Depolarization induces NR2A tyrosine phosphorylation and neuronal apoptosis.

BACKGROUND Cytosol Ca2+ overload plays a vital role in ischemic neuronal damage, which is largely contributed by the Ca2+ influx through L-type voltage-gated calcium channels (L-VGCCs) and N-methyl-D-aspartate (NMDA) type glutamate receptors. In this article, L-VGCCs were activated by depolarization to investigate the cross-talk between NMDA receptors and L-VGCCs. METHODS Depolarization was induced by 20 minutes incubation of 75 mM KCl in cultured rat cortical neuron. Apoptosis-like neuronal death was detected by DAPI staining. Tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A), interactions of Src and NR2A were detected by immunoblot and immunoprecipitation. RESULTS Depolarization induced cortical neuron apoptosis-like cell death after 24 hours of restoration. The apoptosis was partially inhibited by 5 mM EGTA, 100 μM Cd2+, 10 μM nimodipine, 100 μM genistein, 20 μM MK-801, 2 μM PP2 and combined treatment of nimodipine and MK-801. NR2A tyrosine phosphorylation increased after depolarization, and the increase was inhibited by the drugs listed above. Moreover, non-receptor tyrosine kinase Src bound with NR2A after depolarization and restoration. The binding was also inhibited by the drugs listed above. CONCLUSIONS The results indicated that depolarization-induced neuronal death might be due to extracellular Ca2+ influx through L-VGCCs and subsequently Src activationmediated NR2A tyrosine phosphorylation.