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

AbstractCa2+ 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 (τ) of 8.8 ± 0.2 s. Four hours following the start of a 30 min treatment with 200 µm glutamate, a second population of cells emerged with slowed recovery kinetics (τ = 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.