High- and low-affinity alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding sites represent immature and mature forms of AMPA receptors and are composed of differentially glycosylated subunits.

Quantitative alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding autoradiography was performed on frozen-thawed sections 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 selective decrease of [3H]AMPA binding assayed at a low concentration of [3H]AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [3H]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 [3H]AMPA (K(D) approximately 14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K(D) approximately 190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally 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 and endoglycosidase H, clearly indicated that the 103.5-kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors present 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 treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high-affinity [3H]AMPA binding sites represent nonsynaptic, intracellular membrane-bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [3H]AMPA binding/GluR sites.