Abstract
SynGAP, a protein abundant at the postsynaptic density (PSD) of glutamatergic neurons, is known to modulate synaptic strength by regulating the incorporation of AMPA receptors at the synapse. Two isoforms of SynGAP, α1 and α2, which differ in their C-termini, have opposing effects on synaptic strength. In the present study, antibodies specific for SynGAP-α1 and SynGAP-α2 are used to compare the distribution patterns of the two isoforms at the postsynaptic density (PSD) under basal and excitatory conditions. Western immunoblotting shows enrichment of both isoforms in PSD fractions isolated from adult rat brain. Immunogold electron microscopy of rat hippocampal neuronal cultures shows similar distribution of both isoforms at the PSD, with a high density of immunolabel within the PSD core under basal conditions. Application of NMDA promotes movement of SynGAP-α1 as well as SynGAP-α2 out of the PSD core. In isolated PSDs both isoforms of SynGAP can be phosphorylated upon activation of the endogenous CaMKII. Application of tatCN21, a cell-penetrating inhibitor of CaMKII, to hippocampal neuronal cultures blocks NMDA-induced redistribution of SynGAP-α1 and SynGAP-α2. Thus CaMKII activation promotes the removal of two distinct C-terminal SynGAP variants from the PSD.
Highlights
SynGAP is a ras GTPase activating protein (GAP) preferentially located in the postsynaptic density (PSD) of glutamatergic synapses [1,2,3,4,5]
SynGAP-a1 and SynGAP-a2 are Enriched in PSD Fractions Antibodies raised to peptides with sequences corresponding to the C-termini of SynGAP-a1 and SynGAP-a2, each recognized a doublet with close but distinct electrophoretic mobilities (Fig. 1 A), as reported previously [13]
Especially when calmodulin-dependent protein kinase II (CaMKII) activity is blocked, both SynGAP-a1 and SynGAP-a2 are mostly localized within the PSD core
Summary
SynGAP is a ras GTPase activating protein (GAP) preferentially located in the postsynaptic density (PSD) of glutamatergic synapses [1,2,3,4,5]. Previous studies in different laboratories have indicated an inhibitory function of SynGAP on the incorporation of AMPA receptors at the synapse, synaptic strength and spine growth [11,12]. In a previous immunogold electron microscopy study ([5], see corrigendum) we described activityinduced redistribution of SynGAP-a2 away from the PSD core. We again use immunogold electron microscopy to compare the distribution patterns of SynGAP-a1 and SynGAPa2 at the postsynaptic region of hippocampal neurons under basal conditions and following exposure to NMDA
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