Pannexin-1 Couples to Maitotoxin- and Nigericin-induced Interleukin-1β Release through a Dye Uptake-independent Pathway

Pablo Pelegrin,Annmarie Surprenant

doi:10.1074/jbc.m610351200

Abstract

Pannexin-1 is a recently identified membrane protein that can act as a nonselective 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-1beta (IL-1beta) 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 signaling to processing and release of IL-1beta. We have pursued this hypothesis by measuring dye uptake and IL-1beta processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1beta release via the same mechanism. The experiments were carried out over time periods during which no lactate dehydrogenase was released from cells to examine only noncytolytic 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-1beta processing and release in response to all three stimuli. Thus, although pannexin-1 is required for IL-1beta release in response to maitotoxin, nigericin, and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.

Highlights

Ated by inflammatory stimuli such as lipopolysaccharide (LPS), but little or none is released in the absence of a secondary stimulus [2, 3]
To further elucidate Panx1 signaling processes underlying IL-1␤ processing/release, we have examined whether and how Panx1 may be involved in IL-1␤ release evoked by two well known nonphysiological secondary stimuli that have been thought to act via the same mechanism(s) as P2X7 receptor (P2X7R), the marine toxin, maitotoxin, which activates calcium-permeable, cationic currents and leads to ethidium and YoPro1 uptake, and the Kϩ/Hϩ antiport ionophore, nigericin [7, 15,16,17,18,19]
We find that all three stimuli require signaling through Panx1 to induce caspase-1/IL-1␤ processing and release, but Panx1 is involved only with an initial phase of P2X7R-mediated dye uptake, whereas dye-uptake pathways are not involved in IL-1␤ processing/release induced by maitotoxin or nigericin

Summary

Introduction

Ated by inflammatory stimuli such as lipopolysaccharide (LPS), but little or none is released in the absence of a secondary stimulus [2, 3]. We compared dye uptake induced by ATP, maitotoxin, and nigericin in HEK cells stably expressing P2X7R and both dye uptake and caspase-1/IL-1␤ processing/release in mouse J774 macrophage, which natively express P2X7R before and after selective blockade of Panx1.

Results

Conclusion