Pharmacological properties of a pore induced by raising intracellular Ca2+

Abstract

Recent studies on the P2X7receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca2+concentration induces a pore opening similar to P2X7receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca2+concentration is associated to P2X7receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X7receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5–10 μM) increased intracellular free Ca2+concentration and induced pore formation with conductance of 421 ± 14 pS, half-time ( t½) for ethidium bromide uptake of 118 ± 17 s, and t½for Lucifer yellow of 122 ± 11 s. P2X7receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca2+concentration. However, 5-( N, N-hexamethylene)-amiloride, a P2X7receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca2+at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca+2induces a novel membrane pore pharmacologically different from the P2X7associated pore and hemigap-junction pore.