Synaptic SAP97 Isoforms Regulate AMPA Receptor Dynamics and Access to Presynaptic Glutamate

Abstract

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, due to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated α-isoform to the postsynaptic density (PSD) and the L27 domain-containing β-isoform primarily to non-PSD, perisynaptic regions. Consequently, α- and βSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs, and hence their responsiveness to presynaptically released glutamate.

Autism-Associated Mutations in ProSAP2/Shank3 Impair Synaptic Transmission and Neurexin–Neuroligin-Mediated Transsynaptic Signaling

“…After 1-2 h of transfection incubation in Neurobasal medium containing 10 M CNQX and 50 M APV, cells were returned to their original plates with fresh Neurobasal medium containing B27 and L-glutamine. For electrophysiological studies, neurons were transfected at DIV9 using calcium phosphate precipitation (Waites et al, 2009). Briefly, for each 25 mm culture well, 4 g of DNA and 7.5 l of 2 M CaCl 2 in 60 l volume was added dropwise to 60 l of 2ϫ HBS (in mM: 274 NaCl, 10 KCl, 1.4 Na 2 HPO 4 , 15 glucose, and 42 HEPES, pH 7.1) and incubated for 20 min.…”

“…Paired whole-cell patch-clamp recordings to measure synaptic transmission between pyramidal neurons in vitro were performed as described previously (Waites et al, 2009;Li et al, 2011). Briefly, hippocampal neurons from male and female P0 Wistar rat pups were plated on 12 mm round glass coverslips, transferred to a recording chamber mounted on an Olympus BX-51 microscope, and perfused at room temperature with artificial CSF (ACSF) (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO 4 , 2.5 CaCl 2 , 1 Na 2 HPO 4 , 26.2 NaHCO 3 , and 11 glucose).…”

“…Briefly, hippocampal neurons from male and female P0 Wistar rat pups were plated on 12 mm round glass coverslips, transferred to a recording chamber mounted on an Olympus BX-51 microscope, and perfused at room temperature with artificial CSF (ACSF) (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO 4 , 2.5 CaCl 2 , 1 Na 2 HPO 4 , 26.2 NaHCO 3 , and 11 glucose). When recording miniature EPSCs (mEPSCs), the ACSF also contained 1 M tetrodotoxin and 100 M picrotoxin (Waites et al, 2009). Neurons were visualized by infrared differential interference contrast microscopy, and postsynaptic neurons transfected with EGFP-tagged ProSAP2/Shank3 were identified by fluorescence imaging.…”

Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts

“…These included EGFP-tagged versions of SV2, ␣-SAP97, and an mCherry-tagged version of ␤-actin. In the case of SV2 and ␣-SAP97, the EGFP-fusion protein was expressed under the ubiquitin promoter with the FUGW lentiviral vector as previously described (Schlüter et al, 2006;Leal-Ortiz et al, 2008;Waites et al, 2009). With regard to the mCherry-␤-Actin construct, the CMV-enhancer/chicken ␤-actin/␤-globin intron (CAG) promoter driving transgene expression in the lentiviral expression vector pLL4.4 (Kwiatkowski et al, 2007;Kwiatkowski et al, 2009) was replaced with a ubiquitin promoter, creating pLL4.5.…”

The role of SAP97 in synaptic glutamate receptor dynamics

“…Unlike other PSD-MAGUKs, SAP97β, the major isoform of the protein (also known as DLGH1; encoded by Dlgh1), is expressed at presynaptic and postsynaptic sites (2,18) and perisynaptically in dendritic spines (19). SAP97β directly binds to AMPAR GluA1 subunits (20)(21)(22)(23).…”