P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.

• Klíčová slova
• P2X7 receptor, TM1, alanine substitution, dye uptake, membrane current,
• MeSH
• adenosintrifosfát farmakologie   metabolismus   MeSH
• biologický transport MeSH
• HEK293 buňky MeSH
• iontové kanály * metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mutace genetika   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• iontové kanály * MeSH
• purinergní receptory P2X7 * MeSH

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.

• Klíčová slova
• HEK293T cells, P2X7 receptor, deactivation, dye uptake, extracellular vestibule, gating, mutagenesis,
• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kinetika MeSH
• konzervovaná sekvence MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• luminescentní proteiny MeSH
• mutantní proteiny MeSH
• P2rx7 protein, rat MeSH Prohlížeč
• purinergní receptory P2X7 MeSH
• rekombinantní fúzní proteiny MeSH
• yellow fluorescent protein, Bacteria MeSH Prohlížeč

Purinergic P2X receptors (P2XR), activated by extracellular adenosine 5'-triphosphate (ATP), represent a specific type of ligand-gated ion channels. They form functional trimeric homomers or heteromers which are nonselectively cation-permeable after receptor activation. P2X receptors are widely expressed in excitable and nonexcitable tissues and are involved in many physiological and pathophysiological processes such as platelet aggregation, contraction of smooth muscle, immune responses, cell proliferation and apoptosis or neurotransmission. In mammals, seven P2X subunits (P2X1-P2X7) have been identified. They differ mainly in distribution, pharmacological profile and kinetics of ATP-induced responses. The subtype P2X7 is the most specific in the P2X family and widely differs from other P2X subtypes.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• podjednotky proteinů MeSH
• purinergní receptory P2X7 chemie   metabolismus   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• adenosintrifosfát MeSH
• podjednotky proteinů MeSH
• purinergní receptory P2X7 MeSH

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.

• MeSH
• cystein biosyntéza   genetika   fyziologie   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence * MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mutace fyziologie   MeSH
• purinergní receptory P2X7 genetika   metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• cystein MeSH
• purinergní receptory P2X7 MeSH

In the sustained presence of agonist, the opening of P2X7R channel is followed by pore dilatation, which causes an increase in its permeability to larger organic cations, accompanied by receptor sensitization. To explore the molecular mechanisms by which the conductivity and sensitivity are increased, we analyzed the electrophysiological properties and YO-PRO-1 uptake of selected alanine mutants in the first and second transmembrane domains of the rat P2X7R. Substitution of residues Y40, F43, G338, and D352 with alanine reduced membrane trafficking, and the D352A was practically non-functional. The Y40A and F43A mutants that were expressed in the membrane lacked pore dilation ability. Moreover, the Y40A and Y40F displayed desensitization, whereas the Y40W partially recovered receptor function. The G338A/S mutations favored the open state of the channel and displayed instantaneous permeability to larger organic cations. The G338P was non-functional. The L341A and G345A displayed normal trafficking, current amplitude, and sensitization, but both mutations resulted in a decreased pore formation and dye uptake. These results showed that the increase in P2X7R conductivity and sensitivity is critically dependent on residues Y40 and F43 in the TM1 domain and that the region located at the intersection of TM2 helices controls the rate of large pore opening. We investigated the mechanism of the proapoptotic receptor P2X7R's large pore opening and its sensitization. We found that aromatic residues in the upper part of the first transmembrane domain (TM1) are critical for both the P2X7R channel pore opening and receptor sensitization, and residues located at or below the intersection of the second transmembrane domains (TM2) control the rate of pore opening. These findings identify new residues involved in pore formation of P2X7R.

• Klíčová slova
• ATP, BzATP, P2X7R, agonist affinity, pore dilation, purinergic receptors,
• MeSH
• HEK293 buňky MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze cílená MeSH
• purinergní receptory P2X7 chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• purinergní receptory P2X7 MeSH

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

• Klíčová slova
• ATP, Allosteric modulation, Bile acids, Ivermectin, Lithocholic acid, Purinergic P2X receptors,
• MeSH
• adenohypofýza cytologie   účinky léků   fyziologie   MeSH
• gating iontového kanálu účinky léků   MeSH
• HEK293 buňky MeSH
• hypothalamus cytologie   účinky léků   fyziologie   MeSH
• kyselina lithocholová analogy a deriváty   farmakologie   MeSH
• lidé MeSH
• neurony účinky léků   fyziologie   MeSH
• potkani Wistar MeSH
• purinergní receptory P2X - agonisté farmakologie   MeSH
• purinergní receptory P2X - antagonisté farmakologie   MeSH
• purinergní receptory P2X2 fyziologie   MeSH
• purinergní receptory P2X4 fyziologie   MeSH
• purinergní receptory P2X7 fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina lithocholová MeSH
• purinergní receptory P2X - agonisté MeSH
• purinergní receptory P2X - antagonisté MeSH
• purinergní receptory P2X2 MeSH
• purinergní receptory P2X4 MeSH
• purinergní receptory P2X7 MeSH

OBJECTIVE: Genes, involved in the modulation of inflammatory response and bone remodeling, play a role in the development of postorthodontic external apical root resorption (EARR). The aim of our study was to analyze possible associations between seven single nucleotide polymorphisms (SNPs) in interleukin-17A (IL-17), osteopontin (SPP1), purinoreceptor P2X7 (P2RX7), and tumor necrosis factor receptor superfamily member 11B (TNFRSF11B) genes and EARR in children after orthodontic treatment. SUBJECTS AND METHODS: This case-control study comprised 99 orthodontically treated patients (69 controls and 30 subjects with EARR). Genotype determinations of rs2275913, rs11730582, rs9138, rs208294, rs1718119, rs3102735, and rs2073618 were based on polymerase chain reaction using 5' nuclease TaqMan® assays. RESULTS: While no significant differences were observed in allele or genotype frequencies of all seven studied SNPs, specific haplotype of P2RX7 (rs208294 and rs1718119) modified the risk of EARR development (P < 0.05). In addition, the length of treatment with a fixed orthodontic appliance positively correlated with the presence of EARR (P < 0.05). CONCLUSIONS: Although the effect of individual SNPs studied on the EARR development was not confirmed in the Czech population, complex analysis suggested that variability in the P2RX7 gene and the length of orthodontic treatment may be important factors contributing to the etiopathogenesis of postorthodontic EARR.

• Klíčová slova
• gene polymorphism, orthodontic treatment, root resorption,
• MeSH
• frekvence genu genetika   MeSH
• genetická predispozice k nemoci genetika   MeSH
• interleukin-17 genetika   fyziologie   MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• lidé MeSH
• mladiství MeSH
• ortodoncie korekční škodlivé účinky   MeSH
• osteopontin genetika   fyziologie   MeSH
• osteoprotegerin genetika   fyziologie   MeSH
• purinergní receptory P2X7 genetika   fyziologie   MeSH
• resorpce zubního kořene etiologie   genetika   MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• interleukin-17 MeSH
• osteopontin MeSH
• osteoprotegerin MeSH
• P2RX7 protein, human MeSH Prohlížeč
• purinergní receptory P2X7 MeSH
• SPP1 protein, human MeSH Prohlížeč
• TNFRSF11B protein, human MeSH Prohlížeč

The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5'-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP.

• Klíčová slova
• ATP release, P2X7 receptor, P2Y1 receptor, P2Y2 receptor, astrocytes, organotypic cultures, pannexin-1 hemichannel, suprachiasmatic nucleus,
• Publikační typ
• časopisecké články MeSH

P2X4 and P2X7 receptors are ATP-gated ion channels, which play important roles in neuropathic and inflammatory pain, and as such they are important drug targets in diseases of inflammatory origin. While several compounds targeting P2X4 and P2X7 receptors have been developed using traditional high-throughput screening approaches, relatively few compounds have been developed using structure-based design. We initially set out to develop compounds targeting human P2X4, by performing virtual screening on the orthosteric (ATP-binding) pocket of a molecular model of human P2X4 based on the crystal structure of the Danio rerio receptor. The screening of a library of approximately 300,000 commercially available drug-like compounds led to the initial selection of 17 compounds; however, none of these compounds displayed a significant antagonist effect at P2X4 in a Fluo-4 ATP-induced calcium influx assay. When the same set of compounds was tested against human P2X7 in an ATP-stimulated Yo-Pro1 dye uptake assay, one compound (an indeno(1,2-b)pyridine derivative; GP-25) reduced the response by greater than 50% when applied at a concentration of 30 µM. GP-25 displayed an IC50 value of 8.7 μM at human P2X7 and 24.4 μM at rat P2X7, and was confirmed to be active using whole-cell patch clamp electrophysiology and not cytotoxic. Schild analysis suggested that mode of action of GP-25 was orthosteric. Screening of a further 16 commercially available analogues of GP-25 led to the discovery of five additional compounds with antagonist activity at human P2X7, enabling us to investigate the structure-activity relationship. Finally, docking of the R- and S-enantiomers of GP-25 into the orthosteric pocket of molecular models of human P2X4 and human P2X7 revealed that, while both enantiomers were able to make multiple interactions between their carboxyl moieties and conserved positively charged amino-acids in human P2X7, only the S-enantiomer of GP-25 was able to do this in human P2X4, potentially explaining the lack of activity of GP-25 at this receptor.

• Klíčová slova
• P2X4, P2X7, antagonist, structure-based drug design, virtual screen,
• Publikační typ
• časopisecké články MeSH

Periprosthetic osteolysis (OL) is a major long-term complication of the total hip arthroplasty (THA), which can result in aseptic loosening and revision surgery. Purinergic receptor P2X, ligand-gated ion channel 7 (P2RX7) is an important regulator of inflammation and bone turnover. We were therefore interested in whether functional variants of the P2RX7 gene may be associated with OL and risk of THA failure. A total of 205 unrelated Czech patients with cementless-type THA were stratified according to the severity of acetabular OL and revision of THA. Four "loss-of-function" P2RX7 single nucleotide polymorphisms (SNPs), namely Glu496Ala, Ile568Asn, Arg307Gln, and null allele (rs35933842), were genotyped by polymerase chain reaction with sequence-specific primers (PCR-SSP). No significant association of P2RX7 variants with severity of OL was observed. The carriers of rare variants P2RX7 568Asn, 307Gln and null allele, all causing complete loss of P2RX7 function, tended to be overrepresented among patients with THA revision (9.6%) by comparison with those with unrevised functional prosthesis (2.1%, p = 0.09). Furthermore, the carriage of the P2RX7 307Gln allele was associated with greater cumulative hazard of THA revision (p = 0.02). In this preliminary study, we could nominate but not clearly demonstrate rare P2RX7 loss-of-function variants being associated with THA failure. Investigation in large THA cohorts is therefore warranted.

• MeSH
• dospělí MeSH
• genetická predispozice k nemoci * MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus MeSH
• kyčelní protézy * MeSH
• lidé středního věku MeSH
• lidé MeSH
• náhrada kyčelního kloubu * MeSH
• osteolýza genetika   MeSH
• polymerázová řetězová reakce MeSH
• pooperační komplikace etiologie   MeSH
• purinergní receptory P2 genetika   MeSH
• purinergní receptory P2X7 MeSH
• reoperace MeSH
• selhání protézy MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• P2RX7 protein, human MeSH Prohlížeč
• purinergní receptory P2 MeSH
• purinergní receptory P2X7 MeSH