Caspases Mediate Pannexin 1 Channel Activation in Apoptotic Cells

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Pannexins represent a recently discovered family of membrane proteins that form large conductance (400-500 pS) membrane channels permeable to ions and small intracellular signaling molecules. The mechanisms that regulate pannexin channel activity and their physiological roles remain incompletely understood. Here, we used whole cell voltage clamp recordings from Jurkat cells to demonstrate induction of a Pannexin 1 (Panx1)-dependent current only in apoptotic cells; the current was not observed in Jurkat cells that were not undergoing apoptosis, even when Panx1 was overexpressed. The apoptosis-induced currents were attributed to Panx1 through a combination of pharmacological profiling, over-expression, and siRNA knockdown experiments. We found that Panx1 is a target of apoptosis-induced effector caspases (caspases 3 and 7), and that a specific caspase-cleavage site within the channel C-terminus is required for activation of Panx1 currents during apoptosis. Furthermore, expression of a Panx1 construct that is truncated at the C-terminal caspase cleavage site resulted in constitutive Panx1 currents, even in non-apoptotic cells. In addition, uptake of Yo-Pro or To-Pro dyes characteristic of apoptotic cells was altered by these manipulations in direct correspondence with changes in membrane current, suggesting that dye uptake is mediated by Panx1 channels and provides a faithful surrogate for Panx1 currents. Likewise, ATP and UTP release from apoptotic cells was Panx1-dependent. Together, these data reveal a novel mechanism of Panx1 activation by caspase-dependent cleavage in apoptotic cells. Moreover, this work identifies a new physiological role for Panx1 channels in apoptotic cell clearance since Panx1 activation mediates release of ATP and UTP, two nucleotides that provide ‘find-me’ signals for recruitment of phagocytes to apoptotic cells.