Characterization of calpain-mediated proteolysis of GluR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in rat brain

Abstract

Previous results have indicated that GluR1 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C-terminal (C-Ab) and the N-terminal (N-Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen-thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C-Ab and in the formation of a doublet band labeled with N-Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain-induced changes in GluR1 immunological properties were not associated with modifications of [3H]AMPA or 6-cyano-7-[3H]nitroquinoxaline-2,3-dione ([3H]CNOX) binding. Treatment of frozen-thawed brain sections with concentrations of calcium as low as 0.2 mM resulted in a large decrease in the 105-kDa GluR1 band and in the concurrent appearance of the 98-kDa band. This treatment was associated with increased [3H]-AMPA and [3H]CNOX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium-dependent processes regulating the characteristics of receptors in intact tissues.