Effect of Dephosphorylation on Spermine-Dependent Activation of N-Methyl-D-Aspartate (NMDA) Receptor in Normoxic Newborn Piglets. ♦ 343

Abstract

Previous studies have shown that spermine increases the maximal activation of the NMDA receptor ionchannel complex in neuronal membranes of hypoxic newborn piglets. The present study tests the hypothesis that removal of phosphate will change spermine-dependent activation of the NMDA receptor in the newborn piglet brain. 3H-MK-801 binding was used to assess receptor affinity (Kd) and density (Bmax). P2 membranes were prepared from the cerebral cortices of six normoxic piglets and then incubated at 37°C with either 10 units alkaline phosphatase or buffer for 90 min at pH=9. Membranes were rewashed. 3H-MK-801 binding was performed at 32°C in 200μl of medium containing either buffer or 10μM spermine plus 100μM glutamate, 100μM glycine, 10mM HEPES/1mM EDTA buffer (pH 7.0), increasing concentrations of 3H-MK-801 (2.5 to 50nM), and 75μg protein. Saturation curves and Scatchard plots were constructed to determine the Bmax and Kd. Without spermine, the alkaline phosphatase treatment increased the affinity of the channel as indicated by a 33% decrease in Kd(15.64±5.89 without enzyme to 10.89±6.68 nM with enzyme). Similarly, with spermine, the alkaline phosphatase treatment also increased the affinity of the channel as indicated by a 33% decrease in Kd(18.92±10.15 without enzyme to 12.32±5.74 nM with enzyme). The results demonstrate that dephosphorylation changes the affinity of the receptor for MK-801. However, spermine-dependent activation of the NMDA receptor is not affected by dephosphorylation, indicating that the interaction at the polyamine site is phosphorylation independent. Since hypoxia decreases cellular energy metabolism and results in dephosphorylation, we speculate that dephosphorylation due to hypoxia is a potential mechanism for the NMDA receptor ionchannel modification observed in the brain of the newborn piglet.