Shisa6 traps AMPA receptors at postsynaptic sites and prevents their desensitization during synaptic activity

Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs. The Shisa6 C-terminus harbours a PDZ domain ligand that binds to PSD-95, constraining mobility of AMPARs in the plasma membrane and confining them to postsynaptic densities. Shisa6 expressed in HEK293 cells alters GluA1- and GluA2-mediated currents by prolonging decay times and decreasing the extent of AMPAR desensitization, while slowing the rate of recovery from desensitization. Using gene deletion, we show that Shisa6 increases rise and decay times of hippocampal CA1 miniature excitatory postsynaptic currents (mEPSCs). Shisa6-containing AMPARs show prominent sustained currents, indicating protection from full desensitization. Accordingly, Shisa6 prevents synaptically trapped AMPARs from depression at high-frequency synaptic transmission.


(Real-Time) Polymerase Chain Reaction
Primers -Primers for PCR and real-time PCR were generated using Primer3.0. The final sets of primers are listed in Supplementary Table 2.
RNA isolation and cDNA synthesis -RNA from several tissues was extracted as previously described 13  As a bigger Ct-value correlates with a lower expression level, for practical purposes, For HEK293 cell expression, GluA1 isoform 1 and GluA2 isoform 1 were Gatewaycloned into the pTRCGw-IRES2-EGFP vector, yielding respectively GluA1-pTRCGw-IRES2-EGFP and GluA2-pTRCGw-IRES2-EGFP. GluA3 was cloned into the pRK5 vector. GluK2(Q) was cloned into the pcDNA3 vector (Invitrogen) as described previously 15 .
For expression in hippocampal neuronal cultures, N-terminally Flag-tagged Shisa6 was subcloned in the pBI Tet-On vector (Clontech). cDNA corresponding to the GluA subunits was as described previously 16 . Homer 1C::EGFP and Homer 1C::DsRed were generated by subcloning Homer 1C cDNA into the pcDNA3 vector (Invitrogen).
For FLIM experiments, eGFP was inserted at position 253 of PSD-95 as previously described 17 . The mCherry tag was inserted 21 amino acids before the stop codon of Shisa6. All DNA constructs were sequence verified before use.

Antibodies
Anti-Shisa6 antibody was raised in rabbit against sequence DRYRMTKMHSHPSA

Subcellular fractionation
Subcellular fractions were prepared as described previously 9,18 with some modifications.

In-gel Tryptic digestion
The free cysteine residues of Laemmli buffer-eluted proteins were blocked by addition of acrylamide (3.75% final concentration) for 30 minutes at room temperature. Proteins were resolved on a 10% SDS polyacrylamide gel, fixed, and stained with colloidal Coomassie Blue G-250. Sample lanes were cut into 3 segments, destained, and the proteins in-gel digested with Trypsin/Lys-C mix (Promega) during overnight incubation at 37 °C. The peptides were extracted twice with 50% acetonitrile + 0.1% trifluoroacetic acid for 40 minutes, once with 80% acetonitril + 0.1% trifluoroacetic acid for 20 minutes, dried by speedvac, and stored at -80 °C.
Eluates were electrosprayed directly into a TripleTOF 5600+ system (Absciex) operated in Information Dependent Acquisition mode. One full-scan cycle consisted of a precursor ion scan (m/z range of 350 to 1250, charge state +2 to +5, 90 cps

Immunocytochemistry of cell cultures
To study the surface localization of Shisa6, independent of the in-house antibody against Images were collected on an upright Leica DM5000 using epifluorescence and a 40x oil objective with LED light source.

Immunohistochemistry of brain slices
We used the method of Yoneyama et al. (2004) 21 In brief, 6-week-old mice were killed by decapitation after anesthesia with isoflurane (4 minutes, 5% isoflurane per 1.5 L air), brains were dissected and quickly frozen. Sections were cut on a cryostat (15 µm) and fixed in Carnoy's solution containing 6:1 ethanol and acetic acid for 10 minutes at 4 °C.
The sections were incubated 1 h in blocking buffer containing 4% normal goat serum and 0.3% Triton X-100 in PBS, before they were incubated with first antibodies (anti-

Electrophysiological Recordings
Recordings from HEK293 cells: All electrophysiological recordings we made using a