same gel revealed additional bands in the gel showing that purification needs optimisation. Using isolated protein, liposome reconstitution was carried out using DR method. We recorded TRPC6 single channel activity in liposomes by patch clamping. TRPC6 ion channel activity demonstrated outward rectification and calculated conductance was ~44 pS. By applying negative pressure to the patch pipette, we did observe multiple channel activity. TRPC6 was