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

Ca²⁺ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca ²⁺ ATPase (PMCA) plays a major role in clearing Ca²⁺ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca ²⁺ 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 Ca ²⁺-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.