Extrasynaptic Membrane Trafficking Regulated by GluR1 Serine 845 Phosphorylation Primes AMPA Receptors for Long-term Potentiation

Abstract

Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-d-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP. Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-d-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP. At central glutamatergic synapses, AMPAR 2The abbreviations used are: AMPARα-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionte glutamate receptorCaM-KIIcalcium/calmodulin-dependent protein kinase IICPSconsensus phosphorylation sitefEPSPfield excitatory postsynaptic potentialF/Rforskolin/rolipramGluR1AMPA-type glutamate receptor subunit 1LTPlong-term potentiationLTDlong-term depressioncLTPchemical LTPChem-LTDchemical LTDNMDAN-methyl-d-aspartatePKAprotein kinase AGSTglutathione S-transferase. function is dynamically regulated, resulting in bidirectional synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD). Phosphorylation can regulate AMPARs by two distinct mechanisms that affect synaptic transmission (1Lee H.K. Takamiya K. Han J.S. Man H. Kim C.H. Rumbaugh G. Yu S. Ding L. He C. Petralia R.S. Wenthold R.J. Gallagher M. Huganir R.L. Cell. 2003; 112: 631-643Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar): modulation of channel properties (2Derkach V. Barria A. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3269-3274Crossref PubMed Scopus (696) Google Scholar, 3Banke T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar, 4Derkach V.A. Biophys. J. 2003; 84: 1701-1708Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and trafficking of AMPARs to the surface membrane (5Passafaro M. Piech V. Sheng M. Nat. Neurosci. 2001; 4: 917-926Crossref PubMed Scopus (542) Google Scholar, 6Shi S. Hayashi Y. Esteban J.A. Malinow R. Cell. 2001; 105: 331-343Abstract Full Text Full Text PDF PubMed Scopus (888) Google Scholar, 7Hayashi Y. Shi S.H. Esteban J.A. Piccini A. Poncer J.C. Malinow R. Science. 2000; 287: 2262-2267Crossref PubMed Scopus (1253) Google Scholar, 8Shi S.H. Hayashi Y. Petralia R.S. Zaman S.H. Wenthold R.J. Svoboda K. Malinow R. Science. 1999; 284: 1811-1816Crossref PubMed Scopus (1059) Google Scholar, 9Tomita S. Stein V. Stocker T.J. Nicoll R.A. Bredt D.S. Neuron. 2005; 45: 269-277Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Phosphorylation of Ser-831 in GluR1 C terminus by calcium/calmodulin-dependent protein kinase II (CaM-KII) (10Mammen A.L. Kameyama K. Roche K.W. Huganir R.L. J. Biol. Chem. 1997; 272: 32528-32533Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 11Barria A. Derkach V. Soderling T. J. Biol. Chem. 1997; 272: 32727-32730Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar) potentiates single-channel conductance (2Derkach V. Barria A. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3269-3274Crossref PubMed Scopus (696) Google Scholar, 4Derkach V.A. Biophys. J. 2003; 84: 1701-1708Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and phosphorylation of Ser-845 by cAMP-dependent protein kinase (PKA) (12Roche K.W. O'Brien R.J. Mammen A.L. Bernhardt J. Huganir R.L. Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar) increases channel open probability (3Banke T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar). Importantly, Ser-845 phosphorylation can also regulate the surface expression of AMPARs by increasing the pool of GluR1 recycled back to the surface after their endocytosis (13Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). CaM-KII activity is also critical for synaptic incorporation of AMPARs but requires a serine at position 845, which is not phosphorylated by CaM-KII, in GluR1 (14Esteban J.A. Shi S.H. Wilson C. Nuriya M. Huganir R.L. Malinow R. Nat. Neurosci. 2003; 6: 136-143Crossref PubMed Scopus (691) Google Scholar), suggesting an important role of Ser-845 phosphorylation for trafficking of AMPARs in an activity-dependent manner. α-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionte glutamate receptor calcium/calmodulin-dependent protein kinase II consensus phosphorylation site field excitatory postsynaptic potential forskolin/rolipram AMPA-type glutamate receptor subunit 1 long-term potentiation long-term depression chemical LTP chemical LTD N-methyl-d-aspartate protein kinase A glutathione S-transferase. Although these studies indicate that AMPAR phosphorylation and trafficking are coupled (13Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 14Esteban J.A. Shi S.H. Wilson C. Nuriya M. Huganir R.L. Malinow R. Nat. Neurosci. 2003; 6: 136-143Crossref PubMed Scopus (691) Google Scholar), exactly how Ser-845 phosphorylation contributes to trafficking is unclear. Current understanding of the functional role of Ser-845 phosphorylation in GluR1 during synaptic plasticity is limited because previous studies have analyzed only relative changes in phospho-Ser-845. In fact, the functional significance of these changes is determined by the net level of phospho-Ser-845. In this report, we quantified the net Ser-845 phosphorylation and surface expression of GluR1 in hippocampal neurons during bidirectional synaptic plasticity. We observed that receptors phosphorylated at the Ser-845 trafficked specifically to extrasynaptic sites but not to synapses. However, extrasynaptic AMPARs can be incorporated into synapses by synaptic activity. Thus, Ser-845 phosphorylation serves as a “priming” step in enhancing synaptic strength during LTP by enhancing the extrasynaptic delivery of AMPARs. Cultured Hippocampal Neurons and Biochemistry—Hippocampal neurons were cultured from postnatal day 1–2 Sprague-Dawley rats as previously described (15Brewer G.J. J. Neurosci. Methods. 1997; 71: 143-155Crossref PubMed Scopus (367) Google Scholar, 16Atkins C.M. Nozaki N. Shigeri Y. Soderling T.R. J. Neurosci. 2004; 24: 5193-5201Crossref PubMed Scopus (133) Google Scholar) and used on 13–15 days in vitro. For chemical stimulations, cultures were first incubated in ACSF for 30 min at room temperature (in mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 33 d-glucose, and 25 HEPES (pH 7.3; 320 mosm final), followed by stimulation with 50 μm forskolin (Sigma) and 0.1 μm rolipram (Calbiochem) in ACSF (no MgCl2) or 50 μm NMDA. After 10 min of stimulation, neurons were replaced in regular ACSF and then subjected to surface biotinylation at indicated time points. Surface Biotinylation—After F/R or NMDA stimulations, cultured neurons or slices were transferred to ice-cold ASCF for 2 min, followed by biotinylation in 1 mg/ml of biotin (EZ-Link Sulfo-NHS-SS-Biotin; Pierce) for 20 or 45 min with slow agitation for cultured neurons or slices, respectively. This results in complete biotinylation throughout 400-μm hippocampal slices (17Thomas-Crusells J. Vieira A. Saarma M. Rivera C. J. Neurosci. Methods. 2003; 125: 159-166Crossref PubMed Scopus (52) Google Scholar). After three rinses in cold Tris-based ACSF (HEPES replaced with Tris) to quench free biotin, slices were snap frozen in liquid nitrogen. Cultures were immediately scraped without freezing in cold 1% Triton X-100 homogenization buffer (150 μl/35-mm well) (in mm: 50 NaCl, 10 EDTA, 10 EGTA, 1 Na3VO4, 50 NaF, 25 NaPPi, 1 β-glycerophosphate, 1 phenylmethylsulfonyl fluoride, 0.001 microcystine, 1× protease inhibitor mixture tablet (Roche Applied Science), 1× phosphatase inhibitor mixture set I (Calbiochem), and 50 HEPES (pH 7.5). Solubilization was performed in 1% Triton X-100 because this protocol does not solubilize postsynaptic densities (36Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar) and is therefore selective for extrasynaptic AMPARs. Two frozen slices were pooled for each condition and homogenized in 150 μl of cold 1% Triton X-100 homogenization buffer. An additional 150 μl of homogenization buffer was added to obtain 300 μl of total slice homogenate. Homogenates from cultures (2 wells per condition) and slices were centrifuged at 10,000 × g for 20 min to pellet the insoluble fraction. For the total fraction of GluR1 (surface plus internal), 75 μl of the supernatant was mixed and heated with 25 μl of 6× SDS sample buffer. Biotinylated surface proteins in the remaining supernatant (∼225 μl) were immunoprecipitated with 40 μl of 50% avidin-agarose beads (ImmunoPure Immobilized Avidin; Pierce) for >2 h at 4 °C. The beads were pelleted, and 75 μl of the supernatant (internal fraction) was mixed and heated with 25 μl of 6× SDS sample buffer. The beads were then rinsed three times with 1% Triton X-100 homogenization buffer and heated in 100 μl of 2× SDS sample buffer (surface fraction). Equal volumes of the total and internal fractions were subjected to 10% SDS-PAGE, probed for total GluR1, and normalized to tubulin. The surface fraction was calculated as the following: surface = 1 – (internal/total). Quantitative Western Blotting—Primary antibodies were: and and surface GluR1 from cultured neurons and slices were subjected to 10% with a set of on the and every for net of were by a consensus phosphorylation for with in for 30 min at 30 in phosphorylation buffer This phosphorylated Ser-845 GluR1 was mixed with of proteins to obtain and phosphorylated were transferred to membrane and antibodies and were performed on the were to for the in GluR1 between GluR1 and were calculated by the fraction of total GluR1 and then this fraction of of with were used for CaM-KII and phosphorylation of C CaM-KII phosphorylated GluR1 to a of and this was by not GluR1, but Ser-845 into consensus phosphorylation site for phosphorylation whereas a at the CaM-KII site = the two phosphorylation sites that Ser-831 Ser-845 sites are consensus phosphorylation for CaM-KII or with a an at the Western and total GluR1 antibodies of in CaM-KII and phosphorylated results as incorporation and of Hippocampal hippocampal slices were and from Sprague-Dawley rats as previously described T. Soderling T.R. J. Neurosci. 2005; PubMed Scopus Google Scholar, C.M. Derkach V. Soderling T.R. J. Neurosci. 2005; PubMed Scopus Google Scholar). buffer was with (in mm): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, and (pH were at 100 and of were stimulation was every min, and were to for and were each to for between After 20 min of 25 μm forskolin and 0.1 μm rolipram (no MgCl2) were added in slice buffer for 10 min with or without burst stimulation were used as described T. Soderling T.R. J. Neurosci. 2005; PubMed Scopus Google Scholar). After slices were snap for homogenized in 150 μl of ice-cold 1% Triton X-100 homogenization and subjected to 10% with for quantitative Western For and F/R were immediately followed by min of 25 μm NMDA. were replaced in regular slice buffer after stimulation and then subjected to surface biotinylation without at indicated time points. were performed by with or time each In the are as Ser-845 Phosphorylation Surface GluR1 in Cultured Hippocampal net Ser-845 phosphorylation in hippocampal we of GluR1 with phosphorylation This was by in phosphorylation of of the 75 of GluR1 with protein A. Derkach V. Soderling T. J. Biol. Chem. 1997; 272: 32727-32730Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). Using CaM-KII phosphorylation of Ser-831 to a of a of for Ser-845 of the both Ser-831 and Ser-845 that is a for CaM-KII or an in the position the and were and for CaM-KII phosphorylated the the as but phosphorylation of the The results by incorporation were antibodies for Ser-831 and Ser-845 The was phosphorylated by on Ser-845 and mixed with of to These in were on every with of cultured neurons or hippocampal slices and used for Western with to the net Ser-845 phosphorylation of GluR1 in cultured hippocampal neurons. Ser-845 phosphorylation (12Roche K.W. O'Brien R.J. Mammen A.L. Bernhardt J. Huganir R.L. Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar), we used a described chemical stimulation protocol of forskolin plus rolipram that results in NMDA receptor LTP N. L. N. S. S. J. J. 2004; PubMed Scopus Google Scholar) and CaM-KII to N. S. A. J. J. Neurosci. 2004; 24: PubMed Scopus Google Scholar). of hippocampal cultures with F/R for 10 min persistently increased Ser-845 phosphorylation of surface GluR1 to at min which correlated with a increase in surface expression of GluR1 Importantly, there was net increase in Ser-831 phosphorylation during this time suggesting that Ser-845 phosphorylation a role in GluR1 trafficking (13Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 14Esteban J.A. Shi S.H. Wilson C. Nuriya M. Huganir R.L. Malinow R. Nat. Neurosci. 2003; 6: 136-143Crossref PubMed Scopus (691) Google Scholar). The Ser-845 phosphorylation not between the surface and total GluR1 fractions not Ser-845 phosphorylation surface GluR1 delivery was by the and or not The of net normalized surface GluR1 a relationship with an of and of This relationship GluR1 Ser-845 phosphorylation in surface expression of AMPARs. of Ser-845 with a in Surface of NMDA chemical LTD and Ser-845 in hippocampal slices K. Lee H.K. Huganir R.L. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, H.K. Kameyama K. Huganir R.L. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar). We that Ser-845 phosphorylation during F/R stimulation also the surface delivery of AMPARs. of NMDA to cultured hippocampal neurons resulted in a Ser-845 of surface GluR1 and also subsequent Ser-845 phosphorylation Importantly, the in was with a in the surface pool of GluR1 These results that the fraction of GluR1 to the surface was correlated with net (13Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). AMPARs to in Hippocampal functional role of GluR1 Ser-845 phosphorylation was investigated in hippocampal slices by F/R increases phospho-Ser-845, surface GluR1, and synaptic Surface GluR1 was by biotinylation previously to the slice (17Thomas-Crusells J. Vieira A. Saarma M. Rivera C. J. Neurosci. Methods. 2003; 125: 159-166Crossref PubMed Scopus (52) Google Scholar). of AMPARs were on the surface in both slices and cultured that biotinylation was in these not A F/R stimulation resulted in a Ser-845 phosphorylation from a of to that for at 1 h after the of that was with a increase in surface GluR1 fraction with 25 μm NMDA for min min after NMDA normalized = = NMDA = = C.M. Derkach V. Soderling T.R. J. Neurosci. 2005; PubMed Scopus Google Scholar) and Ser-845 phosphorylation and surface delivery of GluR1 Thus, that increased or decreased net increased or decreased the surface pool of GluR1, the that Ser-845 phosphorylation is in the of surface pool of GluR1 in hippocampal slices. We the functional of GluR1 Ser-845 phosphorylation on synaptic strength by from the of hippocampal slices. After 20 min of stimulation was and F/R was to slices for 10 min, followed by stimulation 40 min to changes in synaptic The was to surface GluR1 to increase to 50% a and stimulation was synaptic strength in F/R slices was with the and open This was because the increase in surface GluR1 increase surface AMPARs enhancing synaptic was also as the at during and 40 min after F/R not These that in the of stimulation, F/R was to increase surface AMPA receptors but not synaptic strength that AMPA receptors phosphorylated at Ser-845 were trafficked to extrasynaptic Ser-845 Phosphorylation GluR1 for additional is required to the extrasynaptic receptors into F/R was with synaptic stimulation, the synaptic were resulting in N. L. N. S. S. J. J. 2004; PubMed Scopus Google Scholar) that for 1 h open required synaptic N. L. N. S. S. J. J. 2004; PubMed Scopus Google Scholar). on that synaptic activity is not required for surface expression of AMPARs in to we that Ser-845 phosphorylation AMPARs for synaptic potentiation by trafficking to extrasynaptic sites, followed by synaptic incorporation synaptic activity. extrasynaptic AMPARs regulate synaptic strength during this we hippocampal slices with F/R in the of stimulation to increase extrasynaptic AMPARs, followed by theta burst stimulation to LTP. three theta resulted in LTP in slices with F/R with slices In this and subsequent were not between F/R NMDA and not suggesting a postsynaptic of expression these Furthermore, after in slices with F/R with These results support the that GluR1 Ser-845 phosphorylation increases the of AMPARs for synaptic incorporation (14Esteban J.A. Shi S.H. Wilson C. Nuriya M. Huganir R.L. Malinow R. Nat. Neurosci. 2003; 6: 136-143Crossref PubMed Scopus (691) Google Scholar) by increasing the extrasynaptic surface Ser-845 Phosphorylation of AMPARs for Ser-845 phosphorylation is important for the surface pool of extrasynaptic AMPARs, then Ser-845 phosphorylation also of AMPARs and the corresponding of LTP observed in We this by a of 25 μm that resulted in of Ser-845 and and decreased the surface pool of GluR1 and J. J. Neurosci. 2004; 24: PubMed Scopus Google Scholar). We that NMDA Ser-845 phosphorylation and the of LTP. of NMDA after F/R Ser-845 phosphorylation from in slices with F/R to F/R was followed by NMDA This NMDA also in the of stimulation not NMDA the of F/R on theta burst LTP These results that GluR1 Ser-845 phosphorylation an important role in the of LTP by the of extrasynaptic AMPARs for synaptic incorporation The of this was that GluR1 Ser-845 phosphorylation AMPARs for synaptic incorporation by trafficking AMPARs to extrasynaptic sites on the surface studies (3Banke T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar, 14Esteban J.A. Shi S.H. Wilson C. Nuriya M. Huganir R.L. Malinow R. Nat. Neurosci. 2003; 6: 136-143Crossref PubMed Scopus (691) Google Scholar, H.K. M. Kameyama K. Huganir R.L. Nature. 2000; PubMed Scopus Google Scholar) Ser-845 phosphorylation in GluR1 stimulation but only relative changes were it to the significance of these This is the first to the net phosphorylation of Ser-845 in hippocampal GluR1 during bidirectional synaptic plasticity. This of functional in changes of net and surface GluR1 delivery during bidirectional synaptic plasticity by quantitative surface and slice field of GluR1 was on the surface and only 15% was phosphorylated at of in the of stimulation, increased surface expression and Ser-845 phosphorylation without Ser-831 whereas decreased surface expression and Ser-845 with a functional of synaptic we that Ser-845 surface GluR1 delivery was to extrasynaptic from this indicate that Ser-845 phosphorylation can the of synaptic plasticity by the pool of AMPARs for synaptic Thus, Ser-845 phosphorylation be of several mechanisms that neurons to regulate the of synaptic Although there are mechanisms for GluR1 delivery to the surface membrane during synaptic plasticity, the critical role of Ser-845 phosphorylation is in the of extrasynaptic AMPARs by the between the surface and internal of AMPARs. is that F/R increases in Ser-845 phosphorylation of surface and total GluR1. A model to this is that in the of Ser-845 phosphorylation the between surface and internal AMPARs the internal pool Ser-845 this between surface and endocytosis changes (13Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar), such that of AMPAR between the surface and internal Thus, F/R increase the pool of total surface expression by of was that the surface delivery of AMPARs can be from synaptic that required synaptic activity suggesting that synaptic is required N. L. N. S. S. J. J. 2004; PubMed Scopus Google Scholar). Ser-845 phosphorylation previously to be important for the of GluR1-containing AMPARs for synaptic whereas CaM-KII activity to be critical for synaptic incorporation (14Esteban J.A. Shi S.H. Wilson C. Nuriya M. Huganir R.L. Malinow R. Nat. Neurosci. 2003; 6: 136-143Crossref PubMed Scopus (691) Google Scholar). Our results a for this previous Ser-845 delivery is selective to extrasynaptic The increase in extrasynaptic pool of GluR1 resulted in LTP theta burst stimulation suggesting that synaptic activity can GluR1-containing AMPARs into synapses from the extrasynaptic This was by which the increase in and surface GluR1 M. R.A. J. Biol. Chem. 2005; Google Scholar). results support a two-step model for synaptic delivery of AMPARs where GluR1-containing AMPARs are first to the extrasynaptic sites in a Ser-845 manner. synaptic then an of that the for AMPARs in synapses. Thus, Ser-845 phosphorylation the GluR1-containing AMPARs for synaptic The two-step model for synaptic delivery of AMPARs is with from For studies that extrasynaptic AMPARs in hippocampal are increased during LTP J.C. J. 2004; PubMed Scopus Google Scholar) and extrasynaptic AMPARs are in GluR1 T. R. J.C. J. 2003; PubMed Scopus Google Scholar). 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The of CaM-KII important for this delivery is as it does not to be Ser-831 in GluR1 Y. Shi S.H. Esteban J.A. Piccini A. Poncer J.C. Malinow R. Science. 2000; 287: 2262-2267Crossref PubMed Scopus (1253) Google Scholar). phosphorylation sites, CaM-KII and sites that to be in AMPAR trafficking S. H. M. K. Nicoll R.A. Bredt D.S. Nature. 2005; PubMed Scopus Google Scholar). additional studies are to the mechanisms of synaptic of AMPARs.