Brain area specific regulation of synaptic AMPA receptors by phosphorylation

Abstract

Regulation of synaptic AMPA receptors (AMPARs) is one of the key elements that allow the nervous system to adapt to changes in the sensory environment as well as for memory formation. One way to regulate AMPAR function is by reversible changes in the phosphorylation of its subunits. We recently reported that phosphorylation of the AMPAR subunit GluA1 (or GluR1) on serine-845 (S845) is a pre-requisite step for sensory experience-dependent homeostatic synaptic plasticity in the visual cortex. In particular, increasing GluA1-S845 phosphorylation upregulated cell surface and synaptic AMPAR levels. Here we report that this is rather specific to the visual cortex, in that increasing GluA1-S845 phosphorylation in hippocampal slices only increase cell surface expression, but not synaptic AMPAR function. Our results suggest that depending on the brain region divergent mechanisms may exist to regulate synaptic AMPAR function with phosphorylation.

Keywords: AMPA receptor; GluA1-S845; PKA; isoproterenol; phosphorylation; β-adrenergic receptor.

Figure 1

Isoproterenol increases GluA1-S845 phosphorylation and cell surface AMPAR expression, but does not alter synaptic AMPAR function. (A) Isoproterenol (ISO, 5 µM) treatment for 10, 30 and 60 min increased GluA1-S845 phosphorylation, but not GluA1-S831 phosphorylation. Left: Example immunoblots simultaneously probed with GluA1-S845 phospho-specific (top, pS845) and GluA1 carboxy-terminal specific (bottom, GluA1) antibodies. Middle: Example immunoblots simultaneously probed with GluA1-S831 phospho-specific (top, pS831) and GluA1 carboxy-terminal specific (bottom, GluA1) antibodies. Right: Quantification of the immunoblots. The ratios of pS845/GluA1 and pS831/GluA from isoproterenol treated slices were normalized to controls in each blot and expressed as % of control (CTL). Asterisks show significant difference from CTL: ANOVA, F(3,16) = 3.328, p < 0.05 followed by Fisher's PLSD posthoc test (p < 0.05), n = 5 mice each group. (B) Transient isoproterenol treatment (5 µM, 10 min) results in a sustained increase in GluA1-S845 signal that lasted at least 2 h. Left: Example immunoblots. Right: Quantification of GluA1-S845 phosphorylation. Asterisks note significant difference from CTL: ANOVA, F(4,27) = 5.812, p < 0.01 followed by Fisher's PLSD posthoc test (p < 0.01), n = 5–7 each time point. (C) Isoproterenol treatment (5 µM, 10 min) increased cell surface levels of both GluA1 and GluA2 subunits of AMPAR . Left: Example immunoblots. Different amounts of total homogenate (Input), intracellular fraction (Sup) and cell surface fraction (Biotin) were loaded as indicated in protein amount (µg). The same immunoblot was simultaneously probed for GluA1 and GluA2. The blots were reprobed for tubulin. The absence of tubulin signal in the biotin lanes verifies the specificity of cell surface labeling. Right: Quantification of the cell surface amount of GluA1 and GluA2. Signals from biotin lanes were normalized to input lanes to obtain the cell surface amount of AMPARs as % of total. Asterisks: t-test, p < 0.01, n = 6 mice each group. (D) Isoproterenol treatment (5 µM, 10 min) did not alter mEPSC amplitude or frequency. Left: Comparison of average mEPSC amplitude between control (CTL) slices and isoproterenol (ISO) treated slices. CTL = 8.5 ± 0.3 pA (n = 16), ISO = 8.9 ± 0.3 pA (n = 19). Middle: Comparison of average mEPSC frequency. CTL = 0.6 ± 0.1 Hz (n = 16), ISO = 0.6 ± 0.1 Hz (n = 19). Right: Average mEPSC traces from CTL and ISO groups.

Figure 2

Working model to explain the difference in AMPAR regulation at synapses in layer 2/3 (L2/3) visual cortex and hippocampal CA1 area. (A) In L2/3 visual cortex, increasing GluA1-S845 phosphorylation enhances cell surface expression of both GluA1 (white subunit) and GluA2 (black subunit). These then are free to diffuse into synapses and function as synaptic AMPARs. However, we have evidence that synaptic anchoring of these freely diffusing AMPARs require an additional step. While our model depicts GluA1/GluA2 heteromers, a similar regulatory mechanism may operate for GluA1 homomers. (B) In CA1 of hippocampus, there is an additional perisynaptic compartment that prevents extrasynaptic AMPAR s from freely diffusing into synapses. Perisynaptic to synaptic trafficking may require NMDAR and/or mGluR activation. We have evidence that GluA1 homomers are anchored to perisynaptic sites under basal conditions in the CA1. Materials and methods were the same as in our original article, except all was done in hippocampal slices (400 µm thick) from 3–4 weeks old C57BL6/J mice (Jax lab). All experimental procedures followed the guidelines of the National Institution of Health, and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland.