TY  - JOUR
AU  - Rokić, Miloš
AU  - Stojilković, Stanko S.
AU  - Zemkova, Hana
PY  - 2014
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/764
AB  - P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47 V61 and K326 N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closedopen-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, 1332, 1333, T335, 1337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47 V61 mutants and inhibitory in the majority of reactive K326 N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region.
PB  - Frontiers Research Foundation, Lausanne
T2  - Frontiers in Cellular Neuroscience
T1  - Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating
VL  - 8
DO  - 10.3389/fncel.2014.00003
ER  - 

@article{
author = "Rokić, Miloš and Stojilković, Stanko S. and Zemkova, Hana",
year = "2014",
abstract = "P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47 V61 and K326 N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closedopen-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, 1332, 1333, T335, 1337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47 V61 mutants and inhibitory in the majority of reactive K326 N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region.",
publisher = "Frontiers Research Foundation, Lausanne",
journal = "Frontiers in Cellular Neuroscience",
title = "Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating",
volume = "8",
doi = "10.3389/fncel.2014.00003"
}

Rokić, M., Stojilković, S. S.,& Zemkova, H.. (2014). Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating. in Frontiers in Cellular Neuroscience
Frontiers Research Foundation, Lausanne., 8.
https://doi.org/10.3389/fncel.2014.00003

Rokić M, Stojilković SS, Zemkova H. Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating. in Frontiers in Cellular Neuroscience. 2014;8.
doi:10.3389/fncel.2014.00003 .

Rokić, Miloš, Stojilković, Stanko S., Zemkova, Hana, "Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating" in Frontiers in Cellular Neuroscience, 8 (2014),
https://doi.org/10.3389/fncel.2014.00003 . .

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 . .