Immunohistochemistry of GluR1 subunits of AMPA receptors of rat cerebellar nerve cells.
The surface expression and metabolic turnover time of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are important factors in determining the involvement of these proteins in synaptic function. We used the membrane-impermeant chemical crosslinking reagent BS3 and subsequent immunoblot analysis to show that a large proportion of each of the AMPA receptor subunits GluR1-4 is surface expressed in cultured rat cerebellar granule cells. The apparent molecular mass of the individual subunits was 105 kDa and the surface expressed crosslinked complex was 530 kDa for each of the antibodies tested. These results are consistent with functional ionotropic glutamate receptors being pentameric subunit assemblies. We investigated the time course of AMPA receptor surface expression using the membrane-impermeant biotinylating agent NHS-SS-biotin. The half-life of surface expressed AMPA receptors was found to be 30 h. To estimate the mean degradation rate of AMPA receptors and total trichloracetic acid (TCA)-precipitable protein we used [35S]methionine/cysteine pulse-chase labelling. The half-life of AMPA receptors immunoprecipitated with anti-GluR1 antibody was approximately 48 h and the half-life of total TCA-precipitable protein from the same samples was 37 h.