Abstract
P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.
Highlights
The P2X7 receptor (P2X7) is a ligand-gated ion channel activated by extracellular ATP [1]
In contrast to HEK-hP2X7 cells, ATP-induced dye uptake was reduced by both CBX and probenecid in J774 macrophages (Figure 1C) and both drugs could reduce spontaneous dye uptake in HEK-293 cells transfected with pannexin-1 (Figure S1)
Our data reveal human P2X7 as another target for probenecid and we suggest that caution be used when using this compound to block pannexin-1 in human cells
Summary
The P2X7 receptor (P2X7) is a ligand-gated ion channel activated by extracellular ATP [1]. P2X7 activation opens an ion channel pore allowing permeation of mono- and divalent cations such as Na+, K+ and Ca2+. At least two distinct pathways are thought to exist for uptake of cations and anions into macrophages [3]. Whilst it is still not known what the physiological role of the secondary pore pathway is, it is clear that signals mediated through this pathway play an important role in P2X7 downstream signalling. A mutation in the human P2X7 receptor C-terminus which abolishes the secondary pore pathway impairs induction of IL-1b and IL-18 processing and secretion [5,6]
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