Abstract
The N-methyl-d-aspartate receptor (NMDAR) is a Ca2+-permeable glutamate receptor mediating many neuronal functions under normal and pathological conditions. Ca2+ influx via NMDARs activates diverse intracellular targets, including Ca2+-dependent protease calpain. Biochemical studies suggest that NR2A and NR2B subunits of NMDARs are substrates of calpain. Our physiological data showed that calpain, activated by prolonged NMDA treatment (100 μM, 5 min) of cultured cortical neurons, irreversibly decreased the whole-cell currents mediated by extrasynaptic NMDARs. Animals exposed to transient forebrain ischemia, a condition that activates calpain, exhibited the reduced NMDAR current density and the lower full-length NR2A/B level in a calpain-dependent manner. Disruption of the association between NMDARs and the scaffolding protein postsynaptic density (PSD)-95 facilitated the calpain regulation of synaptic NMDAR responses and NR2 cleavage in cortical slices, whereas inhibition of calcineurin activity blocked the calpain effect on NMDAR currents and NR2 cleavage. Calpain-cleaved NR2B subunits were removed from the cell surface. Moreover, cell viability assays showed that calpain, by targeting NMDARs, provided a negative feedback to dampen neuronal excitability in excitotoxic conditions. These data suggest that calpain activation suppresses NMDAR function via proteolytic cleavage of NR2 subunits in vitro and in vivo, and the susceptibility of NMDARs to calpain cleavage is controlled by PSD-95 and calcineurin.
Footnotes
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This work was supported by grants from American Heart Association and National Institute of Health (to Z.Y.).
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ABBREVIATIONS: NMDAR, N-methyl-d-aspartate receptor; PSD, postsynaptic density; DIV, day in vitro; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid; NMDA, N-methyl-d-aspartate; EPSC, excitatory postsynaptic current; ALLN, Ac-Leu-Leu-Nle-H; FK506, tacrolimus; KN-93, 2-(N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl))amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; aa, amino acid(s); GABAAR, GABAA receptor; siRNA, small interfering RNA; PI, propidium iodide; ANOVA, analysis of variance; PP1/2A, protein phosphatases 1 and 2A; NT, N-terminal; CREB, cAMP response element-binding protein; CaMKII, Ca2+/camodulin-dependent protein kinase II; MAP2, microtubule-associated protein-2.
- Received February 29, 2008.
- Accepted April 29, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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