Synaptic targeting of AMPA receptors is regulated by a CaMKII site in the first intracellular loop of GluA1 Wei Lua,1, Kaname Isozakib,1, Katherine W. Rocheb,2, and Roger A. Nicolla,2 aDepartments of Cellular and Molecular Pharmacology and Physiology, University of California, San Francisco, CA 94143; and bNational Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 Contributed by Roger A. Nicoll, November 5, 2010 (sent for review October 10, 2010) The accumulation of AMPA receptors (AMPARs) at synapses is Results essential for excitatory synaptic transmission. However, the mech- Expression of the GluA1 Loop1 Impairs Synaptic Transmission. In anisms underlying synaptic targeting of AMPARs remain elusive. agreement with previous studies (12), expression of the GluA1 We have now used a molecular replacement approach on an C-tail in hippocampal slice cultures did not impair AMPAR- AMPAR-null background to investigate the targeting mechanisms mediated synaptic transmission (Fig. 1 C and H). We thus ex- necessary for regulating AMPAR trafficking in the hippocampus. plored the possibility that Loop1 (Fig. 1 A and B), an ∼30-aa Although there is an extensive literature on the role of the GluA1 cytoplasmic domain, plays a role in AMPAR trafficking. We first C-tail in AMPAR trafficking, there is no effect of overexpressing expressed GFP tagged GluA1 Loop1 in pyramidal neurons as the C-tail on basal transmission. Instead, we found that the first a potential dominant negative. Next, 2–4 d after biolistic trans- intracellular loop domain (Loop1) of GluA1, a previously over- fection in hippocampal slice cultures, we made simultaneous looked region within AMPARs, is critical for receptor targeting to dual whole-cell recordings from GFP positive pyramidal neurons synapses, but not for delivery of receptors to the plasma mem- and untransfected neighboring neurons, and compared the brane. We also identified a CaMKII phosphorylation site (S567) in amplitudes of AMPAR and NMDAR excitatory postsynaptic the GluA1 Loop1, which is phosphorylated in vitro and in vivo. currents (EPSCs) evoked by a common input. Expression of the Furthermore, we show that S567 is a key residue that regulates GluA1 Loop1 specifically reduced the amplitude of AMPAR Loop1-mediated AMPAR trafficking. Thus, our study reveals a EPSCs by ∼40%, without affecting either NMDAR EPSC am- unique mechanism for targeting AMPARs to synapses to mediate plitude (Fig. 1 D and H) or paired-pulse ratio (Fig. S1), a mea- synaptic transmission. sure of presynaptic release probability. Loop1 from GluA2, which is 72% identical to that of GluA1 (Fig. 1B), also impaired GluA2 | GluR1 | postsynaptic density | hippocampus | pyramidal neurons synaptic transmission (Fig. 1 E and H). In contrast, expression of Loop1 from the kainate receptor subunit GluK2, which is 36% MPA receptors (AMPARs) are tetramers composed of identical to GluA1 Loop1 (Fig. 1B), did not affect synaptic AGluA1-4 (GluR1-4 or GluRA-D) subunits, and mediate the transmission (Fig. 1 F and H). Furthermore, to control for po- majority of fast excitatory synaptic transmission in the brain (1, 2). tential nonspecific effects, we expressed GluA1 Loop1 in pyra- The dynamic regulation of AMPAR trafficking into and out of midal neurons from GluA1 KO mice but observed no effect on synapses is a major mechanism underlying synaptic plasticity (3–6). AMPAR EPSCs (Fig. 1 G and H). Therefore the effect of GluA1 However, the mechanisms underlying AMPAR trafficking to syn- Loop1 expression on synaptic transmission depends on the apses to mediate basal synaptic transmission remain largely elusive presence of GluA1-containing receptors. Taken together, these (3–6). In the hippocampus, the majority of AMPARs are either data show that GluA1 Loop1 functions in AMPAR trafficking. GluA1A2 or GluA2A3 heteromers (7), and associate with their accessory subunits, transmembrane AMPAR regulatory proteins GluA1 Loop1 Is Required for Synaptic Delivery of GluA1 Homomers. fi (TARPs), which are critical for receptor trafficking (8). Further- AMPAR traf cking involves two general steps: delivery of more, GluA1A2 heteromers are the major species at both syn- receptors to the surface and targeting of the receptors to the aptic and extrasynaptic membranes of hippocampal CA1 pyra- synapse. Which step might depend on GluA1 Loop1? To address fi midal neurons (9). Most studies to date have focused on the role of this question, we compared the traf cking of GluA1 to that of AMPAR C-termini in receptor trafficking (3–5). However, in- GluA1/K2, a mutant of GluA1 in which the Loop1 region has been A terference with GluA1 C-tail function has little effect on basal syn- swapped with the loop domain from GluK2 (Fig. 2 ). In – HEK293T cells, agonist-evoked currents from outside-out patches aptic transmission (10 12). For example, basal synaptic transmission fi is not impaired upon overexpression of the GluA1 C-tail (12), nor in showed that GluA1/K2, similar to GluA1, traf cs to the surface, forms homomeric receptors, as judged by the strong inward rec- knockin mice in which GluA1 lacks its C-tail PDZ ligand (10) or key fi A B phosphorylation sites (11). These data suggest that regions other ti cation (Fig. S2 and ), and binds TARPs (Fig. S3). Thus, than the GluA1 C-tail may function in GluA1-containing AMPAR swapping the Loop1 from GluK2 did not change basic biophysical and trafficking properties of GluA1 in heterologous cells. synaptic delivery to maintain synaptic transmission. fi To study AMPAR trafficking, we have generated a triple floxed To study GluA1/K2 traf cking in neurons, we used a molecu- fl/fl lar replacement approach. In hippocampal slice cultures from Gria1-3 mouse line in which expression of the genes encoding fl/fl GRIA1-3 mice, expression of mCherryCre (hereafter Cre) GluA1, A2 and A3 are conditionally regulated (9). Expression of fl/fl Cre recombinase in CA1 pyramidal neurons from Gria1-3 mice leads to the loss of AMPARs (9), creating a null background for Author contributions: W.L., K.W.R., and R.A.N. designed research; W.L. and K.I. the reintroduction of WT or mutant AMPAR subunits that can be performed research; W.L., K.I., K.W.R., and R.A.N. analyzed data; and W.L., K.W.R., and studied independent of native receptors. Using this molecular R.A.N. wrote the paper. replacement approach, we found that delivery of AMPARs to The authors declare no conflict of interest. fi synapses requires the rst cytoplasmic domain (Loop1) of the 1W.L. and K.I. contributed equally to this work. fi AMPAR GluA1 subunit. In addition, we identi ed S567 of GluA1 2To whom correspondence may be addressed. E-mail: [email protected] or nicoll@ Loop1 as a CaMKII phosphorylation substrate, and found that cmp.ucsf.edu. S567 is a critical residue for the regulation of the Loop1- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dependent AMPAR trafficking to synapses. 1073/pnas.1016289107/-/DCSupplemental. 22266–22271 | PNAS | December 21, 2010 | vol. 107 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1016289107 Downloaded by guest on September 25, 2021 A B 558 585 SRFSPYEWHSEEFEEGRDQTTSDQSNEF GluA1 Loop1 SRFSPYEWHTEEFEDGRETQSSESTNEF GluA2 Loop1 ARFSPYEWYNPHPCNPDSDVV-E--NNF GluK2 Loop1 Loop1 C-tail C GluA1 C-tail D GluA1 Loop1 A M P A E P S C NM DA E P S C A M P A E P S C NM DA E P S C - 20 0 15 0 - 80 10 0 A p A ( p ( 1 l i ) p ) t a - 10 0 75 - 40 50 C L o o o L - - 1 1 l u u l G A G A 0 0 0 0 0 - 10 0 - 20 0 0 75 15 0 0 - 40 - 80 0 50 10 0 E GluA2 Loop1 F GluK2 Loop1 - 18 0 12 0 - 80 10 0 A p ( 1 p 1 p ) o L o o o L - - 90 60 - 40 50 L o L 2 A 2 K l u l l u G G - 0 0 0 0 0 - 90 - 18 0 0 60 12 0 0 - 40 - 80 0 50 10 0 Co n t ro l (pA) Co n t ro l (pA) ) ) t 12 0 12 0 t n G GluA1 Loop1 in GluA1 KO H C n C f f % o * * % o - 10 0 15 0 60 60 A M P A ( A ( A ) L o o p 1 i D p M ( 0 0 N O - 50 75 t il 1 1 1 O t a il 1 O K C n a o p n 1 1 - t p o p K L o o p o C t p p K 1 n o o L o o 1 C L o L o o 1 C 1 1 2 L A A 1 L A A 1 2 l u u l u l u A 1 2 2 l l u A A K G l u A l u l u u l u A K G G G A G l u u G i n G l l i n G G p 1 G G G p 1 0 0 o NEUROSCIENCE o o 0 - 50 - 10 0 L L o 0 75 15 0 1 1 A A Co n t ro l (pA) Co n t ro l (pA) l u l u G G Fig. 1. Expression of GluA1 Loop1 impairs synaptic transmission. (A) Topology of the AMPAR subunit with Loop1 and C-tail indicated with arrows. (B)Se- quence alignment of Loop1 domains from GluA1, GluA2, and GluK2. (C–G) Scatter plots show amplitudes of EPSCs for single pairs (○) and mean ± SEM (●), respectively for expression of GluA1 C-tail (C, AMPA: n = 11, P = 0.71; NMDA: n =11,P = 0.90), GluA1 Loop1 (D, AMPA: n =28,*P < 0.001; NMDA: n =25,P = 0.77), GluA2 Loop1 (E, AMPA: n = 36, *P < 0.05; NMDA: n = 36, P = 0.20), GluK2 Loop1 (F, AMPA: n = 16, P = 0.62; NMDA: n = 15, P = 0.21) in WT pyramidal cells, and GluA1 Loop1 in GluA1 KO cells (G, AMPA: n =12,P = 0.89; NMDA: n = 12, P = 0.98).