title: Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Pottorf
authors.forename: Tanner M
authors.initials: TM
authors.lastname: Johanns
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Derrington
authors.forename: Emanuel E
authors.initials: EE
authors.lastname: Strehler
authors.forename: Agnes
authors.initials: A
authors.lastname: Enyedi
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Thayer
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D018698:Glutamic Acid / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006624:Hippocampus / Q000166:cytology*; D007150:Immunohistochemistry; D018613:Microscopy, Confocal / Q000379:methods; D016202:N-Methylaspartate / Q000494:pharmacology; D009433:Neural Inhibition / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D010447:Peptide Hydrolases / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases; D051381:Rats; D013997:Time Factors; D014162:Transfection / Q000379:methods
chemical_list: D000590:Amino Acid Chloromethyl Ketones; C506961:Atp2b2 protein, rat; D027682:Cation Transport Proteins; D004151:Dipeptides; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D049452:Green Fluorescent Proteins; C071482:calpeptin; D018698:Glutamic Acid; D016202:N-Methylaspartate; D010447:Peptide Hydrolases; D000251:Adenosine Triphosphatases; C034688:sodium calcium ATPase; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium; C058076:calpain inhibitor III