Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel
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. Howe...
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doaj-09389b232e4344eb843140547e30e44b2020-11-24T21:10:46ZengMDPI AGInternational Journal of Molecular Sciences1422-00672018-04-01194116110.3390/ijms19041161ijms19041161Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor ChannelMilos B. Rokic0Patricio Castro1Elias Leiva-Salcedo2Melanija Tomic3Stanko S. Stojilkovic4Claudio Coddou5Section on Cellular Signaling, National Institutes of Child Health and Human Development, NIH, Bethesda, MD 20892, USADepartamento de Ciencias Biomédicas, Facultad de Medicina, Universidad Católica del Norte, Coquimbo 1781421, ChileSection on Cellular Signaling, National Institutes of Child Health and Human Development, NIH, Bethesda, MD 20892, USASection on Cellular Signaling, National Institutes of Child Health and Human Development, NIH, Bethesda, MD 20892, USASection on Cellular Signaling, National Institutes of Child Health and Human Development, NIH, Bethesda, MD 20892, USASection on Cellular Signaling, National Institutes of Child Health and Human Development, NIH, Bethesda, MD 20892, USAP2X2 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.http://www.mdpi.com/1422-0067/19/4/1161purinergic receptor channelsdesensitizationATPcalciumallostericcovalent |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Milos B. Rokic Patricio Castro Elias Leiva-Salcedo Melanija Tomic Stanko S. Stojilkovic Claudio Coddou |
spellingShingle |
Milos B. Rokic Patricio Castro Elias Leiva-Salcedo Melanija Tomic Stanko S. Stojilkovic Claudio Coddou Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel International Journal of Molecular Sciences purinergic receptor channels desensitization ATP calcium allosteric covalent |
author_facet |
Milos B. Rokic Patricio Castro Elias Leiva-Salcedo Melanija Tomic Stanko S. Stojilkovic Claudio Coddou |
author_sort |
Milos B. Rokic |
title |
Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel |
title_short |
Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel |
title_full |
Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel |
title_fullStr |
Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel |
title_full_unstemmed |
Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel |
title_sort |
opposing roles of calcium and intracellular atp on gating of the purinergic p2x2 receptor channel |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2018-04-01 |
description |
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. |
topic |
purinergic receptor channels desensitization ATP calcium allosteric covalent |
url |
http://www.mdpi.com/1422-0067/19/4/1161 |
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