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Purinergic P2 Receptors: Structure and Function
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Purinergic P2 Receptors: Structure and Function

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 July 2020) | Viewed by 70421

Special Issue Editor

Dr. Hana Zemkova

E-Mail Website
Guest Editor
Institute of Physiology, Czech Academy of Sciences, 14220 Prague, Czech Republic
Interests: release of neurotransmitters and hormones; ion channels; P2X receptors; molecular structure
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

ATP (adenosine-5´-triphosphate) is a multifunctional intracellular molecule that serves as an energy source in all living cells. Much less is known about its role as an extracellular messenger or neurotransmitter. Extracellular ATP and its metabolic products acts on its plasma membrane receptors termed purinergic receptors, composed of two families: the ion-conducting P2X receptor channels and G protein-coupled P2Y receptors. By mediating depolarization, Ca2+ influx, and Ca2+ mobilization from intracellular stores, the extracellular nucleotide/P2 receptor system has numerous functions in excitable and nonexcitable cells. P2X and P2Y receptors have been documented in many physiological and pathological processes including inflammation, pain, and cancer and are potential therapeutic targets for treatment of these pathologies. This perspective is closely related to understanding the molecular physiology of these receptors that might be activated simultaneously if expressed in the same cell.

This issue aims to present the contemporary research on structural insights into the function of P2X and P2Y receptors, and their physiology and pharmacology. It may include original articles covering experimental reports on some particular receptor, technical development or novel screening technologies, as well as review papers relating to the subjects.

Dr. Hana Zemkova
Guest Editor

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Keywords

  • Extracellular ATP
  • Purinergic signaling
  • P2X receptor
  • P2Y receptor
  • ATP release
  • Molecular structure
  • Molecular pathway

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Editorial

Jump to: Research, Review

4 pages, 205 KiB  
Editorial
Special Issue of International Journal of Molecular Sciences (IJMS) “Purinergic P2 Receptors: Structure and Function”
by Hana Zemkova
Int. J. Mol. Sci. 2021, 22(1), 383; https://doi.org/10.3390/ijms22010383 - 31 Dec 2020
Viewed by 3580
Abstract
This Special Issue of International Journal of Molecular Sciences (IJMS) contains 7 reviews and 12 original research papers written by a panel of experts who highlight recent advances in molecular structure and cellular function of purinergic P2 receptors [...] Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)

Research

Jump to: Editorial, Review

19 pages, 3759 KiB  
Article
Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability
by Marian Rupert, Anirban Bhattacharya, Vendula Tvrdonova Stillerova, Marie Jindrichova, Audrey Mokdad, Eric Boué-Grabot and Hana Zemkova
Int. J. Mol. Sci. 2020, 21(22), 8446; https://doi.org/10.3390/ijms21228446 - 10 Nov 2020
Cited by 4 | Viewed by 2202
Abstract
Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting [...] Read more.
Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49–Y51–Q52) and transmembrane domain 2 (G326–K327–F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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20 pages, 2184 KiB  
Article
New Insights of the Zn(II)-Induced P2 × 4R Positive Allosteric Modulation: Role of Head Receptor Domain SS2/SS3, E160 and D170
by Francisco Andrés Peralta and J. Pablo Huidobro-Toro
Int. J. Mol. Sci. 2020, 21(18), 6940; https://doi.org/10.3390/ijms21186940 - 22 Sep 2020
Cited by 3 | Viewed by 1799
Abstract
P2 × 4R is allosterically modulated by Zn(II), and despite the efforts to understand the mechanism, there is not a consensus proposal; C132 is a critical amino acid for the Zn(II) modulation, and this residue is located in the receptor head domain, forming [...] Read more.
P2 × 4R is allosterically modulated by Zn(II), and despite the efforts to understand the mechanism, there is not a consensus proposal; C132 is a critical amino acid for the Zn(II) modulation, and this residue is located in the receptor head domain, forming disulfide SS3. To ascertain the role of the SS2/SS3 microenvironment on the rP2 × 4R Zn(II)-induced allosteric modulation, we investigated the contribution of each individual SS2/SS3 cysteine plus carboxylic acid residues E118, E160, and D170, located in the immediate vicinity of the SS2/SS3 disulfide bonds. To this aim, we combined electrophysiological recordings with protein chemical alkylation using thiol reagents such as N-ethylmaleimide or iodoacetamide, and a mutation of key amino acid residues together with P2 × 4 receptor bioinformatics. P2 × 4R alkylation in the presence of the metal obliterated the allosteric modulation, a finding supported by the site-directed mutagenesis of C132 and C149 by a corresponding alanine. In addition, while E118Q was sensitive to Zn(II) modulation, the wild type receptor, mutants E160Q and D170N, were not, suggesting that these acid residues participate in the modulatory mechanism. Poisson–Boltzmann analysis indicated that the E160Q and D170N mutants showed a shift towards more positive electrostatic potential in the SS2/SS3 microenvironment. Present results highlight the role of C132 and C149 as putative Zn(II) ligands; in addition, we infer that acid residues E160 and D170 play a role attracting Zn(II) to the head receptor domain. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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17 pages, 6855 KiB  
Article
P2 Receptors Influence hMSCs Differentiation towards Endothelial Cell and Smooth Muscle Cell Lineages
by Yu Zhang, Patrick Babczyk, Andreas Pansky, Matthias Ulrich Kassack and Edda Tobiasch
Int. J. Mol. Sci. 2020, 21(17), 6210; https://doi.org/10.3390/ijms21176210 - 27 Aug 2020
Cited by 11 | Viewed by 2483
Abstract
Background: Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first [...] Read more.
Background: Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first time that purinergic 2 receptors are key players during hMSC differentiation towards adipocytes and osteoblasts. Purinergic 2 receptors play an important role in cardiovascular function when they bind to extracellular nucleotides. In this study, the possible functional role of purinergic 2 receptors during MSC endothelial and smooth muscle differentiation was investigated. Methods and Results: Human MSCs were isolated from liposuction materials. Then, endothelial and smooth muscle-like cells were differentiated and characterized by specific markers via Reverse Transcriptase-PCR (RT-PCR), Western blot and immunochemical stainings. Interestingly, some purinergic 2 receptor subtypes were found to be differently regulated during these specific lineage commitments: P2Y4 and P2Y14 were involved in the early stage commitment while P2Y1 was the key player in controlling MSC differentiation towards either endothelial or smooth muscle cells. The administration of natural and artificial purinergic 2 receptor agonists and antagonists had a direct influence on these differentiations. Moreover, a feedback loop via exogenous extracellular nucleotides on these particular differentiations was shown by apyrase digest. Conclusions: Purinergic 2 receptors play a crucial role during the differentiation towards endothelial and smooth muscle cell lineages. Some highly selective and potent artificial purinergic 2 ligands can control hMSC differentiation, which might improve the use of adult stem cells in cardiovascular tissue engineering in the future. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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18 pages, 3675 KiB  
Article
Prevention of P2 Receptor-Dependent Thrombocyte Activation by Pore-Forming Bacterial Toxins Improves Outcome in A Murine Model of Urosepsis
by Mette G. Christensen, Nanna Johnsen, Marianne Skals, Aimi D. M. Hamilton, Peter Rubak, Anne-Mette Hvas and Helle Praetorius
Int. J. Mol. Sci. 2020, 21(16), 5652; https://doi.org/10.3390/ijms21165652 - 06 Aug 2020
Cited by 4 | Viewed by 2636
Abstract
Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes [...] Read more.
Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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15 pages, 7623 KiB  
Article
Targeting of Intracellular TMEM16 Proteins to the Plasma Membrane and Activation by Purinergic Signaling
by Rainer Schreiber, Jiraporn Ousingsawat and Karl Kunzelmann
Int. J. Mol. Sci. 2020, 21(11), 4065; https://doi.org/10.3390/ijms21114065 - 05 Jun 2020
Cited by 11 | Viewed by 2957
Abstract
Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl− channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and [...] Read more.
Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl− channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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17 pages, 3436 KiB  
Article
The Role of P2X7 Purinergic Receptors in the Renal Inflammation Associated with Angiotensin II-Induced Hypertension
by Rocio Bautista-Pérez, Oscar Pérez-Méndez, Agustina Cano-Martínez, Ursino Pacheco, José Santamaría, Fernando Rodríguez-Sámano, Bernardo Rodríguez-Iturbe, L. Gabriel Navar and Martha Franco
Int. J. Mol. Sci. 2020, 21(11), 4041; https://doi.org/10.3390/ijms21114041 - 05 Jun 2020
Cited by 17 | Viewed by 3045
Abstract
Purinergic receptors play a central role in the renal pathophysiology of angiotensin II-induced hypertension, since elevated ATP chronically activates P2X7 receptors in this model. The changes induced by the P2X antagonist Brilliant blue G (BBG) in glomerular hemodynamics and in tubulointerstitial inflammation resulting [...] Read more.
Purinergic receptors play a central role in the renal pathophysiology of angiotensin II-induced hypertension, since elevated ATP chronically activates P2X7 receptors in this model. The changes induced by the P2X antagonist Brilliant blue G (BBG) in glomerular hemodynamics and in tubulointerstitial inflammation resulting from angiotensin II infusion were studied. Rats received angiotensin II (435 ng kg−1 min−1, 2 weeks) alone or in combination with BBG (50 mg/kg/day intraperitoneally). BBG did not modify hypertension (214.5 ± 1.4 vs. 212.7 ± 0.5 mmHg), but restored to near normal values afferent (7.03 ± 1.00 to 2.97 ± 0.27 dyn.s.cm−5) and efferent (2.62 ± 0.03 to 1.29 ± 0.09 dyn.s.cm−5) arteriolar resistances, glomerular plasma flow (79.23 ± 3.15 to 134.30 ± 1.11 nL/min), ultrafiltration coefficient (0.020 ± 0.002 to 0.036 ± 0.003 nL/min/mmHg) and single nephron glomerular filtration rate (22.28 ± 2.04 to 34.46 ± 1.54 nL/min). Angiotensin II induced overexpression of P2X7 receptors in renal tubular cells and in infiltrating T and B lymphocytes and macrophages. All inflammatory cells were increased by angiotensin II infusion and reduced by 20% to 50% (p < 0.05) by BBG administration. Increased IL-2, IL-6, TNFα, IL-1β, IL-18 and overexpression of NLRP3 inflammasome were induced by angiotensin II and suppressed by BBG. These studies suggest that P2X7 receptor-mediated renal vasoconstriction, tubulointerstitial inflammation and activation of NLRP3 inflammasome are associated with angiotensin II-induced hypertension. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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27 pages, 3389 KiB  
Article
P2X7 Receptor is Involved in Mitochondrial Dysfunction Induced by Extracellular Alpha Synuclein in Neuroblastoma SH-SY5Y Cells
by Anna Wilkaniec, Magdalena Cieślik, Emilia Murawska, Lidia Babiec, Magdalena Gąssowska-Dobrowolska, Ewelina Pałasz, Henryk Jęśko and Agata Adamczyk
Int. J. Mol. Sci. 2020, 21(11), 3959; https://doi.org/10.3390/ijms21113959 - 31 May 2020
Cited by 24 | Viewed by 4361
Abstract
The purinergic P2X7 receptor (P2X7R) belongs to a family of trimeric ion channels that are gated by extracellular adenosine 5′-triphosphate (ATP). Several studies have pointed to a role of P2X7R-dependent signalling in Parkinson's disease (PD)-related neurodegeneration. The pathology of (PD) is characterized by [...] Read more.
The purinergic P2X7 receptor (P2X7R) belongs to a family of trimeric ion channels that are gated by extracellular adenosine 5′-triphosphate (ATP). Several studies have pointed to a role of P2X7R-dependent signalling in Parkinson's disease (PD)-related neurodegeneration. The pathology of (PD) is characterized by the formation of insoluble alpha-synuclein (α-Syn) aggregates—Lewy bodies, but the mechanisms underlying α-Syn-induced dopaminergic cell death are still partially unclear. Our previous studies indicate that extracellular α-Syn directly interact with neuronal P2X7R and induces intracellular free calcium mobilization in neuronal cells. The main objective of this study was to examine the involvement of P2X7R receptor in α-Syn-induced mitochondrial dysfunction and cell death. We found that P2X7R stimulation is responsible for α-Syn-induced oxidative stress and activation of the molecular pathways of programmed cell death. Exogenous α-Syn treatment led to P2X7R-dependent decrease in mitochondrial membrane potential as well as elevation of mitochondrial ROS production resulting in breakdown of cellular energy production. Moreover, P2X7R-dependent deregulation of AMP-activated protein kinase as well as decrease in parkin protein level could be responsible for α-Syn-induced mitophagy impairment and accumulation of dysfunctional mitochondria. P2X7R might be putative pharmacological targets in molecular mechanism of extracellular α-Syn toxicity. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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9 pages, 578 KiB  
Article
Association between P2X7 Polymorphisms and Post-Transplant Outcomes in Allogeneic Haematopoietic Stem Cell Transplantation
by Rachel M Koldej, Travis Perera, Jenny Collins and David S Ritchie
Int. J. Mol. Sci. 2020, 21(11), 3772; https://doi.org/10.3390/ijms21113772 - 27 May 2020
Cited by 8 | Viewed by 1941
Abstract
Allogeneic stem cell transplantation (alloSCT) is a highly effective treatment method for haematologic malignancies. However, infection of acute organ dysfunction and graft versus host disease (GVHD) impact negatively on patient outcomes. Pre-transplant conditioning regimes are associated with high levels of immunogenic cell death [...] Read more.
Allogeneic stem cell transplantation (alloSCT) is a highly effective treatment method for haematologic malignancies. However, infection of acute organ dysfunction and graft versus host disease (GVHD) impact negatively on patient outcomes. Pre-transplant conditioning regimes are associated with high levels of immunogenic cell death and the release of extracellular ATP, which binds to the P2X7 receptor. It has been proposed that signaling through the P2X7 receptor may lead to activation of downstream effectors that influence alloSCT outcome. In this study, we examined the effect of gain-of-function (GOF) or loss-of-function (LOF) P2X7 Single Nucleotide Polymorphisms (SNP) in 453 paired alloSCT donors and recipients and correlated their presence or absence to the major post-transplant outcomes of acute GVHD, relapse free survival and overall survival. The allelic frequency of P2X7 SNP in recipients and donors was not different from those SNP for which there is published population data. The LOF SNP Glu496Ala was overrepresented in recipients who did not develop severe acute GVHD and was associated with improved overall survival in rare homozygous recipients, whereas the LOF SNP Ile568Asn was more common in patients with grade 1–4 GVHD but lost statistical association in patients with grade 2–4 aGVHD, and was associated with reduced overall survival in heterozygotes due to an excess of infection-related deaths. The GOF variant haplotype (homozygous Gln460Arg-Ala348Thr) had no impact on post-alloSCT outcomes. Overall, our data indicate that allelic variations in recipients or donors occurs at the same frequency as the general population and may have a minor, but clinically nominal, impact on post-alloSCT outcomes. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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21 pages, 3030 KiB  
Article
Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts
by Nicholas Mikolajewicz and Svetlana V. Komarova
Int. J. Mol. Sci. 2020, 21(8), 2747; https://doi.org/10.3390/ijms21082747 - 15 Apr 2020
Cited by 10 | Viewed by 3415
Abstract
The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). [...] Read more.
The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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17 pages, 2083 KiB  
Article
Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity
by Maya Popova, Larry Rodriguez, James R. Trudell, Sylvia Nguyen, Michael Bloomfield, Daryl L. Davies and Liana Asatryan
Int. J. Mol. Sci. 2020, 21(7), 2471; https://doi.org/10.3390/ijms21072471 - 02 Apr 2020
Cited by 6 | Viewed by 3034
Abstract
Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol [...] Read more.
Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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18 pages, 6190 KiB  
Article
Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice
by Erandi Velázquez-Miranda, Christian Molina-Aguilar, Adriana González-Gallardo, Olivia Vázquez-Martínez, Mauricio Díaz-Muñoz and Francisco G Vázquez-Cuevas
Int. J. Mol. Sci. 2020, 21(7), 2305; https://doi.org/10.3390/ijms21072305 - 26 Mar 2020
Cited by 10 | Viewed by 2621
Abstract
Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of [...] Read more.
Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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14 pages, 4731 KiB  
Article
Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses
by Anna Miteva, Alexander Gaydukov and Olga Balezina
Int. J. Mol. Sci. 2020, 21(6), 2034; https://doi.org/10.3390/ijms21062034 - 16 Mar 2020
Cited by 9 | Viewed by 2905
Abstract
The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms [...] Read more.
The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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Review

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23 pages, 4182 KiB  
Review
Structural and Functional Basis for Understanding the Biological Significance of P2X7 Receptor
by María Ángeles Martínez-Cuesta, María Amparo Blanch-Ruiz, Raquel Ortega-Luna, Ainhoa Sánchez-López and Ángeles Álvarez
Int. J. Mol. Sci. 2020, 21(22), 8454; https://doi.org/10.3390/ijms21228454 - 10 Nov 2020
Cited by 28 | Viewed by 5412
Abstract
The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels—inexistent in physiological [...] Read more.
The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels—inexistent in physiological conditions—are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capacity of interacting with membrane structural compounds and/or intracellular signaling messengers to mediate flexible transduction pathways. Diverse drugs and a few endogenous molecules have been highlighted as extracellular allosteric modulators of P2X7R. Therefore, studies in human cells that constitutively express P2X7R need to investigate the precise endogenous mediator located nearby the activation/modulation domains of the receptor. Such research could help us understand the possible physiological ATP-mediated P2X7R homeostasis signaling. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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21 pages, 6072 KiB  
Review
P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases
by Derek Strassheim, Alexander Verin, Robert Batori, Hala Nijmeh, Nana Burns, Anita Kovacs-Kasa, Nagavedi S. Umapathy, Janavi Kotamarthi, Yash S. Gokhale, Vijaya Karoor, Kurt R. Stenmark and Evgenia Gerasimovskaya
Int. J. Mol. Sci. 2020, 21(18), 6855; https://doi.org/10.3390/ijms21186855 - 18 Sep 2020
Cited by 24 | Viewed by 4015
Abstract
Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological [...] Read more.
Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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18 pages, 1333 KiB  
Review
eATP/P2X7R Axis: An Orchestrated Pathway Triggering Inflammasome Activation in Muscle Diseases
by Chiara Panicucci, Lizzia Raffaghello, Santina Bruzzone, Serena Baratto, Elisa Principi, Carlo Minetti, Elisabetta Gazzerro and Claudio Bruno
Int. J. Mol. Sci. 2020, 21(17), 5963; https://doi.org/10.3390/ijms21175963 - 19 Aug 2020
Cited by 14 | Viewed by 4166
Abstract
In muscle ATP is primarily known for its function as an energy source and as a mediator of the “excitation-transcription” process, which guarantees muscle plasticity in response to environmental stimuli. When quickly released in massive concentrations in the extracellular space as in presence [...] Read more.
In muscle ATP is primarily known for its function as an energy source and as a mediator of the “excitation-transcription” process, which guarantees muscle plasticity in response to environmental stimuli. When quickly released in massive concentrations in the extracellular space as in presence of muscle membrane damage, ATP acts as a damage-associated molecular pattern molecule (DAMP). In experimental murine models of muscular dystrophies characterized by membrane instability, blockade of eATP/P2X7 receptor (R) purinergic signaling delayed the progression of the dystrophic phenotype dampening the local inflammatory response and inducing Foxp3+ T Regulatory lymphocytes. These discoveries highlighted the relevance of ATP as a harbinger of immune-tissue damage in muscular genetic diseases. Given the interactions between the immune system and muscle regeneration, the comprehension of ATP/purinerigic pathway articulated organization in muscle cells has become of extreme interest. This review explores ATP release, metabolism, feedback control and cross-talk with members of muscle inflammasome in the context of muscular dystrophies. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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25 pages, 1508 KiB  
Review
P2 Purinergic Signaling in the Distal Lung in Health and Disease
by Eva Wirsching, Michael Fauler, Giorgio Fois and Manfred Frick
Int. J. Mol. Sci. 2020, 21(14), 4973; https://doi.org/10.3390/ijms21144973 - 14 Jul 2020
Cited by 25 | Viewed by 4466
Abstract
The distal lung provides an intricate structure for gas exchange in mammalian lungs. Efficient gas exchange depends on the functional integrity of lung alveoli. The cells in the alveolar tissue serve various functions to maintain alveolar structure, integrity and homeostasis. Alveolar epithelial cells [...] Read more.
The distal lung provides an intricate structure for gas exchange in mammalian lungs. Efficient gas exchange depends on the functional integrity of lung alveoli. The cells in the alveolar tissue serve various functions to maintain alveolar structure, integrity and homeostasis. Alveolar epithelial cells secrete pulmonary surfactant, regulate the alveolar surface liquid (ASL) volume and, together with resident and infiltrating immune cells, provide a powerful host-defense system against a multitude of particles, microbes and toxicants. It is well established that all of these cells express purinergic P2 receptors and that purinergic signaling plays important roles in maintaining alveolar homeostasis. Therefore, it is not surprising that purinergic signaling also contributes to development and progression of severe pathological conditions like pulmonary inflammation, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis. Within this review we focus on the role of P2 purinergic signaling in the distal lung in health and disease. We recapitulate the expression of P2 receptors within the cells in the alveoli, the possible sources of ATP (adenosine triphosphate) within alveoli and the contribution of purinergic signaling to regulation of surfactant secretion, ASL volume and composition, as well as immune homeostasis. Finally, we summarize current knowledge of the role for P2 signaling in infectious pneumonia, ALI/ARDS and idiopathic pulmonary fibrosis (IPF). Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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22 pages, 2569 KiB  
Review
P2X7 Receptor at the Crossroads of T Cell Fate
by Elizabeth Rivas-Yáñez, Carlos Barrera-Avalos, Brian Parra-Tello, Pedro Briceño, Mariana V. Rosemblatt, Juan Saavedra-Almarza, Mario Rosemblatt, Claudio Acuña-Castillo, María Rosa Bono and Daniela Sauma
Int. J. Mol. Sci. 2020, 21(14), 4937; https://doi.org/10.3390/ijms21144937 - 13 Jul 2020
Cited by 32 | Viewed by 8010
Abstract
The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium [...] Read more.
The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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17 pages, 4041 KiB  
Review
The Role of Purinergic Receptors in the Circadian System
by Amira A.H. Ali, Gayaneh Avanes Avakian and Charlotte Von Gall
Int. J. Mol. Sci. 2020, 21(10), 3423; https://doi.org/10.3390/ijms21103423 - 12 May 2020
Cited by 9 | Viewed by 3279
Abstract
The circadian system is an internal time-keeping system that synchronizes the behavior and physiology of an organism to the 24 h solar day. The master circadian clock, the suprachiasmatic nucleus (SCN), resides in the hypothalamus. It receives information about the environmental light/dark conditions [...] Read more.
The circadian system is an internal time-keeping system that synchronizes the behavior and physiology of an organism to the 24 h solar day. The master circadian clock, the suprachiasmatic nucleus (SCN), resides in the hypothalamus. It receives information about the environmental light/dark conditions through the eyes and orchestrates peripheral oscillators. Purinergic signaling is mediated by extracellular purines and pyrimidines that bind to purinergic receptors and regulate multiple body functions. In this review, we highlight the interaction between the circadian system and purinergic signaling to provide a better understanding of rhythmic body functions under physiological and pathological conditions. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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10 pages, 636 KiB  
Review
Guanosine-Based Nucleotides, the Sons of a Lesser God in the Purinergic Signal Scenario of Excitable Tissues
by Rosa Mancinelli, Giorgio Fanò-Illic, Tiziana Pietrangelo and Stefania Fulle
Int. J. Mol. Sci. 2020, 21(5), 1591; https://doi.org/10.3390/ijms21051591 - 26 Feb 2020
Cited by 13 | Viewed by 3002
Abstract
Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and nucleotides (nitrogen bases plus ribose and phosphate). These compounds are very common in nature, especially in a phosphorylated [...] Read more.
Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and nucleotides (nitrogen bases plus ribose and phosphate). These compounds are very common in nature, especially in a phosphorylated form. There is increasing evidence that purines are involved in the development of different organs such as the heart, skeletal muscle and brain. When brain development is complete, some purinergic mechanisms may be silenced, but may be reactivated in the adult brain/muscle, suggesting a role for purines in regeneration and self-repair. Thus, it is possible that guanosine-5′-triphosphate (GTP) also acts as regulator during the adult phase. However, regarding GBP, no specific receptor has been cloned for GTP or its metabolites, although specific binding sites with distinct GTP affinity characteristics have been found in both muscle and neural cell lines. Finally, even if the cross regulation mechanisms between the two different purines (ABP and GBP) are still largely unknown, it is now possible to hypothesize the existence of specific signal paths for guanosine-based nucleotides that are capable of modulating the intensity and duration of the intracellular signal, particularly in excitable tissues such as brain and muscle. Full article
(This article belongs to the Special Issue Purinergic P2 Receptors: Structure and Function)
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