Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating
2010
Authors
Rokić, MilošTvrdonova, V.
Vavra, V.
Jindrichova, M.
Obsil, T.
Stojilković, S. S.
Zemkova, H.
Article (Published version)
Metadata
Show full item recordAbstract
Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor functi...on was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.
Keywords:
Purinergic signaling / Ivermectin / Disulfide bonds / Deactivation / ATP-gated receptor-channelsSource:
Physiological Research, 2010, 59, 6, 927-935Publisher:
- Acad Sciences Czech Republic, Inst Physiology, Prague 4
Funding / projects:
- Internal Grant Agency of Academy of Sciences [IAA500110910]
- Grant Agency of the Czech Republic [305/07/0681]
- Academy of Sciences of the Czech Republic [AVOZ 50110509]
- Centrum for Neuroscience [LC554]
- NICHD, NIH
- EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT [ZIAHD000195] Funding Source: NIH RePORTER
DOI: 10.33549/physiolres.931979
ISSN: 0862-8408
PubMed: 20406028
WoS: 000285711400010
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Institution/Community
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Rokić, Miloš AU - Tvrdonova, V. AU - Vavra, V. AU - Jindrichova, M. AU - Obsil, T. AU - Stojilković, S. S. AU - Zemkova, H. PY - 2010 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/403 AB - Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating. PB - Acad Sciences Czech Republic, Inst Physiology, Prague 4 T2 - Physiological Research T1 - Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating EP - 935 IS - 6 SP - 927 VL - 59 DO - 10.33549/physiolres.931979 ER -
@article{ author = "Rokić, Miloš and Tvrdonova, V. and Vavra, V. and Jindrichova, M. and Obsil, T. and Stojilković, S. S. and Zemkova, H.", year = "2010", abstract = "Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.", publisher = "Acad Sciences Czech Republic, Inst Physiology, Prague 4", journal = "Physiological Research", title = "Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating", pages = "935-927", number = "6", volume = "59", doi = "10.33549/physiolres.931979" }
Rokić, M., Tvrdonova, V., Vavra, V., Jindrichova, M., Obsil, T., Stojilković, S. S.,& Zemkova, H.. (2010). Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating. in Physiological Research Acad Sciences Czech Republic, Inst Physiology, Prague 4., 59(6), 927-935. https://doi.org/10.33549/physiolres.931979
Rokić M, Tvrdonova V, Vavra V, Jindrichova M, Obsil T, Stojilković SS, Zemkova H. Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating. in Physiological Research. 2010;59(6):927-935. doi:10.33549/physiolres.931979 .
Rokić, Miloš, Tvrdonova, V., Vavra, V., Jindrichova, M., Obsil, T., Stojilković, S. S., Zemkova, H., "Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating" in Physiological Research, 59, no. 6 (2010):927-935, https://doi.org/10.33549/physiolres.931979 . .