RESUMEN
P2X ion channels have been functionally characterized from a range of eukaryotes. Although these receptors can be broadly classified into fast and slow desensitizing, the molecular mechanisms underlying current desensitization are not fully understood. Here, we describe the characterization of a P2X receptor from the cattle tick Boophilus microplus (BmP2X) displaying extremely slow current kinetics, little desensitization during ATP application, and marked rundown in current amplitude between sequential responses. ATP (EC(50), 67.1 µM) evoked concentration-dependent currents at BmP2X that were antagonized by suramin (IC(50), 4.8 µM) and potentiated by the antiparasitic drug amitraz. Ivermectin did not potentiate BmP2X currents, but the mutation M362L conferred ivermectin sensitivity. To investigate the mechanisms underlying slow desensitization we generated intracellular domain chimeras between BmP2X and the rapidly desensitizing P2X receptor from Hypsibius dujardini. Exchange of N or C termini between these fast- and slow-desensitizing receptors altered the rate of current desensitization toward that of the donor channel. Truncation of the BmP2X C terminus identified the penultimate residue (Arg413) as important for slow desensitization. Removal of positive charge at this position in the mutant R413A resulted in significantly faster desensitization, which was further accentuated by the negatively charged substitution R413D. R413A and R413D, however, still displayed current rundown to sequential ATP application. Mutation to a positive charge (R413K) reconstituted the wild-type phenotype. This study identifies a new determinant of P2X desensitization where positive charge at the end of the C terminal regulates current flow and further demonstrates that rundown and desensitization are governed by distinct mechanisms.
Asunto(s)
Arginina/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores Purinérgicos P2X/metabolismo , Garrapatas/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina/genética , Bovinos , Femenino , Datos de Secuencia Molecular , Mutación/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína/fisiología , Receptores Purinérgicos P2X/genética , Garrapatas/genética , Factores de Tiempo , Xenopus laevisRESUMEN
Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA-E) are present in this organism. However, their roles in purinergic signaling are unclear, because dP2XA proved to have an intracellular localization on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knock-out Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. beta,gamma-Imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na(+) but insensitive to copper and the P2 receptor antagonists pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl(-)-permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knock-out Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB, -C, -D, and -E were found to be intracellularly localized to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium, however, were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signaling.
Asunto(s)
Dictyostelium/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Cobre/metabolismo , Dictyostelium/citología , Oocitos/citología , Oocitos/fisiología , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Purinas/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Suramina/metabolismo , Equilibrio Hidroelectrolítico , Xenopus laevisRESUMEN
BACKGROUND: Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (HdP2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa. RESULTS: ATP (EC50 approximately 44.5 microM) evoked transient inward currents in HdP2X with millisecond rates of activation and desensitisation. HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC50 15.0 microM) and suramin (IC50 22.6 microM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 microM and 19.9 microM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in HdP2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay. CONCLUSION: The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between vertebrates and invertebrates. Furthermore, several characteristics of HdP2X including fast kinetics with low ATP sensitivity, potentiation by ivermectin in a channel with fast kinetics and distinct copper and zinc binding sites not dependent on histidines make HdP2X a useful model for comparative structure-function studies allowing a better understanding of P2X receptors in higher organisms.
Asunto(s)
Cobre/farmacología , Invertebrados/genética , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Zinc/farmacología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Cobre/metabolismo , Ivermectina/farmacología , Cinética , Datos de Secuencia Molecular , Oocitos/metabolismo , Filogenia , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X , Homología de Secuencia de Aminoácido , Xenopus , Zinc/metabolismoRESUMEN
BACKGROUND: P2X1 receptors play an important role in platelet function as they can induce shape change, granule centralization and are also involved in thrombus formation. As platelets have no nuclei, the level of P2X1 expression depends on transcriptional regulation in megakaryocytes, the platelet precursor cell. Since nothing is known about the molecular mechanisms regulating megakaryocytic P2X1 expression, this study aimed to identify and functionally characterize the P2X1 core promoter utilized in the human megakaryoblastic cell line MEG-01. RESULTS: In order to identify cis-acting elements involved in the transcriptional regulation of P2X1 expression, the ability of 4.7 kb P2X1 upstream sequence to drive luciferase reporter gene expression was tested. Low promoter activity was detected in proliferating MEG-01 cells. This activity increased 20-fold after phorbol-12-myristate-13-acetate (PMA) induced differentiation. A transcription start site was detected 365 bp upstream of the start codon by primer extension. Deletion analysis of reporter constructs indicated a core promoter located within the region -68 to +149 bp that contained two Sp1 sites (named Sp1a and Sp1b) and an NF-1 site. Individual mutations of Sp1b or NF-1 binding sites severely reduced promoter activity whereas triple mutation of Sp1a, Sp1b and NF-1 sites completely abolished promoter activity in both untreated and PMA treated cells. Sp1/3 and NF-1 proteins were shown to bind their respective sites by EMSA and interaction of Sp1/3, NF-1 and TFIIB with the endogenous P2X1 core promoter in MEG-01 cells was demonstrated by chromatin immunoprecipitation. Alignment of P2X1 genes from human, chimp, rat, mouse and dog revealed consensus Sp1a, Sp1b and NF-1 binding sites in equivalent positions thereby demonstrating evolutionary conservation of these functionally important sites. CONCLUSION: This study has identified and characterized the P2X1 promoter utilized in MEG-01 cells and shown that binding of Sp1/3 and NF-1 to elements in the direct vicinity of the transcription start site is essential for basal transcription. Targeting the function of these transcription factors in megakaryocytes may therefore provide a basis for the future therapeutic manipulation of platelet P2X1 function.
Asunto(s)
Megacariocitos/citología , Receptores Purinérgicos P2/genética , Factores de Transcripción/fisiología , Transcripción Genética , Sitios de Unión , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Humanos , Neurofibromina 1/fisiología , Regiones Promotoras Genéticas , Receptores Purinérgicos P2X , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3/fisiología , Sitio de Iniciación de la TranscripciónRESUMEN
P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC(50) 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αßmeATP was a weak agonist. BzATP was a partial agonist with an EC(50) of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC(50) values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn(2+) and Cu(2+) ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.
Asunto(s)
Sistema Nervioso Central/metabolismo , Lymnaea/metabolismo , Receptores Purinérgicos P2X2/química , Receptores Purinérgicos P2X2/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cobre/metabolismo , Concentración de Iones de Hidrógeno , Hibridación in Situ , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Agonistas del Receptor Purinérgico P2/farmacología , Antagonistas del Receptor Purinérgico P2/farmacología , Receptores Purinérgicos P2X2/efectos de los fármacos , Receptores Purinérgicos P2X2/genética , Uridina Trifosfato/metabolismo , Zinc/metabolismoRESUMEN
The presence of five P2X-like genes (p2xA-E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca(2+) influx in response to extracellular ATP and ADP (EC(50)=7.5microM and 6.1microM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91+/-0.04s, required extracellular Ca(2+), were inhibited by Gd(3+), modified by extracellular pH and were not affected by deletion of either the single Gbeta or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu(2+) and Zn(2+) inhibited purine-evoked Ca(2+) influx with IC(50) values of 0.9 and 6.3microM, respectively. 300microM Zn(2+) completely abolished the initial large rapid rise in intracellular Ca(2+) revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.
Asunto(s)
Señalización del Calcio , Dictyostelium/metabolismo , Receptores Purinérgicos/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Aequorina/metabolismo , Animales , Apoproteínas/metabolismo , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , AMP Cíclico/farmacología , Dictyostelium/efectos de los fármacos , Dictyostelium/enzimología , Dictyostelium/genética , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Gadolinio/farmacología , Genes Protozoarios , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Fosfolipasas A2/metabolismo , Receptores Purinérgicos/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética/efectos de los fármacos , Zinc/farmacologíaRESUMEN
The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP(3) and Ca(2+), which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP(3) and Ca(2+) oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of G(q/11)alpha protein expression levels in shaping mGlu1/5 receptor-mediated IP(3) and Ca(2+) signals, using RNA interference (RNAi). RNAi-mediated knockdown of G(q/11)alpha almost abolished the single-cell increase in IP(3) caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca(2+) oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca(2+) oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of G(q)alpha enhanced IP(3) signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP(3) and Ca(2+) oscillations in control cells were largely insensitive to agonist concentration, increasing G(q)alpha expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca(2+) signals, up- or down-regulation of G(q/11)alpha expression alters the threshold for the concentration of glutamate at which a measurable Ca(2+) signal could be detected. These experiments indicate that altering G(q/11)alpha expression levels differentially affects spatiotemporal aspects of IP(3) and Ca(2+) signaling mediated by the mGlu1 and mGlu5 receptors.
Asunto(s)
Señalización del Calcio/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Transducción de Señal/fisiología , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN , Humanos , Interferencia de ARN , Receptor del Glutamato Metabotropico 5RESUMEN
P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) labeling of free cysteines were determined. For the majority of single mutants, only a modest decrease (2- to 5-fold) in ATP potency was recorded. For mutants C261A and C270A, the peak current amplitudes were reduced by 93.6 +/- 2.0 and 95.0 +/- 1.0%, respectively; this was a result of low cell-surface expression of these mutant receptors. Wild-type receptors showed no labeling with MTSEA-biotin suggesting that all 10 cysteine residues in the extracellular loop are disulfide-bonded. Mutation of cysteines at positions 126, 132, 149, 159, 217, and 227 resulted in MTSEA-biotinylation of a free cysteine residue created by the disruption of a disulfide bond and provides direct biochemical evidence for at least three disulfide bonds. Based on phenotypic comparisons of single and double cysteine mutants, we propose the following disulfide bond pairs in the human P2X(1) receptor: C117-C165, C126-C149, C132-C159, C217-C227, and C261-C270. None of these bonds are individually essential for channel function. However, trafficking of the receptor to the cell membrane is severely reduced by disruption of the C261-C270 disulfide bond or disruption of C117-C165 together with another bond.
Asunto(s)
Biotina/análogos & derivados , Cisteína/metabolismo , Disulfuros/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/metabolismo , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Transporte Biológico , Biotina/metabolismo , Biotinilación , Secuencia Conservada , Cisteína/genética , Ditiotreitol/farmacología , Electrofisiología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mercaptoetanol/farmacología , Datos de Secuencia Molecular , Mutación , Oocitos , Estructura Terciaria de Proteína , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/efectos de los fármacos , Receptores Purinérgicos P2X , Sustancias Reductoras/farmacología , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The family of ATP-gated P2X receptor channels have a conserved protein kinase C site in the N-terminal intracellular domain. This site was disrupted in human P2X(1) receptors by the mutation T18A. T18A mutants were expressed at normal levels in Xenopus oocytes; however, the peak current amplitude was reduced by >99% and showed approximately 10 fold faster desensitisation in response to ATP than wild type (WT) receptors showed. P2X receptor subunits form functional trimeric channels. Co-expression of T18A and WT receptors (90:10 ratio) produced heteromeric T18A/WT channels with the rapid T18A time-course and an approximately 90-fold increase in peak current amplitude compared to T18A. Similarly, T18A dominated the desensitisation phenotype of heteromeric channels composed of T18A and slowly desensitising K68A mutants. These results suggest that phosphorylation of P2X(1) receptors has a dramatic effect on the time-course of the response and may provide a mechanism for regulating channel function.
Asunto(s)
Activación del Canal Iónico , Proteína Quinasa C/fisiología , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Consenso , Conductividad Eléctrica , Cinética , Datos de Secuencia Molecular , Mutación , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Alineación de Secuencia , Xenopus laevisRESUMEN
Nucleotides are released from bovine chromaffin cells and take part in a feedback loop to inhibit further exocytosis. To identify the nucleotide receptors involved, we measured the effects of a range of exogenous nucleotides and related antagonists on voltage-operated calcium currents (I(Ca)), intracellular calcium concentration ([Ca(2+)](i)), and membrane capacitance changes. In comparative parallel studies, we also cloned the bovine P2Y(12) receptor from chromaffin cells and determined its properties by coexpression in Xenopus laevis oocytes with inward-rectifier potassium channels made up of Kir3.1 and Kir3.4. In both systems, the agonist order of potency was essentially identical (2-methylthio-ATP approximately 2-methylthio-ADP >> ATP approximately ADP > UDP). alphabeta-Methylene-ATP and adenosine were inactive. UTP inhibited I(Ca) in chromaffin cells (pEC(50) = 4.89 +/- 0.11) but was essentially inactive at the cloned P2Y(12) receptor. The relatively nonselective P2 antagonist pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid blocked nucleotide responses in both chromaffin cells and X. laevis oocytes, whereas the P2Y(12)- and P2Y(13)-selective antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP (ARC69931MX) blocked responses to ATP in both chromaffin cells and X. laevis oocytes but not to UTP in chromaffin cells. These results identify the P2Y(12) purine receptor as a key component of the nucleotide inhibitory pathway and also demonstrate the involvement of a UTP-sensitive G(i/o) -coupled pyrimidine receptor.
Asunto(s)
Adenosina Trifosfato/farmacología , Células Cromafines/efectos de los fármacos , Exocitosis/efectos de los fármacos , Proteínas de la Membrana/fisiología , Receptores Purinérgicos P2/fisiología , Uridina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Bovinos , Células Cromafines/fisiología , Clonación Molecular , Electrofisiología , Exocitosis/fisiología , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oocitos/fisiología , ARN Mensajero/análisis , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y12 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The cloning and characterization of a P2X receptor (schP2X) from the parasitic blood fluke Schistosoma mansoni provides the first example of a non-vertebrate ATP-gated ion channel. A number of functionally important amino acid residues conserved throughout vertebrate P2X receptors, including 10 extracellular cysteines, aromatic and positively charged residues involved in ATP recognition, and a consensus protein kinase C site in the amino-terminal tail, are also present in schP2X. Overall, the amino acid sequence identity of schP2X with human P2X(1-7) receptors ranges from 25.8 to 36.6%. ATP evoked concentration-dependent currents at schP2X channels expressed in Xenopus oocytes with an EC(50) of 22.1 microM. 2',3'-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) was a partial agonist (maximum response 75.4 +/- 4.4% that of ATP) with a higher potency (EC(50) of 3.6 microM) than ATP. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid blocked schP2X responses to 100 microm ATP with IC(50) values of 9.6 and 0.5 microM, respectively. Ivermectin (10 microM) potentiated currents to both ATP and Bz-ATP by approximately 60% with a minimal effect on potency (EC(50) of 18.2 and 1.6 microM, respectively). The relative permeability of schP2X expressed in HEK293 cells to various cations was determined under bi-ionic conditions. schP2X has a relatively high calcium permeability (P(Ca)/P(Na) = 3.80 +/- 0.29) and an estimated minimum pore diameter similar to that of vertebrate P2X receptors. SchP2X provides a useful comparative model for the better understanding of human P2X receptor function and may also provide an alternative drug target for treatment of schistosomiasis.