Differential Distribution of Shank and GKAP at the Postsynaptic Density

Abstract

Shank and GKAP are scaffold proteins and binding partners at the postsynaptic density (PSD). The distribution and dynamics of Shank and GKAP were studied in dissociated hippocampal cultures by pre-embedding immunogold electron microscopy. Antibodies against epitopes containing their respective mutual binding sites were used to verify the expected juxtapositioning of Shank and GKAP. If all Shank and GKAP molecules at the PSD were bound to each other, the distribution of label for the two proteins should coincide. However, labels for the mutual binding sites showed significant differences in distribution, with a narrow distribution for GKAP located close to the postsynaptic membrane, and a wider distribution for Shank extending deeper into the cytoplasm. Upon depolarization with high K+, neither the intensity nor distribution of label for GKAP changed, but labeling intensity for Shank at the PSD increased to ~150% of controls while the median distance of label from postsynaptic membrane increased by 7.5 nm. These results indicate a preferential recruitment of Shank to more distal parts of the PSD complex. Conversely, upon incubation in Ca2+-free medium containing EGTA, the labeling intensity of Shank at the PSD decreased to ~70% of controls and the median distance of label from postsynaptic membrane decreased by 9 nm, indicating a preferential loss of Shank molecules in more distal parts of the PSD complex. These observations identify two pools of Shank at the PSD complex, one relatively stable pool, closer to the postsynaptic membrane that can bind to GKAP, and another more dynamic pool at a location too far away to bind to GKAP.

SHANK proteins: roles at the synapse and in autism spectrum disorder

“…On a subcellular level, SHANK proteins are located in a distal area of the PSD that extends up to 120 nm away from the postsynaptic membrane 10,60 . Recent data suggest that there are two pools of SHANK proteins in the PSD complex: one that is closer to the postsynaptic membrane and that seems to be relatively stable and able to bind to SAPAPs, and another, more dynamic, pool that is present at a distal location and probably cannot interact with SAPAPs 61 .…”

Ethanol-Associated Changes in Glutamate Reward Neurocircuitry: A Minireview of Clinical and Preclinical Genetic Findings

“…They are the scaffolding proteins closest to the surface of the postsynaptic membrane and they contain multiple PDZ domains. 67 The PSD-95 protein is one of the most studied MAGUK scaffolding proteins and is involved in postsynaptic stability as well as excitatory receptor insertion. 68 PSD-95 binds to numerous proteins associated with AMPAR and NMDAR complexes.…”

Microtubule‐associated protein 2 mediates induction of long‐term potentiation in hippocampal neurons

“…Consistent with light microscopic data of mature hippocampal cultures (Figures 2 and 3), ultrastructural localization of MAP2 using immunogold electron microscopy revealed a significant increase in MAP2‐positive gold particles in the vicinity of PSD (35 ~ 50 nm away from the postsynaptic membrane) after HFS at Shaffer collateral‐CA1 synapses of the hippocampal slices (Figure 4). This region of the dendritic spine contains various scaffolding proteins, such as Shank2 and GKAP, 34,53 as well as actin‐binding proteins, such as α‐actinin and neurabin, 54,55 all of which are involved in the regulation of spine morphology. While screening experiments to find molecular partners binding to synaptic MAP2 are ongoing, this observation hints at possible functions of MAP2 within the spines.…”

“…The gold particles in individual spine heads were counted using ImageJ software. Distances between gold particles and the postsynaptic membrane were measured from the center of the particle to the outer edge of the postsynaptic membrane as described previously 34 . Postsynaptic density (PSD) area was defined by the postsynaptic membrane and two parallel lines from the edge of the PSD extending 100 nm into the cytoplasm.…”