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Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis

Nature volume 467, pages 863–867 (2010)Cite this article

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Abstract

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes1. The nucleotides ATP and UTP represent one class of find-me signals2, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes3. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

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Figure 1: Release of find-me signals by apoptotic cells is pannexin-1-dependent.
Figure 2: Pannexin 1 expression level correlates with find-me signal release and membrane permeability.
Figure 3: Carbenoxolone-sensitive current induced during apoptosis is pannexin-1-dependent.
Figure 4: Pannexin 1 is a target of effector caspase cleavage during apoptosis.
Figure 5: Caspase-mediated cleavage of pannexin 1 results in channel activation during apoptosis

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Acknowledgements

We thank members of the Ravichandran laboratory for their comments and suggestions. We also thank R. E. Campbell for providing the construct for mBlueberry2. This work was supported by the Pharmacological Sciences Training Grant (F.B.C. and J.K.S.) (NIGMS), a F30 pre-doctoral fellowship from the NHLBI (F.B.C.), a pre-doctoral fellowship from the AHA (J.K.S.), and grants from the National Institutes of Health (K.S.R.). J.M.K. is supported by grants from the American Heart Association (Scientist Development Grant) and the American Cancer Society. K.S.R. is a William Benter Senior Fellow of the American Asthma Foundation.

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Authors and Affiliations

  1. Beirne B. Carter Center for Immunology Research,,

    Faraaz B. Chekeni, Michael R. Elliott, Scott F. Walk, Jason M. Kinchen, Allison J. Armstrong & Kodi S. Ravichandran

  2. Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA,

    Faraaz B. Chekeni, Joanna K. Sandilos & Douglas A. Bayliss

  3. The Center for Cell Clearance, University of Virginia, Charlottesville, 22908, Virginia, USA

    Michael R. Elliott, Scott F. Walk, Jason M. Kinchen, Allison J. Armstrong & Kodi S. Ravichandran

  4. Department of Microbiology, University of Virginia, Charlottesville, 22908, Virginia, USA

    Jason M. Kinchen & Kodi S. Ravichandran

  5. Cardiovascular Research Center and the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, 22908, Virginia, USA

    Brant E. Isakson

  6. Department of Pharmacology, University of North Carolina, Chapel Hill, 27599, North Carolina, USA

    Eduardo R. Lazarowski

  7. Department of Anatomy and Cell Biology, University of Western Ontario, London, N6A 5C1, Ontario, Canada

    Silvia Penuela & Dale W. Laird

  8. Program in Apoptosis and Cell Death Research, The Burnham Institute for Medical Research, La Jolla, 92037, California, USA

    Guy S. Salvesen

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  1. Faraaz B. Chekeni
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Contributions

F.B.C. designed, performed, and analysed most of the experiments in this study with input from K.S.R. M.R.E. performed the migration assays, air pouch experiments, qPCR, and flow cytometry, and provided advice and help on many aspects of this work. J.K.S. performed and analysed the patch-clamp studies with input from D.A.B. S.F.W. generated the PANX1 cleavage-site mutants. J.M.K. conducted phagocytosis assays and aided with flow cytometry of monomeric cyanine dye uptake by transiently transfected cells. Quantification of UTP in supernatants was performed by E.R.L. A.J.A. demonstrated pannexin 1 cleavage in apoptotic thymocytes. S.P., D.W.L. and G.S.S. provided key reagents and intellectual input. B.E.I. performed the scrape assay, and provided intellectual input on these studies. K.S.R. provided overall coordination with respect to conception, design, and supervision of the study. F.B.C. and K.S.R. wrote the manuscript with comments from co-authors.

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Correspondence to Kodi S. Ravichandran.

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Chekeni, F., Elliott, M., Sandilos, J. et al. Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 467, 863–867 (2010). https://doi.org/10.1038/nature09413

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