Mechanism of Modification of the NR1 Subunit of the Cerebral NMDA Receptor during Graded Hypoxia in Newborn Piglets

Abstract 2011 Poster Session II, Sunday, 5/2 (poster 232)

Previous studies have shown that phosphorylation of the serine and tyrosine residues of the NR1 subunit of the cerebral N-methyl-D-aspartate (NMDA) receptor results in receptor activation and increased intracellular Ca⁺⁺ flux during normoxia in isolated neurons. During hypoxia, NMDA receptor activation increases and correlates with the hypoxia-induced decrease in cerebral energy metabolism. The present study tests the hypothesis that phosphorylation of the serine, threonine and tyrosine residues of the NR1 subunit is a mechanism of NMDA receptor modification during hypoxia and that the degree of phosphorylation is a function of the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 15 anesthetized and ventilated newborn piglets. In the hypoxic group (n=9) varying degrees of cerebral energy metabolism were achieved by administration of different concentrations of FiO₂ (5-9%) and were documented biochemically by measuring tissue phosphocreatine (PCr) levels. P₂ membrane proteins were immunoprecipitated with antiphosphoserine, antiphosphotyrosine or antiphosphothreonine antibodies and separated by 8% SDS-PAGE. Proteins and transblotted and probed with NR1 antibodies. The phosphorylated subunit proteins were visualized with horseradish peroxidase conjugate. Protein densities were analyzed by imaging densitometry (GS-700 Bio-Rad) and expressed as absorbance × mm². Results were expressed as the percent of peak density for each gel. An internal standard was used as a reference. During graded hypoxia there was an inverse linear correlation between tissue PCr levels and the % density of the NR1 subunit (r=0.84) and phosphorylated serine residues (r=0.58) but not with phosphorylated threonine residues or phosphorylated tyrosine residues. As tissue PCr values decreased from 3.5 to 0.5 µmol/g brain, the density of the NR1 subunit increased by 82%, phosphorylated serine residues increased by 37%, phosphorylated threonine residues by 10% and phosphorylated tyrosine residues by 10%. Calculating for the increase in density of the NR1 subunit during graded hypoxia, there was an overall 55% decrease in density of phosphorylated serine residues, an 88% decrease in density of phosphorylated threonine residues and an 88% decrease in density of phosphorylated tyrosine residues. The data demonstrate that as cerebral energy metabolism decreases, the ratio of phosphorylated to dephosphorylated residues of the NR1 subunit of the NMDA receptor decreases, with a preferential decrease in percentage of phosphorylated threonine and tyrosine residues. We speculate that the relative increase in density of dephosphorylated NR1 subunits during hypoxia may be due to an increase in NR1 subunits which either have fewer serine, threonine or tyrosine residues or a decreased interaction with protein kinases due to a conformational change of the receptor.

(Funded by NIH HD-20337).