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Characterization of P2X7 isoform B sheds light on the role of pore versus channel activity in P2X7 mediated life and death.
No full textP2X7 isoform B (Gene bank AY847299, P2X7B) is a naturally occurring splice variant of P2X7 full-length receptor (Gene
Bank accession n NT-009775, P2X7A). P2X7B mRNA retains introns between exons 10 and 11 thus causing the loss of the
C terminal tail and incorporation of 18 amminoacids. If compared to P2X7A, P2X7B variant shows a reduced activity as
ion channel and a complete loss of the pore forming ability.
We engineered and stably expressed P2X7B in HEK293 cells were we could confirm that its activity is restricted to that of
small anion channel. P2X7B also loose P2X7A ability of ATP release in the extracellular milieu.
Nevertheless, P2X7B expression confers to HEK293 cells a longer survival in the absence of serum. P2X7B transfectants
also gain an increased competence in growth and migration in serum free soft agar, suggesting a possible role of the receptor
in facilitating tissue infiltration.
This increased resistance to unfavorable colture conditions is dependent on extracellular ATP, as treatment with apyrase
significantly reduces it. P2X7B transfectants show an higher content of intracellular ATP if compared to mock cells,
suggesting an increased metabolic activity. This hypothesis is also supported by a greater content of reticular calcium in
P2X7B cells and by an increased nuclear translocation of the calcineurin activated nuclear factor NFATc1. Moreover, in the
same cells there is and loss of Bz-ATP evoked cell blebbing.
It is tempting to speculate that while P2X7B expression will represent a proliferative advantage for the cell its counterpart,
P2X7A, will also expose it to death. These different phenotypes will be dependent on channel versus pore forming activity
Stable expression in HEK293 cells and characterization of P2X7 isoform B: new hints on P2X7 mediated cell survival.
No full textP2X7 receptor mediated proliferation: a mitochondrial-reticular explanation
No full textThe P2X7 receptor is an ATP gated ion channel, that is endowed with a strong pro-apoptotic activity but also promotes cell survival in the absence of serum. While the pro-apoptotic and pro-necrotic activity of the receptor has been widely investigated, little is known about its growth promoting function. In the last years we focused our attention on the organellar network, fundamental for both cell survival and death, studying the behavior of mitochondria and endoplasmic reticulum (ER) in HEK cells expressing the P2X7 receptor. Thanks to targeted calcium sensing probes we were able to measure increased mitochondrial calcium levels and enhanced reticular calcium uptake in receptor transfected cells versus mock. Experiments with thapsigargin on cell populations allowed us to estimate a total content of reticular calcium significantly higher for P2X7 transfectants. P2X7 expressing clones also resulted more sensitive to carbachol stimulation that caused a burst in both reticular and mitochondrial calcium levels.
This organellar calcium increase correlated with longer survival in the absence of serum and enhanced ATP production. These data allow us to propose a model in which P2X7 expression will determine an increased cellular calcium influx that will be stored in both mitochondria ad ER causing on one hand an higher energy production and on the other stimulating the proliferative pathways involving a release of calcium from the endoplasmic reticulum
Increased reticular calcium content is related to NFATc1 activation and apoptosis resistance due to P2x7R expression
No full textIl recettore purinergico P2X7 come modulatore della fisiologia mitocondriale e del processo apoptotico
No full textTHE N-TERMINAL FATTY AMINO ACID 6-METHYLHEPTANOIC/OCTANOIC-DAB IS CRUCIAL FOR POLYMYXIN B-MEDIATED MODULATION OF P2X7 RECEPTOR FUNCTIONS IN IMMUNE AND NON IMMUNE CELLS
No full text
Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule
No full textBACKGROUND AND PURPOSE: P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown. EXPERIMENTAL APPROACH: We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed. KEY RESULTS: In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion. CONCLUSIONS AND IMPLICATIONS: The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor
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