Related Experiment Videos
Role of NMDA receptor functional domains in excitatory cell death
G A Rameau1, Y Akaneya, L Chiu
1Howard Hughes Medical Institute, Department of Biochemistry, NYU School of Medicine, New York, NY 10016, USA.
Abstract:
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.
Insights
Mutant NMDA receptors (NMDARs) with blocked calcium influx or altered C-terminal domains reduce excitotoxicity. These mutants protect cells by inhibiting endogenous NMDARs, revealing key mechanisms in excitotoxicity.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- NMDA receptors (NMDARs) are crucial for synaptic plasticity but can mediate excitotoxicity.
- Understanding NMDAR-mediated excitotoxicity is vital for neurological disease research.
- The precise roles of NMDAR pore mutations and C-terminal domains in excitotoxicity require further elucidation.
Purpose of the Study:
- To investigate the mechanisms of NMDA receptor (NMDAR) induced excitotoxicity.
- To quantify the cell-killing capacity of wild-type and mutant NMDARs.
- To determine the dominant-negative effects of NMDAR mutants in neuronal cells.
Main Methods:
- Utilized a novel assay measuring firefly luciferase activity to quantify cell killing.
- Expressed wild-type and mutant NMDAR subunits (NR1 pore mutations, C-terminal deletions) in CHO cells and cultured cortical neurons.
- Assessed the dominant-negative function of mutants by co-expressing them with wild-type subunits.
Main Results:
- NR1 subunit pore mutations blocking Ca(2+) influx and C-terminal deletions reduced NMDAR-mediated cell killing.
- Mutant NMDARs exhibited dominant-negative effects, increasing cell survival in CHO cells.
- Mutant NMDARs also conferred dominant-negative protection in primary cortical neurons against excitotoxic death.
Conclusions:
- The selectivity filter and NR1 C-terminal domain play positive roles in Ca(2+)-dependent NMDAR excitotoxicity.
- Mutant NMDARs can co-assemble with endogenous subunits in neurons, inhibiting excitotoxic death.
- These findings offer insights into NMDAR function and potential therapeutic targets for excitotoxicity.