The first identified auxiliary subunit stargazin is

The first identified auxiliary subunit, stargazin, is essential for AMPAR function in cerebellar granule neurons (Hashimoto et al., 1999). Subsequently, a family of six transmembrane AMPAR regulatory proteins (TARPs) were defined that modify channel trafficking, gating, and pharmacology (Kato and Bredt, 2007; Tomita et al., 2003). Cornichons (CNIH-2/3) are a family of AMPAR auxiliary subunits that control export of AMPARs from the endoplasmic reticulum (Harmel et al., 2012; Schwenk et al., 2009) and associate with synaptic AMPARs to modulate channel kinetics (Jackson and Nicoll, 2011; Kato et al., 2010; Schwenk et al., 2009; Yan and Tomita, 2012). Recent proteomic studies have further expanded the complement of AMPAR-associated proteins. The cysteine-knot protein CKAMP44 modulates AMPAR biophysics to attenuate short-term synaptic plasticity in the dentate gyrus (von Engelhardt et al., 2010). The germ cell-specific gene 1-like (GSG1-l) modifies gating and kinetics of receptor DC_AC50 in a subunit-dependent manner (Schwenk et al., 2012; Shanks et al., 2012). Furthermore, more than two dozen proteins occur in AMPAR complexes (Schwenk et al., 2012). These additional AMPAR partners include integral transmembrane, extracellular GPI-anchored, and secreted proteins. Some partners have enzymatic activities; some are cytoskeletal elements; and others are secreted growth factor antagonists. Understanding how this large and diverse protein collection modulates AMPARs is an important challenge. Here, we find that previously well-characterized AMPAR auxiliary subunits TARP, CNIH-2, and GSG1-l dramatically increase GluA1 protein levels in heterologous cells. By systematically evaluating each class of protein found in AMPAR immunoprecipitates (Schwenk et al., 2012), we demonstrate that porcupine (PORCN) and ABHD6 also increase levels of co-transfected GluA1. We find that PORCN controls hippocampal AMPARs, as PORCN knockdown destabilizes AMPAR complexes and thereby diminishes synaptic transmission. AMPAR complexes in PORCN-deficient neurons have deficient TARP γ-8 and show accelerated decay kinetics. This work defines functional roles for AMPAR partners in controlling stability and composition of receptor complexes.
Results
Discussion The stabilizing effect of PORCN on AMPARs also occurs in neurons. Indeed, PORCN knockdown in hippocampal neurons dramatically and specifically decreases levels of all measured components of the AMPAR complex. In parallel, knockout of PORCN in mouse hippocampus results in decreased protein levels of GluA1, GluA2/3, and γ-8. Interestingly, our AMPAR deglycosylation experiments demonstrate that PORCN KO reduces both the EndoH-sensitive and -resistant GluA2/3 populations proportionately, suggesting that PORCN controls the collective AMPAR pool at the level of the ER. This finding is distinct from AMPAR auxiliary subunits like TARPs and CNIHs that preferentially control the EndoH-resistant/mature AMPAR pool (Rouach et al., 2005; Shi et al., 2010). Thus, PORCN is an AMPAR interacting protein that stabilizes the collective AMPAR pool. These findings underscore the unique role of PORCN as an ER chaperone for stabilization and assembly of AMPARs. In addition to reducing extrasynaptic AMPAR components, PORCN KO also significantly decreases synaptic GluA2/3 and γ-8 levels, which likely explains the reduction in synaptic transmission. Nevertheless, these mutant mice are still competent in LTP induction and maintenance. Our subcellular fractionation and EndoH glycosidase experiments suggest that PORCN stabilizes the collective AMPAR pool at the level of the ER. As PORCN knockout reduces both the surface AMPAR pool and the AMPAR pool for LTP, the mutant mice have proportionate decreases of both basal and potentiated transmission. This results in LTP appearing normal in PORCN KO animals. Similar observations were made in the TARP-γ8-Δ4 knockin mice, which also show reduced synaptic transmission, but no changes in LTP (Sumioka et al., 2011). In contrast, knockout of PSD-95 decreases synaptic transmission and increases LTP (Migaud et al., 1998), as PSD-95 anchors synaptic AMPARs but does not regulate the AMPAR pool for LTP. On the other hand, CaMKII is important for induction of LTP but does not control the number of synaptic AMPARs under basal conditions. Accordingly, CaMKII mutant mice show normal basal transmission but decreased LTP (Silva et al., 1992).