Basal levels of AMPA receptor GluA1 subunit phosphorylation at threonine 840 and serine 845 in hippocampal neurons

Abstract

Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are basally phosphorylated at these sites. To examine this question, we used immunoprecipitation/depletion assays to estimate the proportion of GluA1 subunits basally phosphorylated at S845 and T840. Although dephosphorylation of S845 is thought to have a key role in LTD, our results indicate that few GluA1 subunits in hippocampal neurons are phosphorylated at this site. In contrast, ∼50% of GluA1 subunits are basally phosphorylated at T840, suggesting that dephosphorylation of this site can contribute to the down-regulation of AMPAR-mediated synaptic transmission in LTD.

Figure 1.

Schematic of immunoprecipitation/depletion assays using denatured (left) and nondenatured homogenization buffers (right). The starting material for immunoprecipitation (input fraction) consists of either individual subunits obtained from denatured homogenates prepared using 1% sodium dodecyl sulfate (SDS) buffer or intact receptors obtained from nondenatured homogenates prepared in modified radioimmunoprecipitation assay (mRIPA) buffer (Step 1). The input fraction (containing phosphorylated and nonphosphorylated subunits) is incubated with phospho-T840 or phospho-S845-specific antibodies (p-GluA1 Ab) conjugated to protein A coated agarose beads (Step 2). Beads (and attached proteins) are then pelleted by centrifugation, leaving nonphosphorylated subunits/receptors in the supernatant/unbound fraction (Step 3). The pelleted fraction is then resuspended in loading buffer (LB) and boiled to release proteins and antibodies (not shown) from the beads (Step 4). Phosphorylated subunits are collected by taking the supernatant following centrifugation (IP fraction). Western immunoblotting is then used to compare total GluA1 levels in the input and unbound fractions and the depletion of GluA1 levels in the unbound fraction is used to determine the proportion of subunits/receptors basally phosphorylated at each site. For simplicity, only GluA1/2 subunit-containing heteromeric AMPARs are shown.

Figure 2.

Immunoprecipitation of GluA1 from nondenatured and denatured hippocampal homogenates. (A) GluA2 subunits coimmunoprecipitate with GluA1 from nondenatured homogenates. Example immunoblots showing GluA1 and GluA2 levels in the input fraction, immunoprecipitated (IP) fraction (top), and unbound fraction (bottom) after immunoprecipitation with anti-GluA1 or control IgG antibodies. (B) Levels of GluA1 and GluA2 in the unbound fraction expressed as a percentage of levels in input fraction (n = 5, lines connect results from same immunoprecipitation). (C,D) GluA2 subunits do not coimmunoprecipitate with GluA1 from denatured homogenates. Same as in panels A and B except that GluA1 immunoprecipitations were performed using denatured homogenates (n = 5). Bars indicate median values.

Figure 3.

Immunoprecipitation of phosphorylated GluA1 from denatured hippocampal homogenates. (A) Example immunoblots showing total and phosphorylated GluA1 in immunoprecipitated (left) and unbound fractions (right) following immunoprecipitation of T840-phosphorylated GluA1. (B) Plots show levels of total and phosphorylated GluA1 in the unbound fraction following immunoprecipitations using either phospho-T840 GluA1 or control IgG antibodies (n = 5, lines connect results from the same homogenate). GluA1 levels in the unbound fraction were reduced to 51 ± 9% of input following immunoprecipitation of T840-phosphorylated GluA1 (P < 0.01 compared with levels in IgG control unbound fraction) (C,D) Same as in panels A and B except immunoprecipitations were performed using phospho-S845 GluA1 antibodies (n = 4). GluA1 levels in the unbound fraction were 98 ± 8% of the input following immunoprecipitation of S845-phosphorylated GluA1 (P = 0.41 compared with levels in IgG control unbound fraction). Bars in point plots indicate median values.

Figure 4.

Phospho-T840 immunodepletion assays using denatured homogenates prepared from hippocampal slices maintained in vitro. Hippocampal slices obtained from the same animal were either left untreated (UT) or exposed to 20 µM cantharidin (Can) for 1 h (n = 7). (A) Immunoblots showing phospho-T840 GluA1 levels in the input and unbound (UB) fractions from untreated control and cantharidin-treated slices. (B) Levels of T840-phosphorylated GluA1 in the input fractions from untreated control and cantharidin-treated slices. (C) Immunoblots show GluA1 in immunoprecipitated (IP) fraction (top) and unbound fractions (bottom) from untreated control and cantharidin-treated slices. (D) Levels of total GluA1 in the unbound fraction following immunoprecipitation of T840-phosphorylated GluA1 from homogenates prepared from untreated control and cantharidin treated slices. Lines in B and D connect results from the same experiment. Bars indicate median values.

Figure 5.

Depletion of GluA1 following immunoprecipitation of T840-phosphorylated GluA1 from nondenatured homogenates. (A) Example blots showing total GluA1 in the IP fraction (top) and total and T840-phosphorylated GluA1 in the unbound fraction (bottom). (B) Results from all experiments (n = 5) showing levels of T840-phosphorylated (left) and total GluA1 (right) in the unbound fraction. Lines connect results from the same experiment. Bars indicate medians for each group.