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Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity

Nature Neuroscience volume 19, pages 432442 (2016) | Download Citation

Abstract

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca2+-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg2+ or a Ca2+-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

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Acknowledgements

We thank J. Bains (University of Calgary) for critical reading of the manuscript and C. Sank for technical assistance. Plasmids encoding Panx1Y308F, GluN1WT and GluN1N616R were generated by F. Visser in the Hotchkiss Brain Institute Molecular Core Facility. Dominant active/inactive constructs for Src kinase and Src48 peptides were from D. Fujita (University of Calgary) and M. Salter (Sick Kids Research Institute), respectively. Myc-tagged Panx1 constructs were from C. Naus (University of British Columbia). This work was supported by Canadian Institutes for Health Research (CIHR) grants 136812 (R.J.T.), 110967 (I.R.W.), 201022 (M.A.C.) and 130495 (G.C.T.). R.J.T., G.C.T., M.A.C. and I.R.W. received grants from Alberta Innovates–Health Solutions (AI-HS), the Canadian Foundation for Innovation and the Natural Sciences and Engineering Research Council of Canada. R.J.T. and I.R.W. received funds from AI-HS. Additional support was provided to R.J.T. by the Cumming School of Medicine via the Ronald and Irene Ward Foundation and the Gwendolyn McLean Fund. N.L.W. is supported by an AI-HS scholarship and Dr. T. Chen Fong scholarship from the Hotchkiss Brain Institute. A.W.L. is supported by AI-HS and CIHR.

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Affiliations

  1. Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada.

    • Nicholas L Weilinger
    • , Alexander W Lohman
    • , Brooke D Rakai
    • , Evelyn M M Ma
    • , Jennifer Bialecki
    • , Valentyna Maslieieva
    • , Travis Rilea
    • , Lucas Scott
    • , Michael A Colicos
    • , G Campbell Teskey
    •  & Roger J Thompson
  2. Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

    • Nicholas L Weilinger
    • , Alexander W Lohman
    • , Evelyn M M Ma
    • , G Campbell Teskey
    •  & Roger J Thompson
  3. Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Alberta, Canada.

    • Mischa V Bandet
    • , Nathan T Ikuta
    •  & Ian R Winship
  4. Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

    • Lucas Scott
    •  & Michael A Colicos

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Contributions

N.L.W., A.W.L. and R.J.T. designed the experiments. N.L.W. performed and analyzed the electrophysiological and laser-imaging experiments, and wrote the initial draft of the paper that all authors then edited. B.D.R. designed, performed and analyzed the in vivo surgeries, lesion volumes and behavioural tests. A.W.L. performed and analyzed the molecular biology and biochemistry experiments. E.M.M.M. performed and analyzed the calcein dye-efflux experiments. J.B. analyzed behavioural recordings (in a blinded fashion). V.M. and T.R. performed the initial characterization of the phospho-specific pY308Panx1 antibody. M.V.B., N.T.I. and I.R.W. performed and analyzed in vivo cerebral blood flow imaging experiments. L.S. and M.A.C. assisted with the development and maintenance of primary cell cultures. G.C.T. supervised the in vivo stroke experiments. R.J.T. supervised the study and wrote the paper with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Roger J Thompson.

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https://doi.org/10.1038/nn.4236

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