Stimulation of the P2X7receptor 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 P2X7pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7pore 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 P2X7receptor 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 P2X7pore depends on second messengers such as Ca2+and MAP kinases.
Single channel currents activated by gamma-aminobutyric acid, muscimol, and (-)-pentobarbital in cultured mouse spinal neurons
