Our previous studies have demonstrated that hypoxia modifies the glutamate recognition site of the NMDA receptor ion-channel complex resulting in decreased receptor density and increased affinity. The present study tests the hypothesis that hypoxia-induced modification of the glutamate recognition site of the NMDA receptor is due to dephosphorylation. P2 membrane fractions were prepared from normoxic (FiO2 21% n=4) and hypoxic (FiO2 7% n=3) newborn piglet cerebral cortices. Both the normoxic and hypoxic membranes were incubated at 37°C with and without 10 units of alkaline phosphatase for 90 min in 10 mM diethanolamine, 0.25 mM MgCl2 (pH 9.8). Membranes were washed and 3H-glutamate binding was performed in a medium containing 10 mM HEPES/1 mM EDTA (pH 7.0), 25-1000 nM 3H-glutamate and 150 ug protein. NMDA-displaceable specific binding was performed in the presence of 100μM NMDA. Nonspecific glutamate binding was performed in the presence of 1 mM glutamate. Saturation curves and Scatchard plots were made and the characteristics (Bmax=number of receptors, Kd=dissociation constant) were determined. In the normoxic group, enzyme treatment resulted in increased Bmax (untreated: 228±69 fmoles/mg protein vs treated: 446±135 fmoles/mg protein, p<0.025, mean±SD) and Kd (untreated: 228±24 nM vs treated: 427±121 nM, p<0.025). In the hypoxic group, enzyme treatment resulted in increased Bmax (untreated: 358±42 fmoles/mg protein vs treated: 538±64 fmoles/mg protein, p<0.008) while Kd was unaffected (untreated: 336±88 nM vs treated: 496±138 nM, p=ns). The results show that alkaline phosphatase enzyme treatment increases the number of accessible glutamate sites of the NMDA receptor in both normoxic and hypoxic groups and that dephosphorylation affects the affinity of the site in normoxic brain. This data suggest that the interaction of the agonist at the glutamate recognition site is dependent upon phosphorylation/dephosphorylation. We speculate that during hypoxia, the receptor is in a dephosphorylated state and fails to respond to further dephosphorylation by the enzyme. (Funded by NIH# HD-20337)
