Regulation of NR1/NR2C N-Methyl-D-aspartate (NMDA) Receptors by Phosphorylation*

Abstract

NR2C-containing N-methyl-d-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg2+, compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser1244), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser1244 on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser1244 does not influence this PDZ interaction. Furthermore, Ser1244 phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser1244 accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser1244 does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser1244 on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum. NR2C-containing N-methyl-d-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg2+, compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser1244), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser1244 on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser1244 does not influence this PDZ interaction. Furthermore, Ser1244 phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser1244 accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser1244 does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser1244 on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum. NMDA 2The abbreviations used are: NMDA, N-methyl-d-aspartate; AMPA, 1-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PKA, cAMP-dependent protein kinase; CaMKII, Ca2+/calmodulin-dependent protein kinase II; PSD, postsynaptic density; SAP, synapse-associated protein; FACS, fluorescence-activated cell sorting; PVDF, polyvinylidene difluoride; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; WT, wild-type; IRES, internal ribosome entry site; GFP, green fluorescent protein; EGFP, enhanced GFP; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. receptors are glutamate-gated ion channels that are critical for synapse formation, synaptic plasticity, and learning and memory (1Bliss T.V. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9728) Google Scholar, 2Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1426) Google Scholar). They are members of the larger family of ionotropic glutamate receptors that also include AMPA and kainate receptors. Endogenous NMDA receptors are heteromeric channels containing two NR1 subunits combined with additional NR2 (NR2A–D) and NR3 (NR3A-B) subunits (2Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1426) Google Scholar, 3Wisden W. Seeburg P.H. Curr. Opin. Neurobiol. 1993; 3: 291-298Crossref PubMed Scopus (266) Google Scholar). In contrast to the widespread distribution for NR1, NR2 subunits display unique spatiotemporal expression patterns throughout the central nervous system (4Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2930) Google Scholar) and confer distinct pharmacological and functional properties on NMDA receptors (2Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1426) Google Scholar). NMDA receptors in the cerebellum are composed of NR1 and NR2A–C subunits; the NR2C subunit is notably enriched in cerebellar granule cells and is up-regulated developmentally such that in the adult, NR1, NR2A, and NR2C are the predominant NMDA receptor subunits assembling to form functional receptors (2Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1426) Google Scholar, 5Takahashi T. Feldmeyer D. Suzuki N. Onodera K. Cull-Candy S.G. Sakimura K. Mishina M. J. Neurosci. 1996; 16: 4376-4382Crossref PubMed Google Scholar). Moreover, NR2C-containing receptors have unique properties of low conductance and reduced sensitivity to Mg2+ compared with NR2A- or NR2B-containing receptors (2Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1426) Google Scholar, 4Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2930) Google Scholar, 6Farrant M. Feldmeyer D. Takahashi T. Cull-Candy S.G. Nature. 1994; 368: 335-339Crossref PubMed Scopus (264) Google Scholar). Despite this, the specific regulation of NR2C-containing receptors has not been studied extensively. Phosphorylation is an important mechanism regulating glutamate receptors and synaptic plasticity and occurs primarily in the cytosolic COOH-terminal domain of the receptor subunits. Phosphorylation of AMPA receptors by PKA and CaMKII directly affects channel function (7Roche 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 (671) Google Scholar, 8Banke T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar, 9Derkach V. Barria A. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3269-3274Crossref PubMed Scopus (707) Google Scholar), receptor trafficking to synapses (10Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar), and is critical for the expression of long term potentiation and long term depression (11Lee H.K. Barbarosie M. Kameyama K. Bear M.F. Huganir R.L. Nature. 2000; 405: 955-959Crossref PubMed Scopus (910) Google Scholar, 12Lee 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 (654) Google Scholar). Phosphorylation is an important regulator of NMDA receptor function and trafficking (13Crump F.T. Dillman K.S. Craig A.M. J. Neurosci. 2001; 21: 5079-5088Crossref PubMed Google Scholar, 14Lan J.Y. Skeberdis V.A. Jover T. Grooms S.Y. Lin Y. Araneda R.C. Zheng X. Bennett M.V. Zukin R.S. Nat. Neurosci. 2001; 4: 382-390Crossref PubMed Scopus (371) Google Scholar, 15Wenthold R.J. Prybylowski K. Standley S. Sans N. Petralia R.S. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 335-358Crossref PubMed Scopus (295) Google Scholar); however, the direct phosphorylation of NR2C has not been reported. Interestingly, NR2C contains a prototypic PKA phosphorylation site in the distal COOH-terminal region that is not conserved in NR2A and NR2B, suggesting that phosphorylation at this location might play a specific role in regulating NR2C-containing NMDA receptor complexes. In this study, we have investigated the regulation of NR2C by phosphorylation. We demonstrate that Ser1244 is phosphorylated by both PKA and PKC in vitro and also on native NR2C in neurons. This serine is just upstream of the PDZ binding domain, which is conserved in NR2 subunits and is critical for NMDA receptor binding to PSD-95. We demonstrate that NR2C interacts directly with PSD-95; however, we find no effect of Ser1244 phosphorylation on PSD-95 binding to NR2C. Unlike the PKA regulation of AMPA receptors (10Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar), we find no effect of Ser1244 phosphorylation on surface expression of NR1/NR2C NMDA receptors. However, we find that this site alters NMDA receptor channel function, because a phosphomimetic mutation (S1244E) accelerates channel kinetics. Thus, we find that this phosphorylation site in the distal COOH terminus regulates NR2C channel function. DNA Constructs and Site-directed Mutagenesis—The rat NR2C cDNA was obtained from Dr. Peter Seeburg (Max Planck Institute, Heidelberg, Germany). The COOH terminus of NR2C (amino acids 1076–1250) was amplified by PCR using synthetic primers that include flanking EcoRI recognition sequences and subcloned into glutathione S-transferase (GST) fusion vector pGEX-6T-1 (Amersham Biosciences) and the LexA fusion vector pBHA (16Niethammer M. Kim E. Sheng M. J. Neurosci. 1996; 16: 2157-2163Crossref PubMed Google Scholar). Full-length NR2C in the mammalian expression vector pRK5 was epitope tagged with the FLAG epitope (DYKDDDDK) between amino acids 36 and 37 using The was by the DNA of into vector The rat PSD-95 and cDNA obtained from the cDNA PSD-95 (amino acids (amino acids (amino acids (amino acids (amino acids and (amino acids amplified by PCR using synthetic primers that include flanking EcoRI recognition sequences and subcloned into the domain fusion vector NR2C and on fusion LexA fusion and or using the the FLAG epitope and by DNA cells NR2C with containing and and with in for at with containing for at by for and in containing for at 37 of containing to the in a of was by at for The was with for at and with containing by with the cells with and S. J. Standley S. K.W. 2003; PubMed Scopus Google Scholar). HEK-293 cells with containing and with for at cells to and with for at with and with for at and in The cells using a and using a and the with at and and Biosciences) was used to and the and to the of for using and rat with a in and The was at for The was and at for The N. Prybylowski K. Petralia R.S. K. C. S. Wenthold R.J. Nat. 2003; PubMed Scopus Google Scholar) was in containing for at 37 of containing to the in a of was by at for of or was The was with protein for at and with containing by and to The was for at and by with the and in fusion proteins the by the (Amersham Biosciences) with DNA into cells. at 37 to of was to to fusion protein The cells for at and using a in containing and The with of the of (Amersham Biosciences) for at and with The fusion proteins with and reduced and and fusion proteins WT, and phosphorylated in of with of PKA subunit at for PKC in of with of PKC at for CaMKII the by the in of with of at for The phosphorylation by and for The phosphorylated proteins by and by of Phosphorylation and phosphorylation phosphorylated Ser1244 of NR2C by with a synthetic to amino acids of NR2C. and using the was from and FLAG was from HEK-293 cells or cells with NR2C or NR2C using the and the PKA was for at 37 by and to a Phosphorylation of Ser1244 on NR2C COOH-terminal fusion proteins was by with the NR2C Ser1244 phosphorylation of cerebellar granule S. J. PubMed Scopus Google Scholar). the from rat with and in with at a of The cells in with and from The of in the was to The at in in vitro the was with containing with and in vitro, cells in the of for to NR2C expression K. M. Y. S. J. Neurosci. PubMed Scopus Google Scholar). We to NR2C expression by with not with the PKA or the PKC phorbol 12-myristate for at 37 and compared with using by and to Phosphorylation of Ser1244 on NR2C was by with the NR2C Ser1244 phosphorylation was using the H. Cell. PubMed Scopus Google Scholar) to protein-protein in vector pBHA and the domain fusion vector into the in synthetic and and for expression of the in synthetic and cells with and S. J. Standley S. K.W. 2003; PubMed Scopus Google Scholar). and cells used for with cells by using containing BAPTA, The using an at at and and using excised a and in of a system of a from which was from and containing NMDA from the The was the using a of NMDA-evoked currents by a also time the was at The NR2C COOH by PKA in investigate the regulation of NR2C by PKA we first the phosphorylation of the NR2C COOH terminus in vitro, on the distal COOH-terminal The extreme COOH terminus of NR2C contains the PSD-95 binding which is conserved in NR2A and however, the adjacent upstream are a important in trafficking with an K.W. Standley S. J. C. M.D. Wenthold R.J. Nat. Neurosci. 2001; 4: PubMed Scopus Google Scholar) that is conserved in NR2A, is not in NR2C. NR2C contains a PKA consensus in this region which is not conserved in NR2A or NR2B. Therefore, we fusion proteins containing the amino acids of the NR2C COOH-terminal domain (amino acids 1076–1250) of both NR2C and NR2C containing an mutation of Ser1244 We GST, and with PKA and at for to in vitro phosphorylation. The proteins by and phosphorylated proteins by We that the NR2C COOH terminus was phosphorylated by PKA, and mutation of serine 1244 to reduced PKA phosphorylation. However, was PKA phosphorylation of the NR2C COOH terminus with the Ser1244 that or more additional phosphorylated by PKA in the regulation of Ser1244 phosphorylation in vitro and in we a phosphorylation NR2C phosphorylated on We the in vitro phosphorylation of GST, and the proteins by and the proteins to with the NR2C Ser1244 phosphorylation that the the NR2C COOH terminus containing phosphorylated Ser1244 the to the in vitro phosphorylation no specific with this the of this for NR2C phosphorylated on NR2C on Ser1244 in and in NR2C is phosphorylated PKA in cells, we expressed both NR2C and NR2C in HEK-293 cells and the cells with or an of of cell with the NR2C Ser1244 phosphorylation we that was a very low of phosphorylation on NR2C Ser1244 However, phosphorylation of Ser1244 was with was from of cells NR2C with or with the Ser1244 phosphorylation the of this for NR2C phosphorylated on regulation of NR2C Ser1244 phosphorylation was NR2C was expressed in cells mammalian cell investigate the of NR2C phosphorylation in we phosphorylation of NR2C on Ser1244 in cerebellum. We rat NR2C, and with NR2C Ser1244 phosphorylation We that NR2C was phosphorylated on Ser1244 The NR2C on the was specific because no was was used a for NR2C In of the with to the NR2C with the Ser1244 phosphorylation that the was specific for phosphorylated NR2C. NR2C is phosphorylated PKA in we Ser1244 phosphorylation in cerebellar granule cells in the or of In with the in heterologous cells, we phosphorylation of NR2C Ser1244 in to Thus, Ser1244 is on NR2C in is NR2C by consensus for PKA phosphorylation is with consensus for the CaMKII and PKC consensus a amino the phosphorylated Therefore, we PKC and CaMKII also NR2C on We GST, and with PKA, PKC, or CaMKII at for to in vitro phosphorylation. The proteins by to and with Ser1244 phosphorylation PKA and PKC phosphorylated Ser1244 but we no phosphorylation by CaMKII We investigated the of PKC the phosphorylation of NR2C on We cerebellar granule cells in with for We phosphorylation by with NR2C Ser1244 phosphorylation and a in with PKC phosphorylation of NR2C Ser1244 in NR2C with PDZ of Ser1244 phosphorylation site that we on NR2C is because is located just a amino acids upstream of the consensus PSD-95 binding of the PSD-95 family of proteins PDZ which are protein-protein E. Sheng M. Nat. Rev. Neurosci. PubMed Scopus Google Scholar). The PDZ ligand, is conserved in NR2A, NR2B, and NR2C The binding of PSD-95 and proteins to NR2A and has been by however, NR2C is of binding the PSD-95 family of proteins has not been Therefore, we investigated NR2C also directly to PSD-95 and the protein or not phosphorylation of Ser1244 regulates the interaction. this we used a binding a system that has been used to NMDA receptor binding to PDZ domain-containing proteins E. M. A. Sheng M. Nature. PubMed Scopus Google Scholar, G. J. Lee K.W. 2003; PubMed Scopus Google Scholar). We with the NR2C COOH terminus and PSD-95 or and a with expression of a that on in to direct of and on containing was used a for of the NR2C to the of PSD-95 and which the PDZ In NR2C not to PSD-95 or COOH-terminal This is with the of the NR2A and with the PSD-95 family of proteins (16Niethammer M. Kim E. Sheng M. J. Neurosci. 1996; 16: 2157-2163Crossref PubMed Google Scholar, U. S. S. Huganir R.L. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In we investigated a role for Ser1244 phosphorylation in protein binding by two Ser1244 in the mutation of Ser1244 on NR2C to to phosphorylation or mutation to glutamate to phosphorylation. In these we no in binding to that phosphorylation of Ser1244 does not binding of NR2C for PDZ domain-containing PKA Phosphorylation of Ser1244 on NR2C NMDA in distal COOH terminus of NMDA receptor subunits is an important of receptor for the in this region of affects trafficking and of the binding of PSD-95 G. J. Lee K.W. 2003; PubMed Scopus Google Scholar). this we investigated PKA phosphorylation of NR2C affects receptor trafficking or surface expression in heterologous cells. PKA NR2C Ser1244 in vitro and PKA Ser1244 phosphorylation in heterologous we the effect of on NR2C trafficking and surface expression in HEK-293 cells. NMDA receptors of NR1 and NR2 subunits; we the surface expression of NR2C with NR1 in HEK-293 cells using expression vector containing the sequences of NR2C and was used to cells We that the of cells containing surface expression of NR2C with NR1 was of the cells with to to phosphorylation of NR2C on Ser1244 in no effect on NR2C surface expression Furthermore, of NR2C in and cells suggesting that PKA does not surface expression of NR2C. In to we used a to surface expression of a cell surface of NR2C. We HEK-293 cells NR2C and NR1 for at proteins with and with to of a cytosolic was used a for surface the of this we obtained that with that the of NR2C expressed on the was low and with PKA We also NR2C phosphorylation on Ser1244 in the surface of receptors to investigate surface NR2C is in a in Ser1244 the phosphorylation not with in expression and the of surface expressed NR2C was the using the phosphorylation or an of NR2C of these are with Ser1244 phosphorylation a role distinct from regulating NR2C trafficking to the Ser1244 on NR2C phosphorylation of AMPA receptors has been to channel function in a distinct from role in receptor trafficking Huganir R.L. Neurosci. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we investigated phosphorylation of Ser1244 regulate the channel function of NR2C-containing NMDA receptors. We used patches combined with to the kinetics of NR1/NR2C receptors to be obtained from HEK-293 cells NR1/NR2C or NR1/NR2C a phosphomimetic of patches and NMDA in the of was using a have investigated the kinetics of NR1/NR2C receptors using a of low surface expression S. J. PubMed Scopus Google Scholar); the kinetics of NR1/NR2C receptors have not been for excised we that HEK-293 cells NR1/NR2C exhibit very NMDA receptor-mediated however, we currents in a of excised In these patches we that currents from NR1/NR2C cells more and decay kinetics for NR1/NR2C properties such and not between the and cells not that phosphorylation of this distal COOH-terminal phosphorylation site the kinetics of NR2C-containing NMDA receptors. we a of NMDA receptor kinetics to phosphorylation of NR2C. The NR2 subunit of NMDA receptors a of important receptor properties channel and synaptic NR2 subunits have unique expression patterns throughout the and throughout at synapses in the central nervous NR2C is an NMDA receptor subunit that is highly enriched in the suggesting a unique role in cerebellar have been the phosphorylation of ionotropic glutamate no such have been on the NR2C subunit of NMDA receptors. phosphorylation is such an important mechanism regulating glutamate receptor function, and synaptic plasticity, we have the phosphorylation and regulation of NR2C-containing receptors. NR2 NR2C has a long COOH-terminal NR2A and are phosphorylated by and this COOH and phosphorylation regulates NMDA receptor and synaptic of NR2C a PKA consensus site at the distal COOH terminus of the receptor adjacent to the consensus for PDZ domain This extreme COOH-terminal region is known to be important in glutamate receptor trafficking and in NMDA receptor subunits K.W. Standley S. J. C. M.D. Wenthold R.J. Nat. Neurosci. 2001; 4: PubMed Scopus Google Scholar, G. J. K.W. J. Neurosci. PubMed Scopus Google Scholar); however, the PDZ on NR2C the consensus PKA site has been We the first of NR2C phosphorylation. In the current study, we have the phosphorylation of NR2C in and investigated the functional role of phosphorylation on PSD-95 NMDA receptor and channel function. an in vitro phosphorylation we that NR2C is directly phosphorylated on Ser1244 by PKA and PKC in We PKA phosphorylation of NR2C expressed in heterologous cells. we very little phosphorylation of Ser1244 on NR2C expressed in HEK-293 cells or cells we a in Ser1244 phosphorylation the cells with to Furthermore, we that NR2C is phosphorylated in cerebellar and NR2C phosphorylation on Ser1244 is PKA or PKC in cerebellar granule cell This that Ser1244 is phosphorylated by at two distinct kinase NMDA receptors directly with PDZ domain-containing proteins such PSD-95. The binding of PDZ domain-containing proteins is by phosphorylation the PDZ on receptors or PKC phosphorylation of the AMPA receptor subunit on the PDZ with PDZ domain-containing proteins J. X. Huganir R.L. J. Neurosci. 2000; 20: PubMed Google Scholar). In PKA phosphorylation of the channel on a serine the PDZ binding to PSD-95 Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). phosphorylation of by kinase on the PDZ binding to PSD-95 and regulates of NMDA receptors Huganir R.L. J. Neurosci. PubMed Scopus Google Scholar). is the that phosphorylation of a residue a amino acids upstream of the PDZ regulates with PDZ domain-containing proteins and affects AMPA receptor surface expression T. Huganir R.L. J. Neurosci. PubMed Scopus Google Scholar). we have a phosphorylation site on NR2C located just a amino acids upstream of the consensus PSD-95 binding NR2C the consensus for binding to PSD-95 conserved in NR2A and NR2B, this has been Therefore, we used the and that NR2C interacts with both PSD-95 and and that the is specific for the of these which contains the PDZ binding We also the effect of Ser1244 phosphorylation on protein binding by Ser1244 to a phosphomimetic glutamate (S1244E) or to We no in PSD-95 binding with suggesting that phosphorylation of this site does not regulate these protein-protein receptor phosphorylation regulate receptor trafficking and surface expression by PDZ binding Huganir R.L. J. Neurosci. PubMed Scopus Google Scholar) but also regulate trafficking by of PDZ proteins (10Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, C. M.D. J. Neurosci. 2001; 21: PubMed Google Scholar, Y. S. T. Nat. Neurosci. PubMed Scopus Google Scholar). The trafficking of glutamate receptors has been a of in the for has from the that AMPA receptor trafficking in synaptic Phosphorylation of AMPA receptors by PKA regulates surface channel function, and long term to that PKA phosphorylation of NR2C-containing NMDA receptors might also surface Therefore, using we investigated the role of PKA phosphorylation of Ser1244 on NR2C on protein we that a of NR1/NR2C receptors are expressed on the and that was no in surface expression of NR2C with to PKA and We also cell surface to surface expression of NR2C. with the we that little NR2C was on the and that was no in surface expression with that phosphorylation of Ser1244 on NR2C does not trafficking or surface expression of NR2C expressed in heterologous cells. We also of NR1 and NR2C and no in the of NR1/NR2C receptor with not However, phosphorylation of on NR2C might regulate Furthermore, is that NR2C phosphorylation on Ser1244 a specific role in regulating synaptic expression of NMDA receptors in not in heterologous cells. Therefore, to this be In to protein binding and protein the phosphorylation of glutamate receptors directly channel function. PKA and CaMKII phosphorylation of the AMPA receptor subunit directly affects channel properties by the T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar) or channel conductance V. Barria A. Soderling T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3269-3274Crossref PubMed Scopus (707) Google Scholar), Therefore, we also the NMDA receptor channel kinetics of NR2C-containing receptors in excised patches from HEK-293 cells. We that the phosphomimetic NR2C NR2C accelerates the kinetics of the that phosphorylation of this distal COOH-terminal phosphorylation site regulates ion channel This is a not such a distal region of the COOH terminus to regulate ion channel a in kinetics the of NMDA receptor-mediated synaptic currents by NR2C-containing for in cerebellar granule cells. This of the kinetics is to NMDA receptors and may the of long term synaptic Therefore, into the of the regulation of NR2C-containing NMDA receptors in be of In we for the first time the phosphorylation of the NMDA receptor subunit NR2C, which is highly enriched in the cerebellum. NR2C is phosphorylated on Ser1244 of PKA or Ser1244 is located in the distal COOH terminus adjacent to the PDZ ligand, we find no effect on PDZ binding or protein trafficking but find an and in channel kinetics of NR1/NR2C NMDA receptors. NR1/NR2C channels have low channel conductance and reduced sensitivity to compared with the more commonly studied NR2A- and NR2B-containing a unique role in and synaptic that PKA and PKC directly NR2C, that this site is phosphorylated in and that regulates channel kinetics to the unique functional regulation of this of NMDA receptors and that this may play an important role in defining the properties of NR2C-containing receptors that are enriched in the cerebellum. We for Dr. and Dr. for in cerebellar granule Dr. for and in the of and and for DNA