TY  - JOUR
AU  - Rokić, Miloš
AU  - Stojilković, Stanko S.
AU  - Vavra, Vojtech
AU  - Kuzyk, Pavlo
AU  - Tvrdonova, Vendula
AU  - Zemkova, Hana
PY  - 2013
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/625
AB  - The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a beta-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.
PB  - Public Library Science, San Francisco
T2  - PLoS One
T1  - Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor
IS  - 3
VL  - 8
DO  - 10.1371/journal.pone.0059411
ER  - 

@article{
author = "Rokić, Miloš and Stojilković, Stanko S. and Vavra, Vojtech and Kuzyk, Pavlo and Tvrdonova, Vendula and Zemkova, Hana",
year = "2013",
abstract = "The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a beta-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.",
publisher = "Public Library Science, San Francisco",
journal = "PLoS One",
title = "Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor",
number = "3",
volume = "8",
doi = "10.1371/journal.pone.0059411"
}

Rokić, M., Stojilković, S. S., Vavra, V., Kuzyk, P., Tvrdonova, V.,& Zemkova, H.. (2013). Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor. in PLoS One
Public Library Science, San Francisco., 8(3).
https://doi.org/10.1371/journal.pone.0059411

Rokić M, Stojilković SS, Vavra V, Kuzyk P, Tvrdonova V, Zemkova H. Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor. in PLoS One. 2013;8(3).
doi:10.1371/journal.pone.0059411 .

Rokić, Miloš, Stojilković, Stanko S., Vavra, Vojtech, Kuzyk, Pavlo, Tvrdonova, Vendula, Zemkova, Hana, "Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor" in PLoS One, 8, no. 3 (2013),
https://doi.org/10.1371/journal.pone.0059411 . .

TY  - 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 . .