The Phosphorylation State of GluR1 Subunits Determines the Susceptibility of AMPA Receptors to Calpain Cleavage

Wenhua Liu,Eunice Y. Yuen,Zhen Yan

doi:10.1074/jbc.m701283200

Wenhua Liu, Eunice Y. Yuen + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m701283200

Copy DOI

Abstract

The alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) is an ionotropic glutamate receptor that governs most of excitatory synaptic transmission in neurons. In vitro biochemical assay has shown that calpain, a Ca2+-activated protease, can cleave AMPAR GluR1 subunits. Our physiological study found that calpain, which was activated by prolonged stimulation of the N-methyl-D-aspartate receptor (100 microM, 10 min), caused a substantial suppression of AMPAR currents in cortical neurons. Since the phosphorylation sites of GluR1 by several protein kinases are located in close proximity to the calpain cleavage sites, we investigated the effect of phosphorylation on the susceptibility of GluR1 to calpain cleavage. Interestingly, we found that the calpain regulation of AMPAR currents was diminished by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) but was augmented by inhibition of protein phosphatase 1/2A (PP1/2A). In agreement with this, in vitro assay showed that the calpain-induced proteolytic cleavage of GluR1 C-terminal fusion protein was strongly potentiated by adding the purified active CaMKII, and GluR1 phosphorylated at Ser831 by CaMKII is much more sensitive to calpain cleavage. Taken together, our data suggest that calpain activation suppresses AMPA receptor currents via proteolytic cleavage of GluR1 subunits, and the susceptibility of AMPARs to calpain cleavage is determined by the phosphorylation state of GluR1 subunits, which is mediated by CaMKII-PP1/2A activity.

Highlights

Another one is to change the trafficking of Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR), thereby increasing or decreasing the number of functional AMPARs located at the synaptic membrane [1]
In vitro biochemical assay show that AMPAR GluR1 subunit is a potential substrate of the Ca2ϩ-activated protease calpain [5, 6], suggesting that AMPAR function could be regulated by another mechanism, i.e. calpain-mediated proteolysis
These results suggest that calpain activated by prolonged stimulation of NMDA receptors induces the specific proteolysis of GluR1 subunits, which could account for the down-regulation of AMPAR currents

Summary

EXPERIMENTAL PROCEDURES

Acute Dissociation Procedure and Primary Neuronal Culture— Cortical neurons from 3– 4-week-old Sprague-Dawley rats were acutely dissociated as what we described previously [10, 22]. We examined the regulation of AMPAR currents by calpain, which is activated by prolonged NMDA treatment, in cortical neurons transfected with the calpain siRNA. When endogenous calpain was activated by prolonged NMDA treatment (100 ␮M, 10 min), AMPAR currents was substantially reduced in neurons transfected with a scrambled siRNA (48.2 Ϯ 2.0%, n ϭ 7, Fig. 1C) or FIGURE 1. B, plot of normalized peak AMPAR currents (IAMPA) with a prolonged NMDA application (100 ␮M, 10 min) in GFP-positive neurons transfected with calpain siRNA or a scrambled siRNA. GFP alone (44.7 Ϯ 1.5%, n ϭ 9, Fig. 1C), while this effect was significantly abolished in neurons transfected with the calpain siRNA (10.2 Ϯ 2.1%, n ϭ 6, Fig. 1C) These data suggest that calpain, which is activated by prolonged stimulation of NMDA receptors, bated overnight at room temperature in a phosphorylation down-regulates AMPAR function in cortical neurons. Samples were denatured with SDS- with the calpain siRNA or a scrambled siRNA were treated with sample buffer and resolved by SDS-PAGE

RESULTS

Findings

DISCUSSION