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This Article Full Text Full Text (PDF) All Versions of this Article: 297/1/C28    most recent 00476.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Faria, R.X. Articles by Alves, L.A. PubMed PubMed Citation Articles by Faria, R.X. Articles by Alves, L.A.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Pharmacological properties of a pore induced by raising intracellular Ca²⁺

¹Laboratory of Cellular Communication and ²Laboratory of Cell Biology, Oswaldo Cruz Institute, Fiocruz; and ³Program in Neurobiology, Institute of Biophysics, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Submitted 19 September 2008 ; accepted in final form 19 March 2009

Recent studies on the P2X₇ receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca²⁺ concentration induces a pore opening similar to P2X₇ receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca²⁺ concentration is associated to P2X₇ receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X₇ receptor (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 (t) for ethidium bromide uptake of 118 ± 17 s, and t for Lucifer yellow of 122 ± 11 s. P2X₇ receptor 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 P2X₇ receptor 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⁺² induces a novel membrane pore pharmacologically different from the P2X₇ associated pore and hemigap-junction pore.

P2X₇ receptor; pore formation; second messenger

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