Role of domain calcium in purinergic P2X2 receptor channel desensitization

Coddou, Claudio; Yan, Zonghe; Stojilković, Stanko S.

doi:10.1152/ajpcell.00399.2014

Cited by 8 publications

(14 citation statements)

References 42 publications

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“…For this purpose, we studied the patterns of currents generated by P2X2aR activation using electrophysiological techniques and tested Cdk5-mediated changes in those currents. As we have previously demonstrated, the P2X2R has a unique property, an increase in receptor desensitization rate after repetitive ATP applications that is termed use-dependent desensitization (UDD) [14; 27]. In HEK293 cells expressing the P2X2aR and measured under the whole-cell configuration, receptor desensitization increased after repetitive 10 μM ATP applications in the presence of extracellular Ca 2+ (Figure 4A).…”

Section: Resultsmentioning

confidence: 79%

“…We found that the activation of endogenous Cdk5, by the expression of its activator p35, significantly prevented P2X2aR desensitization after repetitive ATP applications. We have previously shown that the P2X2aR increases its desensitization rates after successive ATP applications, arising a Ca 2+ -dependent process termed UDD [14; 27]. The most plausible explanation for this phenomenon is that Cdk5-phosphorylation favors a non-desensitized phenotype and protects the receptor for the Ca 2+ -dependent UDD.…”

Section: Discussionmentioning

confidence: 99%

“…The most plausible explanation for this phenomenon is that Cdk5-phosphorylation favors a non-desensitized phenotype and protects the receptor for the Ca 2+ -dependent UDD. We have previously showed that UDD requires the presence of Ca 2+ in a nanomolar or less concentration to be observed in whole-cell recordings [14] and this could reflect the loss of some relevant cytosolic factor to determine P2X2aR desensitization. This can explain the differences observed in the recordings performed in HEK293 cells and Xenopus oocytes, though future experiments will help to completely clarify the mechanism of P2X2aR desensitization.…”

Section: Discussionmentioning

confidence: 99%

“…the shorter variant P2X2bR, lacks a 69 amino acids in the intracellular C-terminal domain and desensitizes faster than the full length P2X2aR [5; 28; 48]. Compared to other P2XR subtypes, P2X2Rs exhibit slow desensitization profile, which depends on agonist and Ca 2+ concentration, and in N- and C-terminal residues/domains [13; 14; 27]. The gating properties of P2X2R are also affected by protons, divalent metals [11; 34], reactive oxygen species [12], steroid hormones [18], and membrane phosphoinositides [22], all these molecules act as allosteric regulators.…”

Section: Introductionmentioning

confidence: 99%

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Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Coddou

Sandoval

Castro

et al. 2017

PAIN

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The purinergic P2X2 receptor (P2X2R) is an ATP-gated ion channel widely expressed in the nervous system. Here, we identified a putative Cdk5 phosphorylation site in the full-size variant P2X2aR (372TPKH375), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine-phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5-activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells, and P2X2/3R mediated increases of intracellular Ca2+ in trigeminal neurons were Cdk5-dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

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Section: Resultsmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Coddou

Sandoval

Castro

et al. 2017

PAIN

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“…26460693] ABBREVIATIONS: 17-AAG, 17-N-allylamino-17-demethoxygeldanamycin; 17-DMAG, 17-dimethylaminoethylamino-17-demethoxygeldanamycin; Bz-ATP, 29 (39)-O-(4-benzoylbenzoyl)adenosine-59-triphosphate; HEK, human embryonic kidney; HSP, heat shock protein; NMDG 1 , N-methyl-transfer across the membrane (called "current facilitation") (Yan et al, 2008(Yan et al, , 2010Khadra et al, 2013;Rokic and Stojilkovic, 2013). In contrast, the long applications of ATP needed to induce the permeability changes in P2X2Rs result in a decrease in charge transfer because the receptor desensitizes (Khakh et al, 1999;Coddou et al, 2015). The different time-dependent effects of ATP on the size of P2X2R and P2X7R currents suggest that chimeras of these channels may be useful tools to determine the locus of the HSP90 effect on P2X7R function.…”

Section: Introductionmentioning

confidence: 99%

HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus

Migita

Ozaki

Shimoyama

et al. 2016

Molecular Pharmacology

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P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-D-glucamine 1 (NMDG 1 ). The effects of genetic modification of the C-terminus on NMDG 1 permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATPgated current measured under bi-ionic NMDG 1 /Na 1 condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus.

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Rehabilitation of the P2X5 receptor: a re-evaluation of structure and function

King

2022

Purinergic Signalling

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Of the extended family of ATP-gated P2X ion-channels, the P2X5 receptor has received comparatively little attention since first cloned over 25 years ago. Disinterest in studying this P2X subtype stems from two commonly held beliefs: (i) canonical human P2X5 is non-functional because the P2X5 subunit is truncated (hP2X5A, 422 aa) and missing the critical peptide sequence (22 aa) encoded by exon 10; (ii) rat and mouse P2X5 subunits are fully formed (455 aa) but the receptor is only weakly functional, and successive ATP responses rapidly run down in amplitude. However, newer studies have re-evaluated these notions. First, a low proportion (around 10%) of humans possess full-length P2X5 subunits (444 aa) and can form competent P2X5 receptors. Full-length P2X5 has been identified only in black Americans, but may occur in a wider population as more ethnicities are screened. Second, replacement of one of three amino acids in rat P2X5 subunits with corresponding residues in human P2X5 subunits (V67I, S191F, or F195H) significantly improves the responsiveness of rat P2X5 to ATP. Replaced residues exert an allosteric action on the left flipper, allowing the docking jaw for ATP to flex the lower body of the subunit and fully open the ion pore. This proposed action may drive the search for naturally occurring modulators which act allosterically on wildtype rat P2X5. This review collates the available information on the structure and function of human and rat P2X5 receptors, with the view to rehabilitating the reputation of these ATP-gated ion channels and stimulating future lines of research.

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Role of domain calcium in purinergic P2X2 receptor channel desensitization

Cited by 8 publications

References 42 publications

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus

Rehabilitation of the P2X5 receptor: a re-evaluation of structure and function

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