Stoichiometry and phosphoisotypes of hippocampal AMPA-type glutamate receptor phosphorylation

Abstract

It has been proposed that the AMPAR phosphorylation regulates trafficking and channel activity, thereby playing an important role in synaptic plasticity. However, the actual stoichiometry of phosphorylation, information critical to understand the role of phosphorylation, is not known because of the lack of appropriate techniques for measurement. Here, using Phos-tag SDS-PAGE, we estimated the proportion of phosphorylated AMPAR subunit GluA1. The level of phosphorylated GluA1 at S831 and S845, two major sites implicated in AMPAR regulation, is almost negligible. Less than 1% of GluA1 is phosphorylated at S831 and less than 0.1% at S845. Considering the number of AMPAR at each synapse, the majority of synapses do not contain any phosphorylated AMPAR. Also, we did not see evidence of GluA1 dually phosphorylated at S831 and S845. Neuronal stimulation and learning increased phosphorylation, but the proportion was still low. Our results impel us to reconsider the mechanisms underlying synaptic plasticity.

Figure 1. Separation and quantification of phosphoisotypes of GluA1 using Phos-tag SDS-PAGE

(A) HEK293T cells expressing GluA1 were incubated with or without 1 μM okadaic acid (OA) for 4 hrs. The homogenate was separated using conventional (left) or Phos-tag (right) SDS-PAGE and blotted with an antibody against the C-terminus of GluA1. (B) Pre-treatment of the sample with λ protein phosphatase (λ PPase) abolishes the band shift. (C) Wild type (WT) GluA1, 4 alanine mutants (4A) containing mutations in all but one out of five known sites or a mutant where all known phosphorylation sites were mutated to alanine (5A) were expressed in HEK293T cells. To maximally induce phosphorylation, a constitutively active CaMKII was cotransfected and the cells were further treated with forskolin, phorbol 12-myristate 13-acetate, cyclosporine A and okadaic acid for 2 hours. The cell homogenate was separated using Phos-tag SDS-PAGE and blotted with antibodies against GluA1 C-terminus, pS831, pT840 and pS845. (D) Detection of dually phosphorylated GluA1. 4A and 3 alanine (3A) mutants were expressed in HEK293T cells and phosphorylated as in (C). The numbers above the blot indicate phosphorylatable sites. (E) Sensitivity of C-terminus antibody to phosphorylated GluA1. HEK293T cells expressing indicated mutants were treated as in (C) but for a longer duration (6 hours) to ensure complete phosphorylation. Then either unphosphorylated (left) or phosphorylated (right) AMPARs were separated using conventional SDS-PAGE (top) or Phos-tag SDS-PAGE (bottom) and blotted with the C-terminus antibody. (F) GluA1 samples containing different amounts of pS831 GluA1 (on a 4A background) made by mixing different amounts of immunoprecipitated GluA1 from transfected HEK293T cells untreated or treated with okadaic acid. The mixtures were separated on Phos-tag SDS-PAGE and blotted with the C-terminus antibody. The proportion of pS831 GluA1 in each sample is shown. The same samples were subjected to AQUA (Fig. S1B–D). (G) Correlation of the result of Phos-tag SDS-PAGE (F) and AQUA (Fig. S1D) in the stoichiometry of pS831 GluA1. N=3, each.

Figure 2. Endogenous GluA1 in neurons is mostly unphosphorylated

(A) Phosphoisotypes of endogenous GluA1 in adult rat hippocampus. Hippocampal extract was subjected to conventional SDS-PAGE (top) or Phos-tag SDS-PAGE (bottom) and blotted with antibodies against GluA1 C-terminus, pS831, pT840 and pS845. (B) The densitometoric profile of blots of hippocampal extract. The positions of pS831, pT840 and pS845 phosphoisotypes are shown by red, green and blue lines respectively. (C) Quantification of the population of each phosphoisotype. The pS831 was below the level where it could be confidently quantified (<1%) and pS845 was not detected. (D) Blots of the dilution series of maximally phosphorylated 4A mutants on a conventional SDS-PAGE. Endogenous GluA1 from adult rat hippocampus was loaded on the right lane. Only one rat out of six is shown. A blot with the C-terminus antibody (bottom) was used to adjust the total amount of GluA1. (E) Calibration of band density and amount of phosphorylated protein. The densitogram reading was normalized by the 10% phosphorylated sample. Squares are averages of 6 samples. (F) GluA1 phosphorylation in different subcellular fractions. The fractions from adult rat hippocampi were subjected to SDS-PAGE or Phos-tag SDS-PAGE and blotted with an anti-GluA1 C-terminus. The amount of total GluA1 is preadjusted by performing a separate blot (not shown). (G) Comparison of intracellular and neuronal surface GluA1 phosphoisotypes. The surface fraction was obtained from dissociated neuronal cultures by surface biotinylation and avidin pull down. The remaining fraction was used as the intracellular fraction. (H) Ontogeny of GluA1 phosphorylation from P1 to adult hippocampus. Hippocampal homogenate from animals at various ages was separated by conventional and Phos-tag SDS-PAGE and blotted with indicated antibodies. We used the N-terminus antibody because the C-terminus antibody produced a non-specific band in the P1 sample (not shown). Also, the uppermost band with * in the pT840 blot represents cross-reactivity as it was not detected with the N-terminus antibody. See Fig. S2C for identification of phosphoisotypes. The amount of total GluA1 is preadjusted by performing a separate blot (not shown). (I) Quantification of phosphorylation in P1 hippocampus. The level is expressed as adult levels as one. The original image is in Fig. S2B. The level of pT840 may be an overestimation due to cross-reactivity shown with * in the pT840 blot in Fig. S2B. N=5.

Figure 3. Glycine-induced chemical LTP increases phosphorylation of cell surface GluA1 but the absolute proportion of the phosphorylated population is low

(A) Dissociated neuronal cultures were treated with glycine to induce chemLTP in the absence (−) or presence of okadaic acid and cyclosporine A (OA+CysA). Surface GluA1 was obtained by surface biotinylation and avidin pull down. The samples were subjected to SDS-PAGE (top) or Phos-tag SDS-PAGE (bottom) and blotted with indicated antibodies. Each Phos-tag SDS-PAGE blot had a different exposure time and therefore cannot be cross-compared. (B) Quantification of the surface GluA1 confirms successful induction of chemLTP. (C) Amount of phosphorylation normalized by the amount of surface GluA1. The level was determined from the density of conventional SDS-PAGE in (A). (D) The densitometoric profile of Phos-tag SDS-PAGE. (E) Quantification of data in (C). The peaks at S831 and S845 are below the amount that can be confidently quantified. (F) The amount of phosphorylated GluA1 before and after chemLTP induction plotted on calibration line of the dilution series. *: p<0.05, **: p<0.01, ***: p<0.001 (two-tailed t-test). n=6 for (B) to (F).

Figure 4. Inhibitory avoidance conditioning increases GluA1 phosphorylation but the absolute proportion of phosphorylated population is low

Rats were subjected to the inhibitory avoidance paradigm. 5 or 30 min after conditioning, dorsal hippocampi were dissected and the fraction insoluble to 1% Triton-X 100 was isolated. (A) Blotting in SDS-PAGE (top) and Phos-tag SDS-PAGE (bottom). One rat from each group is shown. The top bands (arrowhead) of the pS845 blot (A) most likely represent a dually phosphorylated population at T840 and S845 but because the amount was small (<0.01%), they were not analyzed. (B) Quantification of phosphorylation using a phospho-specific antibody normalized to the level observed in naïve animals. Blotting in conventional SDS-PAGE was used for the quantification. (C) The densitometoric profile of Phos-tag SDS-PAGE. (D) Quantification of data in (C). (E) The amount of phosphorylated GluA1 before and after inhibitory avoidance learning is plotted on calibration line of the dilution series. *: p<0.05, **: p<0.01 (two-tailed t-test). A total of 9 rats were separated into three groups.