Are second messengers crucial for opening the pore associated with P2X₇ receptor?

Abstract

Stimulation of the P2X7 receptor by ATP induces cell membrane depolarization, increase in intracellular Ca2+ concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X7, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X7 pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7 pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X7 receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca2+ as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca2+ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X7 pore depends on second messengers such as Ca2+ and MAP kinases.

Pharmacological properties of a pore induced by raising intracellular Ca2+

“…Altogether, our data indicate that the pore formed by ionomycin has conductance, permeability, and cutoff similar to a pore induced by ATP (16,28). To test whether ionomycin-induced pore is the same pore formed by P2X 7 receptor, ionomycin was applied in the presence of different blockers of the P2X 7 receptor.…”

“…Ferrari and colleagues (31) showed that polimixin B, which is a natural peptide used as an antibiotic, potentializes ATP effects in the membrane permeabilization and cytotoxic effects induced by extracellular Ca 2ϩ influx mediated by P2X 7 receptor. Recently, Faria and colleagues (28) demonstrated that the ATP-induced pore can be potentialized by Ca 2ϩ ionophores.…”

“…Macrophages obtained from the intraperitoneal cavity of Swiss mice were prepared in our laboratory as previously described (28). Cells were incubated at 37°C in a 5% CO 2 humidified atmosphere.…”

“…In the other, P2X 7 channel is formed upon activation, inducing the production or release of a second messenger that may autoactivate P2X 7 receptor or an independent pore-forming membrane protein (16,61). Recently, we have demonstrated that the action of second messengers is necessary to form the pore (28). Moreover, our data indicated that there was actually a pore associated to P2X 7 , i.e., the P2X 7 channel is different from the low-selective pore.…”

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

“…p38 MAPK and caspase inhibitors have been reported to impair P2X7-induced pore formation [33,34]. To exclude the possibility that the above compounds impaired P2X7-induced apoptosis of MEL cells by directly inhibiting P2X7, cells were pre-incubated in the presence of DMSO, SB202190, SB203580 or Z-VAD-FMK (as above), and the P2X7-induced ethidium + uptake assessed.…”