Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons

J Neurochem. 2006 Sep;98(5):1646-56. doi: 10.1111/j.1471-4159.2006.04063.x.


Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Amino Acid Chloromethyl Ketones / pharmacology
  • Animals
  • Calcium / metabolism
  • Calcium-Transporting ATPases / metabolism*
  • Cation Transport Proteins / metabolism*
  • Cells, Cultured
  • Dipeptides / pharmacology
  • Dose-Response Relationship, Drug
  • Embryo, Mammalian
  • Enzyme Inhibitors / pharmacology
  • Excitatory Amino Acid Agonists / pharmacology
  • Flow Cytometry / methods
  • Glutamic Acid / pharmacology*
  • Green Fluorescent Proteins / biosynthesis
  • Hippocampus / cytology*
  • Immunohistochemistry
  • Microscopy, Confocal / methods
  • N-Methylaspartate / pharmacology
  • Neural Inhibition / drug effects
  • Neurons / drug effects*
  • Peptide Hydrolases / metabolism*
  • Plasma Membrane Calcium-Transporting ATPases
  • Rats
  • Time Factors
  • Transfection / methods


  • Amino Acid Chloromethyl Ketones
  • Atp2b2 protein, rat
  • Cation Transport Proteins
  • Dipeptides
  • Enzyme Inhibitors
  • Excitatory Amino Acid Agonists
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • Green Fluorescent Proteins
  • calpeptin
  • Glutamic Acid
  • N-Methylaspartate
  • Peptide Hydrolases
  • Adenosine Triphosphatases
  • sodium calcium ATPase
  • Plasma Membrane Calcium-Transporting ATPases
  • Calcium-Transporting ATPases
  • Calcium
  • calpain inhibitor III