Effects Of Gap Junction Blockers On The P2x7 Receptor

Abstract

Peritoneal macrophages express the P2X7 receptor, which opens a pore in the membrane after long exposure with ATP, allowing passage of molecules up to 900 Daltons. It has been argued that activation of P2X7 receptor leads to opening of an independent pore entity, not structurally related to the P2X7 receptor. Based on results with connexin knock outs and pharmacological manipulation with known gap junction blockers, some groups have included connexins and pannexins, the gap junction-forming proteins in vertebrates, as reliable candidates to provide for the large permeation pores associated with P2X7 activation.In the present study we performed electrophysiological (whole cell patch clamping recordings) and permeabilization assays (optical analysis and FACS analysis) in which both efficacy and specificity of some gap junction blockers were tested at conditions of putative P2X7R activation by ATP. ATP generated a current in a nA levels that was blocked by well know P2X blockers as BBG, KN-62 and oxidized ATP, in contrast the junction blockers did not interfere with these effect. More than that, the up take assays showed similar results to the patch clamp experiments, none of the junction blockers was able to block the up take of ethidium bromide or propidium iodide. Our results indicate that well-known gap junction pharmacological blockers do not interfere with current generation or dye uptake after activation of P2X7 receptor.Taken together, our data strongly suggest that the high permeability pore evident at prolonged P2X7 activation does not correspond to connexin or pannexin hemichannels in peritoneal macrophages.