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Stanko S. Stojilkovic - One of the best experts on this subject based on the ideXlab platform.
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Characterization of novel Pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels.
General and comparative endocrinology, 2011Co-Authors: Melanija Tomić, Stanko S. StojilkovicAbstract:Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with Purinergic P2X Receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 Receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.
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Activation and Regulation of Purinergic P2X Receptor Channels
Pharmacological reviews, 2011Co-Authors: Claudio Coddou, Zonghe Yan, Tomas Obsil, J. Pablo Huidobro-toro, Stanko S. StojilkovicAbstract:Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of Receptor desensitization and/or pore dilation and are critical for the regulation of Receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of Receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.
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Molecular dissection of Purinergic P2X Receptor channels.
Annals of the New York Academy of Sciences, 2005Co-Authors: Stanko S. Stojilkovic, Melanija Tomić, Zonghe Yan, Taka-aki Koshimizu, Hana ZemkovaAbstract:The P2X Receptors (P2XRs) are a family of ATP-gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. P2XRs are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X 1 -P2X 7 . All subunits possess intracellular N-and C-termini, two transmembrane domains, and a relatively large extracellular ligand-binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of extracellular ATP leads to deactivation and resensitization of Receptors. Activated P2XRs generate inward currents caused by Na + and Ca 2 + influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage-gated calcium entry in excitable cells. No crystal structures are available for P2XRs and these Receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of Receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of P2XRs, including heteromerization, chimerization, mutagenesis, and biochemical studies.
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Intracellular calcium measurements as a method in studies on activity of Purinergic P2X Receptor channels
American journal of physiology. Cell physiology, 2003Co-Authors: Hana Zemkova, Taka-aki Koshimizu, Melanija Tomić, Stanko S. StojilkovicAbstract:Extracellular nucleotide-activated Purinergic Receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subs...
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Dependence of Purinergic P2X Receptor Activity on Ectodomain Structure
The Journal of biological chemistry, 2003Co-Authors: Hana Zemkova, Stanko S. StojilkovicAbstract:Abstract Purinergic Receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a Receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using Receptors with variable sensitivity to ATP in order: P2X4R > P2X2aR = P2X2bR ≫ P2X7R. Chimeras having the ectodomain Ile66–Tyr310sequence of P2X2R and Val61–Phe313sequence of P2X7R in the backbone of P2X4R were expressed but were non-functioning channels. P2X2a + X4R and P2X2b + X4R chimeras having the Val66–Tyr315 ectodomain sequence of P2X4R in the backbones of P2X2aR and P2X2bR were functional and exhibited increased sensitivity to ligands as compared with both parental Receptors. These chimeras also desensitized faster than parental Receptors and in a ligand-nonspecific manner. However, like parental P2X2bR and P2X2aR, chimeric P2X2b + X4R desensitized more rapidly than P2X2a + X4R, and the rate of desensitization of P2X2a+X4R increased by substituting its Arg371–Pro376intracellular C-terminal sequence with the Glu376–Gly381 sequence of P2X4R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and Receptor activation. Furthermore, the ligand potency positively correlates with the rate of Receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.
Matthew Drill - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Purinergic P2X Receptor 7 P2X7r decreases granulocyte macrophage colony stimulating factor gm csf expression in u251 glioblastoma cells
Scientific Reports, 2020Co-Authors: Matthew Drill, Kim L Powell, Liyen Katrina Kan, Nigel C Jones, Terence J Obrien, John A Hamilton, Mastura MonifAbstract:Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12-15 months. The P2X Receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.
Mastura Monif - One of the best experts on this subject based on the ideXlab platform.
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inhibition of Purinergic P2X Receptor 7 P2X7r decreases granulocyte macrophage colony stimulating factor gm csf expression in u251 glioblastoma cells
Scientific Reports, 2020Co-Authors: Matthew Drill, Kim L Powell, Liyen Katrina Kan, Nigel C Jones, Terence J Obrien, John A Hamilton, Mastura MonifAbstract:Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12-15 months. The P2X Receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma.
Kenton J. Swartz - One of the best experts on this subject based on the ideXlab platform.
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Physical basis of apparent pore dilation of ATP-activated P2X Receptor channels
Nature Neuroscience, 2015Co-Authors: Mufeng Li, Gilman E. S. Toombes, Shai D. Silberberg, Kenton J. SwartzAbstract:The prevailing view for Purinergic P2X Receptor channels is that their ion conduction pores dilate upon prolonged activation. This study finds that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. The selectivity of ion channels is fundamental for their roles in electrical and chemical signaling and in ion homeostasis. Although most ion channels exhibit stable ion selectivity, the prevailing view of Purinergic P2X Receptor channels, transient Receptor potential V1 (TRPV1) channels and acid-sensing ion channels (ASICs) is that their ion conduction pores dilate upon prolonged activation. We investigated this mechanism in P2X Receptors and found that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. We derived a physical model to calculate ion concentration changes during patch-clamp recordings, which validated our experimental findings and provides a quantitative guideline for effectively controlling ion concentration. Our results have fundamental implications for understanding ion permeation and gating in P2X Receptor channels, as well as more broadly for using patch-clamp techniques to study ion channels and neuronal excitability.
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Physical basis of apparent pore dilation of ATP-activated P2X Receptor channels
Nature neuroscience, 2015Co-Authors: Gilman E. S. Toombes, Shai D. Silberberg, Kenton J. SwartzAbstract:The prevailing view for Purinergic P2X Receptor channels is that their ion conduction pores dilate upon prolonged activation. This study finds that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions.
Hana Zemkova - One of the best experts on this subject based on the ideXlab platform.
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Molecular dissection of Purinergic P2X Receptor channels.
Annals of the New York Academy of Sciences, 2005Co-Authors: Stanko S. Stojilkovic, Melanija Tomić, Zonghe Yan, Taka-aki Koshimizu, Hana ZemkovaAbstract:The P2X Receptors (P2XRs) are a family of ATP-gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. P2XRs are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X 1 -P2X 7 . All subunits possess intracellular N-and C-termini, two transmembrane domains, and a relatively large extracellular ligand-binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of extracellular ATP leads to deactivation and resensitization of Receptors. Activated P2XRs generate inward currents caused by Na + and Ca 2 + influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage-gated calcium entry in excitable cells. No crystal structures are available for P2XRs and these Receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of Receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of P2XRs, including heteromerization, chimerization, mutagenesis, and biochemical studies.
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Intracellular calcium measurements as a method in studies on activity of Purinergic P2X Receptor channels
American journal of physiology. Cell physiology, 2003Co-Authors: Hana Zemkova, Taka-aki Koshimizu, Melanija Tomić, Stanko S. StojilkovicAbstract:Extracellular nucleotide-activated Purinergic Receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subs...
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Dependence of Purinergic P2X Receptor Activity on Ectodomain Structure
The Journal of biological chemistry, 2003Co-Authors: Hana Zemkova, Stanko S. StojilkovicAbstract:Abstract Purinergic Receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a Receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using Receptors with variable sensitivity to ATP in order: P2X4R > P2X2aR = P2X2bR ≫ P2X7R. Chimeras having the ectodomain Ile66–Tyr310sequence of P2X2R and Val61–Phe313sequence of P2X7R in the backbone of P2X4R were expressed but were non-functioning channels. P2X2a + X4R and P2X2b + X4R chimeras having the Val66–Tyr315 ectodomain sequence of P2X4R in the backbones of P2X2aR and P2X2bR were functional and exhibited increased sensitivity to ligands as compared with both parental Receptors. These chimeras also desensitized faster than parental Receptors and in a ligand-nonspecific manner. However, like parental P2X2bR and P2X2aR, chimeric P2X2b + X4R desensitized more rapidly than P2X2a + X4R, and the rate of desensitization of P2X2a+X4R increased by substituting its Arg371–Pro376intracellular C-terminal sequence with the Glu376–Gly381 sequence of P2X4R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and Receptor activation. Furthermore, the ligand potency positively correlates with the rate of Receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.
