Evidence for two conductive pathways in P2X7 receptor: Differences in modulation and selectivity

Institute of Biophysics, National Research Council, Genoa, Italy.
Journal of Neurochemistry (Impact Factor: 4.28). 02/2010; 113(3):796-806. DOI: 10.1111/j.1471-4159.2010.06649.x
Source: PubMed

ABSTRACT The P2X(7) receptor (P2X(7)R) is an ATP-gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated by divalent cations and organic messengers such as arachidonic acid (AA). In this study, we analysed the differential modulation of magnesium (Mg(2+)) and AA on P2X(7)R by measuring whole-cell currents and intracellular Ca(2+) ([Ca(2+)](i)) and Na(+) ([Na(+)](i)) dynamics in HEK293 cells stably expressing full-length P2X(7)R and in cells endowed with the P2X(7)R variant lacking the entire C-terminus tail (trP2X(7)R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X(7)R- and trP2X(7)R-mediated [Ca(2+)](i) rise but did not affect the ionic currents in both conditions. Extracellular Mg(2+) reduced the P2X7R- and trP2X(7)R-mediated [Ca(2+)](i) rise in a dose-dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X(7)R-mediated ionic current and [Na(+)](i) rise were strongly dependent on Mg(2+) concentration but occurred in a non-competitive manner. In contrast, in cells expressing the trP2X(7)R, the small ionic currents and [Na(+)](i) signals were totally insensitive to Mg(2+). Collectively, these results support the tenet of a functional structure of P2X(7)R possessing at least two distinct conductive pathways one for Ca(2+) and another for monovalent ions, with the latter which depends on the presence of the receptor C-terminus.

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Available from: Stefano Ferroni, Sep 29, 2015
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    • "In our context it is important that P2X7 receptor inhibition activates neuritogenesis in a CaMKII-dependent pathway (Leon et al., 2006; Gomez-Villafuertes et al., 2009) in neuronal cells. As Mg 2þ demonstrates an antagonistic effect on P2X7 receptor activation, as demonstrated in a variety of cells (Jiang, 2009; Alloisio et al., 2010; Lee et al., 2011) (Fig. 2), this points to a "
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    ABSTRACT: Unlabelled: The glutamatergic mechanism of antidepressant treatments is now in the center of research to overcome the limitations of monoamine-based approaches. There are several unresolved issues. For the action of the model compound, ketamine, NMDA-receptor block, AMPA-receptor activation and BDNF release appear to be involved in a mechanism, which leads to synaptic sprouting and strengthened synaptic connections. The link to the pathophysiology of depression is not clear. An overlooked connection is the role of magnesium, which acts as physiological NMDA-receptor antagonist: 1. There is overlap between the actions of ketamine with that of high doses of magnesium in animal models, finally leading to synaptic sprouting. 2. Magnesium and ketamine lead to synaptic strengthening, as measured by an increase in slow wave sleep in humans. 3. Pathophysiological mechanisms, which have been identified as risk factors for depression, lead to a reduction of (intracellular) magnesium. These are neuroendocrine changes (increased cortisol and aldosterone) and diabetes mellitus as well as Mg(2+) deficiency. 4. Patients with therapy refractory depression appear to have lower CNS Mg(2+) levels in comparison to health controls. 5. Experimental Mg(2+) depletion leads to depression- and anxiety like behavior in animal models. 6. Ketamine, directly or indirectly via non-NMDA glutamate receptor activation, acts to increase brain Mg(2+) levels. Similar effects have been observed with other classes of antidepressants. 7. Depressed patients with low Mg(2+) levels tend to be therapy refractory. Accordingly, administration of Mg(2+) either alone or in combination with standard antidepressants acts synergistically on depression like behavior in animal models. Conclusion: On the basis of the potential pathophysiological role of Mg(2+)-regulation, it may be possible to predict the action of ketamine and of related compounds based on Mg(2+) levels. Furthermore, screening for compounds to increase neuronal Mg(2+) concentration could be a promising instrument to identify new classes of antidepressants. Overall, any discussion of the glutamatergic system in affective disorders should consider the role of Mg(2+).
    Journal of Psychiatric Research 03/2013; 47(7). DOI:10.1016/j.jpsychires.2013.02.015 · 3.96 Impact Factor
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    • "ase We recently addressed P2X7 receptor gating behavior by comparing full - length and truncated rP2X7 receptors expressed in HEK293 cells . The model predicted two distinct conductive pathways : one for Ca 2+ , which still functioned in the truncated receptor lacking the C - tail ; the other for NMDG + , which was lost in the truncated receptor ( Alloisio et al . 2010 ) . Taken together , our previous ( Marcoli et al . 2008 ) and present data indicate that these two rP2X7 pathways may be linked to glutamate release : the pathway conductive for Ca 2+ being associated with the receptor function as a Ca 2+ channel coupled to glutamate exocytosis ; the pathway permeable to NMDG + being associated with th"
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    ABSTRACT: P2X7 receptors trigger Ca(2+) -dependent exocytotic glutamate release, but also function as a route for non-exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full-length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18-amino acid cysteine-rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non-competitive way by extracellular Mg(2+) , did not require the recruitment of pore-forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine-rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca(2+) elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca(2+) -dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non-exocytotic glutamate efflux. © 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12143.
    Journal of Neurochemistry 01/2013; 124(6). DOI:10.1111/jnc.12143 · 4.28 Impact Factor
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    • "Moreover, even if chemical crosslinking and blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of recombinant receptors have revealed a trimeric structure of P2X ion channels, which has been confirmed by atomic force microscopy and by crystallography, the P2X7 functional structure is still far to be determined [18] [19]. Recently, we proposed a possible functional and structural model of the P2X7R, in which the receptor could be composed of two conductive pathways: the first with the ATPbinding sites and is characterized by sensitivity to extracellular Mg 2+ and selective permeability to Ca 2+ (P2X7R-A); the second one permeable to cations and with activation and divalent cation sensitivity controlled by the first pathway (P2X7R-B) [20]. Other recent works have investigated the functional properties of P2X7R by studying the dynamics of the currents evoked by BzATP [21] [22] [23]. "
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    ABSTRACT: The P2X7 receptor (P2X7R) induces ionotropic Ca²⁺ signalling in different cell types. It plays an important role in the immune response and in the nervous system. Here, the mechanisms underlying intracellular Ca²⁺ variations evoked by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist of the P2X7R, in transfected HEK293 cells, are investigated both experimentally and theoretically. We propose a minimal model of P2X7R that is capable of reproducing, qualitatively and quantitatively, the experimental data. This approach was also adopted for the P2X7R variant, which lacks the entire C-terminus tail (trP2X7R). Then we introduce a biophysical model describing the Ca²⁺ dynamics in HEK293. Our model gives an account of the ionotropic Ca²⁺ influx evoked by BzATP on the basis of the kinetics model of P2X7R. To explain the complex Ca²⁺ responses evoked by BzATP, the model predicted that an impairment in Ca²⁺ extrusion flux through the plasma membrane is a key factor for Ca²⁺ homeostasis in HEK293 cells.
    Physical Biology 04/2012; 9(2):026001. DOI:10.1088/1478-3975/9/2/026001 · 2.54 Impact Factor
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