Role of NMDA receptor functional domains in excitatory cell death.
The mechanisms by which the NMDA receptor (NMDAR) induces excitotoxicity were investigated using a novel assay. We quantitated the capacity of wild type and mutant receptors for cell killing in CHO cells and cultured cortical neurons by measuring the activity of a co-transfected firefly luciferase expression plasmid. NR1 subunit pore mutations that block Ca(2+) influx, and deletion of the NR1 cytoplasmic C-terminal domain, which functions in Ca(2+) regulation of receptor currents, decreased NMDAR mediated cell killing. We also transfected the NR1 pore mutants and C-terminal truncations in the presence of co-expressed exogenous wild type subunits. The pore and C-terminal truncation mutants acted in a dominant negative fashion and increased the survival of NMDAR-expressing CHO cells. Although physiological studies of similar NMDA receptor mutants have been carried out in heterologous cell lines, their functions in neurons remain relatively unknown. We show that expression of pore mutants and specific C terminal truncation mutants in cultured cortical neurons also exerts dominant negative function and protects these primary cells from endogenous receptor induced excitotoxic death. These results implicate positive actions of the selectivity filter and of the NR1 C-terminal domain in a Ca(2+)-dependent mechanism for NMDAR excitotoxicity. They also indicate that the mutant receptors which show diminished excitotoxicity and dominant negative action in heterologous cells can co-assemble with endogenous subunits in primary neurons and block NMDAR-dependent excitotoxic death.
Rameau
G A
GA
Howard Hughes Medical Institute, Department of Biochemistry, NYU School of Medicine, New York, NY 10016, USA.
Akaneya
Y
Y
Chiu
L
L
Ziff
E B
EB
D000818:Animals; D016466:CHO Cells; D016923:Cell Death / Q000502:physiology; D002470:Cell Survival; D002540:Cerebral Cortex / Q000166:cytology; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D018846:Excitatory Amino Acids / Q000633:toxicity*; D008156:Luciferases / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D018846:Excitatory Amino Acids; D016194:Receptors, N-Methyl-D-Aspartate; D004247:DNA; D008156:Luciferases