HIGH-AFFINITY AND LOW-AFFINITY [H-3]AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID ([H-3]AMPA) BINDING-SITES REPRESENT IMMATURE AND MATURE FORMS OF AMPA RECEPTORS AND ARE COMPOSED OF DIFFERENTIALLY GLYCOSYLATED SUBUNITS

Quantitative [H-3]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ( [H-3]AMPA) binding autoradiography was performed on frozen-thawed sect ions from rat brain after preincubation at 0 or 35 degrees C for 1 h. Preincubation at 35 degrees C instead of 0 degrees C resulted in a sel ective decrease of [H-3]AMPA binding assayed at a low concentration of [H-3]-AMPA (50 nM) and an enhancement of binding at a high concentrat ion (500 nM). The decrease in [H-3]AMPA binding after preincubation at 35 degrees C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [H-3]AMPA (K-D similar to 14 nM), whereas heavier organelles exhi bited lower affinity for AMPA (K-D similar to 190 nM). This small subp opulation of AMPA/GluR receptors contained almost exclusively a struct urally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti-GluR2/3, C-terminal antibodies) . Experiments using two deglycosylating enzymes, N-glycopeptidase F an d endoglycosidase H, clearly indicated that the 103.5-kDa species repr esented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors pr esent in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N-terminal domain of GluR2(4). The same enzymatic trea tment indicated that GluR1 subunits also exhibited a partially glycosy lated form. These data indicate that high-affinity [H-3]AMPA binding s ites represent nonsynaptic, intracellular membrane-bound AMPA receptor s that differ from synaptic receptors by at least the glycosylation st ate of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and struct urally distinct [H-3]AMPA binding/GluR sites.