Pannexin-1 couples to maitotoxin and nigericin- induced il-1β release through a dye-uptake independent pathway

Abstract

Pannexin-1 is a recently identified membrane protein that can act as a non-selective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X7 receptor (P2X7R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1β (IL-1β) in response to P2X7R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X7R to provide the conduit for dye uptake and downstream signalling to processing and release of IL-1β. We have pursued this hypothesis by measuring dye uptake and IL-1β processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1β release via the same mechanism. Experiments were carried out over time periods during which no LDH was released from cells in order to examine only non-cytolytic pathways. P2X7R activation evoked dye-uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1- independent phase. Maitotoxin evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1β processing and release in response to all three stimuli. Thus, while pannexin-1 is required for IL-1β release in response to maitotoxin, nigericin and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.