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Stanko S Stojilkovic - One of the best experts on this subject based on the ideXlab platform.
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opposing roles of calcium and intracellular atp on gating of the Purinergic P2X2 Receptor channel
International Journal of Molecular Sciences, 2018Co-Authors: Milos B Rokic, Stanko S Stojilkovic, Melanija Tomic, Patricio A Castro, Elias Leivasalcedo, Claudio CoddouAbstract:P2X2 Receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates Receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in Receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates Receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.
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role of domain calcium in Purinergic P2X2 Receptor channel desensitization
American Journal of Physiology-cell Physiology, 2015Co-Authors: Claudio Coddou, Zonghe Yan, Stanko S StojilkovicAbstract:Activation of P2X2 Receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as Receptor desensiti...
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Purinergic P2X2 Receptor desensitization depends on coupling between ectodomain and c terminal domain
Molecular Pharmacology, 2002Co-Authors: Takaaki Koshimizu, Melanija Tomic, Stanko S StojilkovicAbstract:The wild-type P2X2 Purinergic Receptor (P2X2aR) and its splice form lacking the intracellular Val370-Gln438 C-terminal sequence (P2X2bR) respond to ATP stimulation with comparable EC50 values and peak current/calcium responses but desensitize in a Receptor-specific manner. P2X2aR desensitizes slowly and P2X2bR desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X2aR and P2X2bR. Both Receptors showed similar EC50values (estimated from the peak calcium response) and IC50values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP ≤ ATP ≤ ATPγS
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expression of Purinergic P2X2 Receptor channels and their role in calcium signaling in pituitary cells
Biochemistry and Cell Biology, 2000Co-Authors: Stanko S Stojilkovic, Melanija Tomic, Fredrick Van Goor, Takaaki KoshimizuAbstract:Pituitary cells express Purinergic Receptor-channels (P2XR), the activation of which by ATP is associated with the facilitation of Ca2+ influx. Pharmacological, RT-PCR, and nucleotide sequence anal...
Claudio Coddou - One of the best experts on this subject based on the ideXlab platform.
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opposing roles of calcium and intracellular atp on gating of the Purinergic P2X2 Receptor channel
International Journal of Molecular Sciences, 2018Co-Authors: Milos B Rokic, Stanko S Stojilkovic, Melanija Tomic, Patricio A Castro, Elias Leivasalcedo, Claudio CoddouAbstract:P2X2 Receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates Receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in Receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates Receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.
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cyclin dependent kinase 5 modulates the P2X2a Receptor channel gating through phosphorylation of c terminal threonine 372
Pain, 2017Co-Authors: Claudio Coddou, Milos B Rokic, Patricio A Castro, Rodrigo Sandoval, Pablo Lazcano, Maria Jose Hevia, Bradford Hall, Anita Terse, Christian Gonzalezbillault, Ashok B KulkarniAbstract:The Purinergic P2X2 Receptor (P2X2R) is an adenosine triphosphate-gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (TPKH), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.
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role of domain calcium in Purinergic P2X2 Receptor channel desensitization
American Journal of Physiology-cell Physiology, 2015Co-Authors: Claudio Coddou, Zonghe Yan, Stanko S StojilkovicAbstract:Activation of P2X2 Receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as Receptor desensiti...
Melanija Tomic - One of the best experts on this subject based on the ideXlab platform.
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opposing roles of calcium and intracellular atp on gating of the Purinergic P2X2 Receptor channel
International Journal of Molecular Sciences, 2018Co-Authors: Milos B Rokic, Stanko S Stojilkovic, Melanija Tomic, Patricio A Castro, Elias Leivasalcedo, Claudio CoddouAbstract:P2X2 Receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates Receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in Receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates Receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.
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Purinergic P2X2 Receptor desensitization depends on coupling between ectodomain and c terminal domain
Molecular Pharmacology, 2002Co-Authors: Takaaki Koshimizu, Melanija Tomic, Stanko S StojilkovicAbstract:The wild-type P2X2 Purinergic Receptor (P2X2aR) and its splice form lacking the intracellular Val370-Gln438 C-terminal sequence (P2X2bR) respond to ATP stimulation with comparable EC50 values and peak current/calcium responses but desensitize in a Receptor-specific manner. P2X2aR desensitizes slowly and P2X2bR desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X2aR and P2X2bR. Both Receptors showed similar EC50values (estimated from the peak calcium response) and IC50values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP ≤ ATP ≤ ATPγS
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expression of Purinergic P2X2 Receptor channels and their role in calcium signaling in pituitary cells
Biochemistry and Cell Biology, 2000Co-Authors: Stanko S Stojilkovic, Melanija Tomic, Fredrick Van Goor, Takaaki KoshimizuAbstract:Pituitary cells express Purinergic Receptor-channels (P2XR), the activation of which by ATP is associated with the facilitation of Ca2+ influx. Pharmacological, RT-PCR, and nucleotide sequence anal...
Takaaki Koshimizu - One of the best experts on this subject based on the ideXlab platform.
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Purinergic P2X2 Receptor desensitization depends on coupling between ectodomain and c terminal domain
Molecular Pharmacology, 2002Co-Authors: Takaaki Koshimizu, Melanija Tomic, Stanko S StojilkovicAbstract:The wild-type P2X2 Purinergic Receptor (P2X2aR) and its splice form lacking the intracellular Val370-Gln438 C-terminal sequence (P2X2bR) respond to ATP stimulation with comparable EC50 values and peak current/calcium responses but desensitize in a Receptor-specific manner. P2X2aR desensitizes slowly and P2X2bR desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X2aR and P2X2bR. Both Receptors showed similar EC50values (estimated from the peak calcium response) and IC50values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP ≤ ATP ≤ ATPγS
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expression of Purinergic P2X2 Receptor channels and their role in calcium signaling in pituitary cells
Biochemistry and Cell Biology, 2000Co-Authors: Stanko S Stojilkovic, Melanija Tomic, Fredrick Van Goor, Takaaki KoshimizuAbstract:Pituitary cells express Purinergic Receptor-channels (P2XR), the activation of which by ATP is associated with the facilitation of Ca2+ influx. Pharmacological, RT-PCR, and nucleotide sequence anal...
Milos B Rokic - One of the best experts on this subject based on the ideXlab platform.
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opposing roles of calcium and intracellular atp on gating of the Purinergic P2X2 Receptor channel
International Journal of Molecular Sciences, 2018Co-Authors: Milos B Rokic, Stanko S Stojilkovic, Melanija Tomic, Patricio A Castro, Elias Leivasalcedo, Claudio CoddouAbstract:P2X2 Receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates Receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in Receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates Receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.
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cyclin dependent kinase 5 modulates the P2X2a Receptor channel gating through phosphorylation of c terminal threonine 372
Pain, 2017Co-Authors: Claudio Coddou, Milos B Rokic, Patricio A Castro, Rodrigo Sandoval, Pablo Lazcano, Maria Jose Hevia, Bradford Hall, Anita Terse, Christian Gonzalezbillault, Ashok B KulkarniAbstract:The Purinergic P2X2 Receptor (P2X2R) is an adenosine triphosphate-gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (TPKH), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.
