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The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells
M Iizuka1, S Nishimura, M Wakamori
1Department of Molecular and Cellular Biology, Nippon Boehringer Ingelheim Co., Ltd Kawanishi Pharma Research Institute, 3-10-1, Yato, Kawanishi, 666-0193, Japan.
Abstract:
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) -type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1-GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+-permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2-lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip-mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor-mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.
Insights
The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor GluR2flip/GluR4flip subunit combination causes cell death by prolonged sodium influx, not calcium. This finding clarifies excitotoxicity mechanisms in brain injury.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are crucial in excitotoxicity following acute brain insults.
- AMPA receptors comprise GluR1-GluR4 subunits, with splice variants (flip/flop), influencing channel properties and calcium permeability.
Purpose of the Study:
- To investigate the specific lethal effects of different AMPA receptor subunit and variant combinations.
- To elucidate the ion flux mechanisms underlying AMPA receptor-mediated excitotoxicity.
Main Methods:
- HEK293 cell coexpression of AMPA receptor subunits (GluR2flip/GluR4flip).
- Patch clamp recordings and calcium imaging to analyze ion permeability and flux.
- Assessment of lethality and potentiation by inhibiting sodium extrusion mechanisms (ouabain, benzamil).
Main Results:
- Coexpression of GluR2flip and GluR4flip subunits induced cell lethality, unlike other combinations.
- The GluR2flip/GluR4flip receptor showed low calcium permeability but prolonged sodium influx upon AMPA stimulation.
- Cell lethality was exacerbated by inhibiting cellular sodium extrusion.
Conclusions:
- AMPA receptor-mediated excitotoxicity involving the GluR2flip/GluR4flip subunit combination is primarily driven by excessive sodium influx and overload.
- This contrasts with the established role of calcium influx in excitotoxicity via GluR2-lacking channels.
