Initial studies analyzing the NT2 cell line as a model for human N-methyl-D-aspartate (NMDA) receptor function demonstrated that these cells express ligand (MK-801) sensitive receptors with a receptor affinity comparable to values for the human brain and ten times lower than those of animal models. The present study determines the binding characteristics of the NMDA receptor in this human cell line in the presence of the biogenic amine spermine. NT2 cells derived from a human teratocarcinoma cell line can be induced to terminally differentiate into a stable neuronal phenotype simulating the late embryonic CNS neuron. In the present studies[3H]MK-801 binding was used as an index of NMDA receptor channel activity. Membranes were prepared from terminally differentiated NT2 cells which were lysed and washed 6 times prior to performing binding assays. Spermine-dependent activation of [3H]MK-801 binding was performed at spermine concentrations of 10 μM, 30 μM and 100 μM and was carried out at 32°C for 3 hrs (in 10 mM HEPES buffer pH 7.0, 0.1 mM K+-EDTA), with 150 μg membrane protein and 75 nM [3H]MK-801 in the presence or absence of glutamate and glycine (100 μM). Non-specific binding was carried out in the presence of 625 μM unlabelled MK-801. Results show in the absence of glutamate and glycine, 10 μM spermine activated NMDA receptors (enhanced MK-801 binding) to levels 131% above baseline. Above 10 μM, however, spermine decreased binding to below baseline (87% and 49% at 30 μM and 100 μM respectively). In the presence of glutamate and glycine, spermine had no effect on [3H]MK-801 binding at 10 μM concentration and inhibited binding at 30 μM and 100 μM (73% and 64% respectively). The data demonstrate that the response to spermine seen in these human NMDA receptors differs markedly from that seen in other species, but supports human post-mortem studies showing inhibition in fetal/neonatal samples and activation in older specimens. As the NMDA receptor has been shown to play a central role in learning, it may be these properties, unique to the human NMDA receptor, that underly the differences between human and non-human brain. Funded by NIH-HD-20337, MOD #6-FY94-0135, UCPR06-93.
