Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Opposite effects of zinc on human and rat P2X2 receptors.

ABSTRACT: P2X(2) receptors from rats show potentiation when a submaximal concentration of ATP is combined with zinc in the range of 10-100 microM. Alignment of the amino acid sequences of human P2X(2) (hP2X(2)) and rat P2X(2) (rP2X(2)) indicated that only one of two histidines essential for zinc potentiation in rP2X(2) is present at the homologous position in hP2X(2) (H132), with the position homologous to rat H213 instead having an arginine (R225). When expressed in Xenopus oocytes, mouse P2X(2a) and P2X(2b) receptors showed zinc potentiation indistinguishable from rat P2X(2a), but hP2X(2b) receptors were inhibited by zinc. The extent of zinc inhibition of hP2X(2b) varied with the ATP concentration, with an IC(50) of 8.4 microM zinc when ATP was applied at 10% of maximal and 87 microM zinc when ATP was applied at 99% of maximal. Site-directed mutagenesis showed that none of the nine histidines in the extracellular domain of hP2X(2b) were required for zinc inhibition, although inhibition was attenuated in the H204A and H209A mutations. Mutating R225 to a cysteine was sufficient to confer zinc potentiation onto hP2X(2b), and zinc potentiation was absent in the hP2X(2b)H132A/R225C double mutant. This suggests that zinc potentiation in the mutant hP2X(2b) uses the same mechanism as zinc potentiation in wild-type rP2X(2a). Because of the species-specific modulation by zinc, evidence for an in vivo role of P2X(2) receptors based on studies conducted on genetically modified mice needs to be viewed with caution when extrapolations are made to the function of the human nervous system.

SUBMITTER: Tittle RK

PROVIDER: S-EPMC2586956 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism.

Project description:Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2-100 μM, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μM. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nM) to very low (>100 μM) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.

| S-EPMC3381168 | biostudies-literature

Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP.

Project description:Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C(5)-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.

| S-EPMC3480388 | biostudies-literature

ATP analogues with modified phosphate chains and their selectivity for rat P2X2 and P2X2/3 receptors.

Project description:1. Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to alphabeta-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the alphabeta and/or betagamma oxygen atoms were replaced by other moieties (including -CH2-, -CHF-, -CHCl-, -CHBr-, -CF2-, -CCl2-, -CBr2-, -CHSO3-, -CHPO3-, -CFPO3-, -CClPO3-, -CH2-CH2-, C triple bond C, -NH-, -CHCOOH-). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except betagamma-difluoromethylene-ATP, which was a weak agonist. 5. At P2X2/3 receptors, betagamma-imido-ATP, betagamma-methylene-ATP, and betagamma-acetylene-ATP were weak agonists, whereas alphabeta,betagamma- and betagamma,gammadelta-bismethylene-AP4 were potent full agonists. betagamma-Carboxymethylene-ATP and betagamma-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 microm). 6. The results indicate (a). that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b). that the heteromeric P2X2/3 receptor is much more tolerant of alphabeta and betagamma substitution; and (c). that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the betagamma-methylene.

| S-EPMC1574118 | biostudies-other

Responses of rat P2X2 receptors to ultrashort pulses of ATP provide insights into ATP binding and channel gating.

Project description:To gain insight into the way that P2X(2) receptors localized at synapses might function, we explored the properties of outside-out patches containing many of these channels as ATP was very rapidly applied and removed. Using a new method to calibrate the speed of exchange of solution over intact patches, we were able to reliably produce applications of ATP lasting <200 micros. For all concentrations of ATP, there was a delay of at least 80 micros between the time when ATP arrived at the receptor and the first detectable flow of inward current. In response to 200-micros pulses of ATP, the time constant of the rising phase of the current was approximately 600 micros. Thus, most channel openings occurred when no free ATP was present. The current deactivated with a time constant of approximately 60 ms. The amplitude of the peak response to a brief pulse of a saturating concentration of ATP was approximately 70% of that obtained during a long application of the same concentration of ATP. Thus, ATP leaves fully liganded channels without producing an opening at least 30% of the time. Extensive kinetic modeling revealed three different schemes that fit the data well, a sequential model and two allosteric models. To account for the delay in opening at saturating ATP, it was necessary to incorporate an intermediate closed state into all three schemes. These kinetic properties indicate that responses to ATP at synapses that use homomeric P2X(2) receptors would be expected to greatly outlast the duration of the synaptic ATP transient produced by a single presynaptic spike. Like NMDA receptors, P2X(2) receptors provide the potential for complex patterns of synaptic integration over a time scale of hundreds of milliseconds.

| S-EPMC2151634 | biostudies-literature

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors.

Project description:The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of ?,?-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.

| S-EPMC3340182 | biostudies-literature

Regulation of P2X2 receptors by the neuronal calcium sensor VILIP1.

Project description:Extracellular adenosine triphosphate (ATP) activates P2X receptors, which are involved in diverse physiological functions. Using a proteomic approach, we identified the neuronal calcium sensor VILIP1 as interacting with P2X2 receptors. We found that VILIP1 forms a signaling complex in vitro and in vivo with P2X2 receptors and regulates P2X2 receptor sensitivity to ATP, peak response, surface expression, and diffusion. VILIP1 constitutively binds to P2X2 receptors and displays enhanced interactions in an activation- and calcium-dependent manner owing to exposure of its binding segment in P2X2 receptors. VILIP1-P2X2 interactions are also enhanced in hippocampal neurons during conditions of action potential firing known to trigger P2X2 receptor activation. Our data thus reveal a previously unrecognized function for the neuronal calcium sensor protein VILIP1 and a mechanism for regulation of ATP-dependent P2X receptor signaling by neuronal calcium sensors.

| S-EPMC3523710 | biostudies-literature

Molecular determinants for agonist recognition and discrimination in P2X2 receptors.

Project description:P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding. P2XRs contribute to synaptic transmission and are involved in pain and inflammation, thus representing valuable drug targets. Recent crystal structures have confirmed the findings of previous studies with regards to the amino acid chains involved in ligand recognition, but they have also suggested that backbone carbonyl atoms contribute to ATP recognition and discrimination. Here we use a combination of site-directed mutagenesis, amide-to-ester substitutions, and a range of ATP analogues with subtle alterations to either base or sugar component to investigate the contributions of backbone carbonyl atoms toward ligand recognition and discrimination in rat P2X2Rs. Our findings demonstrate that while the Lys69 backbone carbonyl makes an important contribution to ligand recognition, the discrimination between different ligands is mediated by both the side chain and the backbone carbonyl oxygen of Thr184. Together, our data demonstrate how conserved elements in P2X2Rs recognize and discriminate agonists.

| S-EPMC6605687 | biostudies-literature

Coexpression of rat P2X2 and P2X6 subunits in Xenopus oocytes.

Project description:Transcripts for P2X(2) and P2X(6) subunits are present in rat CNS and frequently colocalize in the same brainstem nuclei. When rat P2X(2) (rP2X(2)) and rat P2X(6) (rP2X(6)) receptors were expressed individually in Xenopus oocytes and studied under voltage-clamp conditions, only homomeric rP2X(2) receptors were fully functional and gave rise to large inward currents (2-3 microA) to extracellular ATP. Coexpression of rP2X(2) and rP2X(6) subunits in Xenopus oocytes resulted in a heteromeric rP2X(2/6) receptor, which showed a significantly different phenotype from the wild-type rP2X(2) receptor. Differences included reduction in agonist potencies and, in some cases (e.g., Ap(4)A), significant loss of agonist activity. ATP-evoked inward currents were biphasic at the heteromeric rP2X(2/6) receptor, particularly when Zn(2+) ions were present or extracellular pH was lowered. The pH range was narrower for H(+) enhancement of ATP responses at the heteromeric rP2X(2/6) receptor. Also, H(+) ions inhibited ATP responses at low pH levels (<pH 6.3). The pH-dependent blocking activity of suramin was changed at this heteromeric receptor, although the potentiating effect of Zn(2+) on ATP responses was unchanged. Thus, the rP2X(2/6) receptor is a functionally modified P2X(2)-like receptor with a distinct pattern of pH modulation of ATP activation and suramin blockade. Although homomeric P2X(6) receptors function poorly, the P2X(6) subunit can contribute to functional heteromeric P2X channels and may influence the phenotype of native P2X receptors in those cells in which it is expressed.

| S-EPMC6772291 | biostudies-literature

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers.

Project description:Background and purposeThe P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7.Experimental approachWe used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging.Key resultsBoth pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation.Conclusions and implicationsWe conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.

| S-EPMC3130952 | biostudies-literature

Rat and Drosophila synaptotagmin 4 have opposite effects during SNARE-catalyzed membrane fusion.

Project description:Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca(2+). In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca(2+). Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca(2+) and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca(2+) when the conserved Asp-to-Ser Ca(2+) ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca(2+) sensor capable of coupling Ca(2+) to membrane fusion.

| S-EPMC2945570 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data