Membrane Lipid Compositions Control Dye-Permeable Pore of the P2X7 Receptor

Abstract

The P2X7 receptor is an extracellular ATP-gated cation channel. Activation of P2X7 leads to the opening of a dye permeable pore, which causes cell lysis and eventually results in various health problems including chronic pain, neurodegeneration, and tumor growth. However, it remains controversial what constitutes this dye-permeable pore and how it opens. Two prevailing hypotheses are: 1) P2X7 dilates its transmembrane channel to form a larger pore and 2) P2X7 activation recruits other large conductance channels. To clarify whether P2X7 itself constitutes a dye permeable pore, we purified and reconstituted P2X7 into liposomes and measured pore activity. We observed strong dye uptake from P2X7 reconstituted liposomes, suggesting that P2X7 can form a dye permeable pore in the absence of other cellular components. To our surprise, dye uptake was independent of the C-terminal domain, which has been considered to be absolutely necessary for pore formation. Furthermore, dye uptake was immediately observed after ATP application, indicating that pore dilation is unlikely. We also discovered that the P2X7-dependent pore was facilitated by phosphatidylglycerol and sphingomyelin but strongly inhibited by cholesterol through direct interactions with the transmembrane domain of P2X7. In combination with a fatty acid incorporation assay, our results suggest that palmitoylation at the C-terminal domain counteracts the inhibitory effect of cholesterol by facilitating P2X7 channel opening.