Driving AMPA Receptors into Synapses by LTP and CaMKII: Requirement for GluR1 and PDZ Domain Interaction
Tóm tắt
Từ khóa
Tài liệu tham khảo
R. Malinow et al. in Imaging Living Cells R. Yuste A. Lanni A. Konnerth Eds. (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1999) pp. 58-1–58-9.
Construction of GluR1-GFP was described in (3). Point mutants of GluR1-GFP were generated by QuickChange mutagenesis kit (Stratagene). CaMKII cDNA was isolated from a rat forebrain cDNA library mutated so that initiation methionine overlaps the Nco I site (ccATGg) and truncated after the 290th amino acid by adding a stop codon. This truncated CaMKII (tCaMKII) cDNA was cloned after the IRES sequence derived from encephalomyocarditis virus so that the initiation methionine matches the 12th ATG of IRES. The resulting IRES-tCaMKII was transferred downstream to GluR1-GFP mutant constructs. The tCaMKII-GFP fusion protein construct was made by inserting polymerase chain reaction–amplified tCaMKII fragment into pEGFP-N1 vector (Clontech). Sindbis virus containing each of these constructs was produced as described (3 4).
Electrophysiological recordings from CA1 pyramidal neurons were carried out in rat hippocampal organotypic slice cultures prepared as described (3). Neurons were infected as described (3) and recordings were made 24 to 36 hours later. External solution contained 119 mM NaCl 2.5 mM KCl 2 mM CaCl 2 4 mM MgCl 2 26.2 mM NaHCO 3 1 mM NaH 2 PO 4 11 mM glucose 100 μM picrotoxin 20 μM bicuculline 100 μM APV 1 μM 3-(( R S )-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and 4 to 10 μM 2-chloroadenosine (to reduce polysynaptic excitation) and was gassed with 5% CO 2 and 95% O 2 at ambient temperature. Internal solution consisted of 115 mM cesium methanesulfonate 20 mM CsCl 10 mM Hepes 2.5 mM MgCl 2 4 mM Na 2 -ATP (adenosine triphosphate) 0.4 mM Na-GTP (guanosine triphosphate) 10 mM sodium phosphocreatine 0.6 mM EGTA and 0.1 mM spermine (pH 7.2). For recordings from cell pairs two cells with cell bodies within ∼20 μm were selected one cell showing and the other not showing GFP fluorescence. The stimulus electrode was placed in stratum radiatum ∼50 μm from stratum pyramidale. Recordings were first generally made from an infected cell and the stimulus level was set to produce a synaptic response of ∼30 pA. Upon termination of that recording a whole-cell recording was immediately obtained from the nearby control cell with the same location and intensity of stimulus. Ratio of amplitude of synaptic response at –60 and +40 mV (average of 50 to 100 traces each) was used as a measure of rectification throughout this study. Response at 0 mV was also measured to ensure reversal potential of response and quality of voltage clamping. As expected rectification is independent of the absolute amplitude of the response [Web figure 1 (26)]. Thus recordings that were not carried out in a paired fashion were also included in the calculation of the average rectification. However comparisons were restricted to those among cells recorded on the same day. LTP was induced as described in (3). Baseline recordings were limited to about 2 min due to faster washout of LTP in slice culture. Recording from HEK293 cells was carried out as described (3).
Six hours after infection with respective virus BHK cells (2 × 10 5 cells per 35-mm dish) were solubilized in 50 mM Hepes-NaOH (pH 7.4) 1 mM EGTA 0.5 mM dithiothreitol 50 mM NaCl 10% glycerol 40 mM NaF 0.1 mM phenylmethylsulfonyl fluoride (PMSF) 2 μg/ml CLAP (cocktail of chymostatin leupeptin pepstatin A and antipain) and 1% NP40. The Ca 2+ and calmodulin-independent phosphorylation was determined in a mixture of 2 μg of protein 10 mM Hepes-NaOH (pH 7.4) 5 mM MgCl 2 10 mM EGTA 50 μM [γ- 32 P]ATP (Amersham) 20 μM autocamtide-2 (Calbiochem) 40 mM NaF and 0.5 mM dithiothreitol in a final volume of 20 μl. The reaction was carried out at 30°C for 4 min and the mixture was spotted onto P81 phosphocellulose paper. The paper was immediately dropped into 1% phosphoric acid to terminate the reaction. After washing with the same solution three times the paper was dried and the radioactivity was measured.
Y. Hayashi S.-H. Shi J. A. Esteban R. Malinow unpublished results.
For collection and analysis of electrophysiological data in which there was expression of GluR1 and its mutants in approximately half of the experiments the experimenter was blind to the genotype of Sindbis virus vectors. Resultant data were not significantly different from nonblind experiments and thus were pooled. For assessment of statistical significance to the difference in means we used Wilcoxon nonparametric test (for change in amplitude between a pair of infected and uninfected cells) and the nonpaired version the Mann-Whitney nonparametric test (for rectification). The two-tailed P values are indicated in each graph. Student's t test on raw data or on log-normalized data gave similar results. Error bars indicate SEM.
This increase in AMPA-R–mediated response is not due to change in electrotonic properties caused by morphological change in dendrite because the NMDA receptor–mediated component was not changed by expression of tCaMKII. In uninfected cells NMDA current amplitude was 10.0 ± 1.6 pA whereas in tCaMKII-GFP infected cells it was 7.5 ± 1.2 pA. The difference was not statistically significant (Mann-Whitney test: P = 0.28; t test: P = 0.21 n = 25 each).
After 1.5 days of infection slices were pooled and solubilized in homogenization buffer (100 μl per slice) composed of 10 mM Hepes-NaOH 0.5 M NaCl 10 mM sodium pyrophosphate 10 mM NaF 10 mM EDTA 4 mM EGTA 0.1 mM PMSF 2 μg/ml CLAP and 1% Triton X-100. The solution was cleared by centrifugation at 10000 g for 5 min at 4°C. To supernatant (0.5 mg of protein per 0.5 ml for each reaction) protein G–Sepharose (40 μl 50% volume in homogenization buffer) was added to preabsorb nonspecific resin binding and the solution was again centrifuged at 5000 g for 1 min at 4°C. After reaction with antibody to GFP (anti-GFP monoclonal 10 μg per sample Boehringer Mannheim) or anti-GluR1 (polyclonal 1 μg per sample Chemicon International) at 4°C for 2 hours the immunocomplex was absorbed onto protein G–Sepharose resin (40 μl) at 4°C for 2 hours. Finally the resin was washed three times with homogenization buffer subjected to SDS-PAGE and blotted with anti-GFP (polyclonal Clontech) anti-GluR1 and anti-GluR2 (polyclonal Chemicon International).
This response was sensitive to 3 μM NBQX an AMPA-R antagonist.
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr. X indicates any residue.
It is possible that phosphorylation of GluR1 at Ser 831 and another protein are both necessary for AMPA-R delivery. In this case S831A may mimic phosphorylation of Ser 831 possibly by preventing a protein-protein interaction mediated by the hydroxyl group of Ser. The marked synaptic potentiation seen in cells expressing GluR1(S831A)-GFP-IRES-tCaMKII supports this view.
The phosphorylation of Thr 887 by CaMKII is not likely because in the case of GluR1 phosphorylation stimulated by either neuronal activity or CaMKII occurs exclusively on the Ser residue in both endogenous and recombinant protein but not on the Thr residue [
; A. Barria thesis Vollum Institute Portland OR (1998)].
The depression by GluR1(T887A)-GFP of transmission may be explained in the following manner. Normally there is a pool of GluR1-containing AMPA-Rs outside the synapse. Upon activation of CaMKII-dependent plasticity these receptors are incorporated into a delivery pathway in which PDZ proteins play a critical role. This delivery process may contain elements used in a separate constitutive delivery process; such a process appears to act on AMPA-Rs containing GluR2 and requires N -ethylmaleimide–sensitive fusion protein (NSF). The mutant receptor appears to be recruited upon CaMKII activation or LTP into an interaction site where it can block this constitutive process; the time-course of its effects on transmission is similar to the effect of peptides that block the interaction between GluR2 and NSF [
Supplemental material is available at www.sciencemag.org/feature/data/1046986.shl
We thank C. Sano for help in DNA construction N. Dawkins-Pisani for technical assistance and H. Schulman M. Hollmann and S. F. Heineman for cDNA clones. Y.H. was supported by Japan Society for the Promotion of Science and Uehara Memorial Foundation J.A.E. by Alzheimer Association and National Alliance for Research on Schizophrenia and Depression and J.-C.P. by the Human Frontier Science Program Organization. This study was supported by NIH and the Mathers Foundation (to R.M).