Stargazin regulates synaptic targeting of AMPA ... - Semantic Scholar

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to the surface membrane of granule cells, whereas its binding with PSD-95 and related .... site for association with certain synaptic PDZ proteins such as PSD-.
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Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms Lu Chen*², Dane M. Chetkovich²³§, Ronald S. Petraliak, Neal T. Sweeney³, Yoshimi Kawasaki³, Robert J. Wentholdk, David S. Bredt³ & Roger A. Nicoll*³ Departments of * Cellular and Molecular Pharmacology, ³ Physiology, and § Neurology, University of California, San Francisco, California 94143, USA k Laboratory of Neurochemistry, NIDCD, National Institutes of Health, Bethesda, Maryland 20892, USA ² These authors contributed equally to this work

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Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (a-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZbinding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system. Excitatory synapses in the central nervous system (CNS) release glutamate onto a number of receptor subtypes. The principal ionotropic glutamate receptors include AMPA receptors (AMPARs) and NMDARs (N-methyl-D-aspartate receptors). AMPARs mediate moment-to-moment signalling, whereas NMDARs initiate synaptic plasticity. Recent studies emphasize remarkable differences in synaptic expression of these receptors. NMDARs are relatively ®xed components of the postsynaptic density (PSD), whereas AMPARs are more loosely associated and their density at synapses is tightly controlled by neuronal activity1±8. That the number of AMPARs at the synapse is regulated independently of NMDARs raises intriguing questions concerning mechanisms involved in synaptic targeting of glutamate receptors and the role that this plasticity plays in learning and memory. This independent regulation of synaptic AMPARs versus NMDARs is clearly manifested in the stargazer mutant mouse, which exhibits seizures and cerebellar ataxia. Among the defects identi®ed in the cerebellum is the lack of functional AMPARs on granule cells9,10. The defective protein stargazin was recently identi®ed as a relative of the g-1 subunit of the skeletal muscle calcium channel11. More recently, a family of g-like subunits has been identi®ed, each with distinct expression patterns in the brain12,13. Why stargazin is necessary for functional AMPARs in cerebellar granule cells has remained mysterious and is the focus of this study.

stg/stg granule cells lack AMPAR synaptic currents

We ®rst determined whether the defect in AMPARs in the stargazer mouse results from abnormal cerebellar circuitry or alternatively is an autonomous granule cell defect. We therefore evaluated AMPARs in granule cell cultures, which lack the normal excitatory mossy ®bre input, as well as Purkinje cells, the primary target for granule cells. In +/stg cultures, spontaneous rapid inward currents are routinely recorded from individual granule cells, presumably generated by synapses between granule cells (Fig. 1a). These events are largely mediated by the action potential dependent release of glutamate onto AMPARs, as their frequency is greatly reduced by tetrodotoxin (TTX) and they are abolished by the AMPAR antagonist CNQX (data not shown). In striking contrast, stg/stg neurons exhibit essentially no spontaneous activity (Fig. 1b). The average 936

size of events in +/stg cells was 18.3 6 5.1 pA (n = 9), whereas the average size in stg/stg cells was 4.8 6 0.4 pA (n = 18), close to the threshold value of 4 pA used to detect these events (Fig. 1c, see broken line in graph). Furthermore, the frequency of events was 1.7 6 0.7 Hz (n = 9) in +/stg and 0.1 6 0.1 Hz (n = 18) (P , 0.01) in stg/stg (data not shown). The stg/stg neurons clearly form excitatory synaptic connections as NMDAR currents can be recorded when Mg2+ is removed from, and CNQX and glycine are added to, the solution (compare Fig. 1d and e). As expected for NMDARs, these currents are considerably slower than those mediated by AMPAR currents and are blocked by the NMDAR antagonist AP5 (D(-)-amino-5-phosphonovaleric acid (data not shown). Figure 1f shows that there was no difference in the amplitude of NMDAR-mediated events in +/stg cells (15.4 6 1.2 pA, n = 20) and stg/stg cells (14.1 6 2.4 pA, n = 9), or in the frequency of these events (+/stg = 0.5 6 0.1 Hz, n = 20; stg/stg = 0.4 6 0.1 Hz, n = 9; P . 0.1).

Localization of AMPAR subunits in granule cells

In cerebellar cultures from +/stg mouse, the AMPAR subunit GluR4 forms discrete synaptic puncta that colocalize with the presynaptic marker synaptophysin1 (Fig. 1g). By contrast, few synaptic GluR4 puncta are evident in stg/stg cells. Despite lacking synaptic GluR4 puncta, the cultured stg/stg granule cells are decorated with presynaptic synaptophysin1 puncta. To assess the synaptic localization of AMPAR subunits at mossy ®bre synapses in the intact cerebellar glomeruli, we used post embedding immunogold electron-microscopic analysis. Granule cell synapses in stg/stg cerebellum are virtually devoid of GluR2/3 labelling, whereas these synapses in +/stg are labelled abundantly (Fig. 2a, b). Similar results were obtained with an antibody to the GluR4 subunit; most synapses in the +/stg mouse are GluR4-positive (Fig. 2d), whereas synapses in the stg/stg mouse are rarely labelled (Fig. 2c). A `blind' quantitative analysis showed roughly a 10-fold decrease in the number of GluR2/ 3-reactive particles at stg/stg synapses (Table 1). In contrast to the defect in synaptic GluR 2/3 labelling, the NMDAR subunit NR1 labelling was increased in the stg/stg mutant (gold particles per synapse: +/stg, 0.40; stg/stg, 0.84; P , 1 ´ 10-5). The cytoplasmic GluR2/3 labelling was low in both +/stg

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articles and stg/stg granule cells, making quanti®cation dif®cult, but no obvious difference was noted. The presence of cytoplasmic staining indicates that AMPARs are present in the stg/stg granule cells, in agreement with western blot analysis10. Finally, there was no obvious difference in the pre- or postsynaptic morphology of the mossy ®bre to granule cell synapse between the stg/stg mice and +/stg mice. For example, the length of postsynaptic densities averaged 149 nm for stg/stg (n = 227) and 143 nm for +/stg (n = 242) (P . 0.1).

Calcium currents are normal in stg/stg granule cells

On the basis of its weak homology to the g-1 calcium channel subunit11, we looked for altered calcium currents in stg/stg granule cells. In cultured cerebellar granule cells, voltage-dependent calcium currents can be well clamped and are largely mediated by P/Q-, Land N-type channels14. There was no difference in the peak amplitude (control = 83.9 6 10.3 pA, n = 11; stg/stg = 88.7 6 9.3, n = 9; P . 0.5) (data not shown), the activation curve for the calcium currents (Fig. 3a), or the steady-state inactivation (Fig. 3b) between stg/stg and +/stg cells. Together with the absence of AMPAR currents, these data make it most unlikely that the primary defect in stg/stg granule cells is altered calcium channel function. a +/stg

Stargazin interacts with AMPARs and PDZ proteins

As stargazer granule cells lack AMPAR currents, we next determined whether stargazin associates with GluR subunits. When co-transfected into COS cells, stargazin and GluR4 co-immunoprecipitate (Fig. 4a). Notably, stargazin also interacts with co-transfected GluR1 or 2 subunits (Fig. 4b). These stargazin interactions with GluR subunits appear speci®c, as stargazin does not bind to NMDAR (NR1-4b) (Fig. 4c) or Kv1.4 (data not shown). In brain homogenates, stargazin is enriched in Triton X-100 insoluble postsynaptic density fractions together with GluR4, NR1 and postsynaptic density-95 (PSD-95) (Fig. 4g). Although we were unable to co-immunoprecipitate stargazin with GluR from brain extracts (data not shown), the harsh conditions required for solubilization of the PSD disrupt many protein complexes and often preclude detection of interactions15,16. Stargazin is predicted to contain four putative transmembrane domains with intracellular amino- and carboxy-terminal tails11. We noticed that its C-terminal region contains a type I PDZ-binding site for association with certain synaptic PDZ proteins such as PSD95. We found that stargazin interacts with PSD-95 (Fig. 4d) and SAP-97 (Fig. 4e), as well as with PSD-93 and SAP-102 (data not Table 1 Summary of immunogold labelling for GluR2/3 at cerebellar glomeruli

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............................................................................................................................................................................. * The percentage of labelled synapse, the average number of particles per synapse and the average number of particles per labelled synapse were signi®cantly different (t-test) between +/stg and stg/ stg. No signi®cant difference was found between two animals (no. 1 and 2) within each group.

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Figure 1 Functional AMPARs are absent at stg/stg granule cell synapses in culture. a, b, Spontaneous AMPAR EPSCs are abundant in +/stg granule cells, but are absent in stg/stg granule cells. Asterisk, individual event displayed in the inset on the right. c, Mean amplitude of AMPAR EPSCs from +/stg and stg/stg granule cells (P , 0.0005). The detection threshold, 4 pA (dashed line), was assigned to cells in the stg/stg group that did not show any synaptic AMPAR responses during the recording period. d, e, Spontaneous NMDAR EPSCs from +/stg and stg/stg granule cells in the presence of CNQX (10 mM), glycine (20 mM) and 0 Mg2+. f, Mean amplitude of NMDAR EPSCs from +/stg and stg/stg granule cells (P . 0.5). g, Immunostaining of GluR4 and synaptophysin1 in methanol®xed 8-DIV granule cell cultures. +/stg cells, in contrast to stg/stg cells, exhibit dendritic GluR4 puncta that colocalized with synaptophysin1. Scale bar, 5 mm. NATURE | VOL 408 | 21/28 DECEMBER 2000 | www.nature.com

Figure 2 AMPAR immunogold labelling of cerebellar mossy ®bre-granule cell synapses. The examples illustrate the low labelling of stg/stg synapses compared with that of +/stg synapses. a, No GluR2/3-positive gold particles are present at the four asymmetric synapses (arrow heads) made by a single mossy ®bre terminal in stg/stg cerebellum. b, Typical synaptic labelling with the GluR2/3 antibody in +/stg mice. c, d, Examples of synapses in the stg/stg (c) and +/stg (d) mouse labelled for GluR4. Scale bar, 0.2 mm.

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articles shown). Deleting the ®nal four amino acids of stargazin (stargazinDC) disrupts interaction with PSD-95 (data not shown), although stargazinDC does retain binding to GluR4 (Fig. 4f). Because PSD-95 can mediate clustering of ion channels17, we wondered whether its interactions with stargazin might cluster GluRs in heterologous cells. Co-expression of GluR4 with PSD-95 (Fig. 4h, top row), or with stargazin (middle row) results in diffuse, overlapping distributions for these proteins, which generally resemble the localization of GluR4 expressed alone (data not shown). However, transfecting the three together causes a remarkable redistribution of the proteins to patch-like clusters (Fig. 4h, bottom row). This clustering requires interaction of stargazin with the PDZ domains from PSD-95, as clustering is not observed in co-transfections with stargazinDC (data not shown). The stargazin/PSD-95induced clusters of GluR4 occur at the cell surface, as they are labelled in non-permeabilized cells by an antibody to the extracellular green ¯uorescent protein (GFP) tag on GluR4 (data not shown).

AMPAR responses to glutamate in stg/stg granule cells

The absence of spontaneous activity in stg/stg granule cells suggests a

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that stargazin mediates synaptic targeting, but does not address whether extrasynaptic receptors are altered. We therefore analysed granule cell responses to rapid application of glutamate. Responses were ®rst recorded at a membrane potential of -80 mV, which isolates AMPAR responses, and these were compared to responses evoked at +60 mV, where both AMPAR and NMDAR currents are recorded (Fig. 5a, b). In +/stg neurons, a considerable current is generated at -80 mV (77 6 8 pA, n = 7), and this is largely abolished by NBQX (10 6 0.3 pA, n = 4), con®rming that at -80 mV this current is carried by AMPARs. In striking contrast, stg/stg neurons show very little current at -80 mV (9 6 3 pA, n = 5). The small NBQX resistant component, similar in size to that recorded in stg/stg granule cells, is presumably generated by NMDARs, which contain the NR2C subunit in granule cells and have an incomplete Mg2+ blockade at negative potential18. At +60 mV, a large outward current is recorded in +/stg neurons (177 6 32 pA, n = 7), which is not signi®cantly different from the current recorded in stg/stg neurons (257 6 57 pA, n = 5). NBQX has little effect on the +/stg responses recorded at +60 mV (162 6 22 pA, n = 4), because the low concentration of glutamate used (100 mM) more effectively activates NMDARs compared with AMPARs. These results illustrate the complete and selective absence of functional AMPARs in stg/stg granule cells.

Stargazin rescues AMPAR responses in stg/stg cells

Transfecting stargazin in stg/stg neurons restores synaptic AMPAR function as stargazin±GFP expressing cells display a large number of spontaneous inward currents (15.8 6 2.7 pA, n = 7) as compared with GFP-expressing cells (4.8 6 0.9 pA, n = 3) or untransfected cells (4.8 6 0.4 pA, n = 19) (Fig. 6a, b). Stargazin transfection increased the frequency of events from 0.1 6 0.1 Hz (n = 19) to 1.3 6 0.3 Hz (n = 7) (P , 0.0005) (data not shown). By contrast, expression of stargazinDC failed to restore synaptic responses (5.0 6 0.7 pA, n = 8). We also found that glutamate-evoked responses were restored by stargazin transfection. First, to assess any possible role for voltagedependent calcium channels (VDCCs) in the AMPAR phenotype, we transfected stargazin into 5-DIV (days in vitro) stg/stg cultures that had all VDCCs blocked by 10 mM cadmium from the time of transfection. Despite the continuous absence of calcium currents (data not shown), glutamate-evoked responses were restored 24 h after transfection (control, 4.8 6 1.7 pA, n = 3; transfected, 44.4 6 7.0 pA, n = 6; P , 0.005). Second, we examined the surfacereceptor response in stargazinDC-transfected stg/stg cultures. Unexpectedly, stargazinDC fully restores responses in stg/stg cells at -80 mV (stg/stg = 14.6 6 4.4 pA, n = 5; versus stg/stg plus stargazinDC = 205.7 6 56.6 pA, n = 7), but had no signi®cant effect on responses recorded at +60 mV (Fig. 6c, d). In wild-type cells, stargazinDC has little effect on AMPAR responses (-80 mV, untransfected: 104.3 6 10.1 pA, n = 26; transfected: 141 6 36.0 pA, n = 10; P . 0.1) or on the NMDAR responses (+60 mV, untransfected, 205.1 6 25.5 pA, n = 18; transfected, 306.0 6 91.1 pA, n = 10; P . 0.1) to glutamate applications (Fig. 6e). Although stargazinDC has no effect on the AMPAR response to glutamate application in wild-type neurons (Fig. 6e), it markedly reduced the amplitude (control = 14.4 6 0.7 pA, n = 22; stargazinDC = 7.0 6 0.7 pA, n = 18; P , 1 ´ 10-7) and frequency (control = 1.1 6 0.2 Hz, n = 22; stargazinDC = 0.4 6 0.2 Hz, n = 18; P , 0.05) of spontaneous AMPAR synaptic events (Fig. 6f), indicating that stargazinDC disrupts synaptic localization of AMPARs.

StargazinDC downregulates hippocampal AMPAR EPSCs

Why is the AMPAR defect in stargazer mice restricted to cerebellar granule cells9,10? Perhaps the same mechanism applies to all excitatory synapses, but stargazin isoforms are expressed in other regions of the brain. Indeed, studies have shown that a family of three stargazin related proteins is expressed throughout the CNS13. In situ

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articles hybridization shows that stargazin (g-2) is expressed in cerebellar granule cells, cerebellar Purkinje cells and hippocampal pyramidal cells (Fig. 7a, top). The stargazin related gene g-3 is not expressed in cerebellar granule cells but does occur in cerebellar Purkinje cells and hippocampal pyramidal cells (Fig. 7a, bottom). We evaluated possible functions for stargazin proteins in forebrain by transfecting cultured hippocampal cells. Transfected stargazin has a punctate distribution in hippocampal neurons (Fig. 7b, left), whereas stargazinDC is diffuse (Fig. 7b, right). The stargazin clusters are synaptic as shown by colocalization with GluR1 (Fig. 7c), SV2 and synaptophysin1 (data not shown). Stargazin appears restricted to excitatory synapses, as it does not colocalize with inhibitory boutons stained for glutamic-acid decarboxylase 65 (GAD 65) (Fig. 7d). We wondered whether stargazinDC might disrupt AMPAR EPSCs in hippocampal neurons by preventing interaction between AMPARs and other members of the stargazin family. StargazinDC

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This study de®nes an essential role for stargazin in synaptic expression of glutamate receptors in the CNS. Previous studies have focused on two possible roles for stargazin. Homology to the g-1 subunit of the calcium channel has suggested that stargazin may be a

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had no effect on glutamate (1 mM) induced currents at -80 mV (control = 413 6 170 pA, n = 11; stargazinDC = 425 6 109 pA, n = 8), or at +60 mV (control = 770 6 174 pA, n = 11; stargazinDC = 700 6 146 pA, n = 8) (data not shown). However, stargazinDC expression decreased markedly the amplitude of AMPAR miniature EPSCs (mEPSCs) (control = 20.2 6 1.1 pA, n = 15; stargazinDC = 10.1 6 1.0 pA, n = 14) when compared with neighbouring untransfected neurons (Fig. 7e, left). In addition, the frequency of mEPSCs was reduced greatly (control = 15.7 6 4.2 Hz, n = 15; stargazinDC = 1.6 6 0.9 Hz, n = 14) (Fig. 7e, right).

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synaptophysin Figure 4 Interactions between Stargazin, GluR and PDZ proteins. a, b, Coimmunoprecipitation of stargazin and AMPAR subunits from COS cells. Cells were transfected with stargazin±GFP and GluR4 (a) or stargazin±GFP and GluR1, GluR2 or GluR4 (b). c, Stargazin fails to immunoprecipitate Myc-tagged NR1. d, e, Coimmunoprecipitation of stargazin with PSD-95 (d) and with SAP-97 (e). f, Deletion of the C-terminal PDZ-binding domain from stargazin (starDC±GFP) does not prevent the interaction of stargazin with GluR4. g, Stargazin is enriched in the PSD fraction resistant to NATURE | VOL 408 | 21/28 DECEMBER 2000 | www.nature.com

solubilization with Triton X-100. Antibodies against GluR4, PSD-95 and NR1 were used as controls for postsynaptic proteins, and an antibody against synaptophysin was used as a presynaptic control. h, COS cells were transfected with different combinations of GFP± GluR4, PSD-95 and stargazin±HA as indicated. After permeabilization and ®xation, the distribution of transfected proteins was visualized by indirect immuno¯uorescence. The protein speci®cally visualized is indicated by coloured text.

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articles calcium-channel subunit in the CNS11. Alternatively, de®cits in cerebellar function9,10 suggested that stargazin may regulate AMPARs. The homology of stargazin with the g-1 subunit is weak (less than 25%), and the physiological effects of stargazin on calcium channels are modest11,13. We were unable to detect any difference between the calcium channel currents recorded in stg/stg or +/stg granule cells. Furthermore, AMPAR currents were rescued

despite blockade of voltage-dependent calcium channels with cadmium, indicating that stargazin effects on AMPAR function are independent of calcium channels. Our work shows that stargazin has a direct interaction with AMPARs. Stargazin co-immunoprecipitates with GluR1, -2 and -4 in heterologous cells. Ultrastructural studies using immunogold labelling found a marked decrease in synaptic GluR2/3 and GluR4

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Figure 6 Stargazin rescues AMPAR responses in stg/stg granule cells. a, b, Transfection of stargazin±GFP, but not GFP or stargazinDC, restores synaptic AMPAR responses in stg/stg granule cells (asterisk, P , 0.0001). Dashed line in b indicates the detection threshold (4 pA): cells without synaptic events during the recording period were assigned a response amplitude of 4 pA. c, d, StargazinDC restores glutamate-evoked responses at -80 mV in stg/stg cells (asterisk, P , 0.05). Responses at +60 mV are unaltered 940

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(P . 0.1). e, In +/stg granule cells, stargazinDC transfection does not signi®cantly alter the amplitude of glutamate-evoked responses at either -80 mV (P . 0.1) or + 60 mV (P . 0.1). f, Transfection of stargazinDC into +/stg granule cells downregulates synaptic AMPAR responses (P , 1 x 10-7). Cells were recorded in pairs in an attempt to control for differences in connectivity that may occur across cover slips and culture batches. WT, wild type.

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articles labelling, but no decrease in NR1 labelling of cerebellar mossy ®bre granule cell synapses in stg/stg mice. Expressing stargazin in stg/stg granule cells rescues AMPAR responses to synaptically released glutamate. Finally, when transfected into cultured hippocampal neurons, stargazin±GFP was found to cluster at synapses and colocalize with GluR1. Further studies indicated that the PDZ-binding domain of stargazin is required for a subset of its actions. Removing the Cterminal PDZ binding domain from stargazin disrupts binding to PSD-95 and presumably other PDZ proteins in heterologous cells, but has no effect on AMPAR binding. This stargazinDC mutant rescues AMPAR responses to exogenous glutamate in stg/stg granule cells, but fails to restore synaptic responses. Finally, stargazinDC does not cluster at synapses and severely impairs AMPAR synaptic responses in hippocampal neurons. These ®ndings suggest that stargazin has two distinct roles in controlling AMPAR function. First, stargazin regulates delivery of AMPARs to the membrane surface and this function does not require the PDZ-binding domain. Second, stargazin mediates synaptic targeting of AMPARs and this function does require the PDZ-binding C terminus. Moreover, we propose that stargazin and the related proteins g-3 and g-4 mediate AMPAR targeting throughout the brain12,13. That stargazin clusters at hippocampal synapses and that stargazinDC acts as a dominant-negative support a general role for stargazin-like proteins in synaptic targeting of

AMPARs. A class of PDZ-domain-containing proteins, including GRIP (glutamate receptor interacting protein) 1 and 2, ABP (AMPAR-binding protein), PICK1 (protein interacting with C kinase 1) and SAP-97 (synapse-associated protein) have been implicated in synaptic targeting/clustering of AMPARs19±21. A recent study suggests that the association of GluR2 with ABP/ GRIP is not essential for synaptic targeting, but is required for maintaining the synaptic surface accumulation of AMPARs22. Together, it seems that synaptic targeting/insertion and synaptic stabilization of AMPARs may be mediated by several mechanisms. Whereas the interaction between stargazin and PSD-95 or related PDZ proteins is crucial for the initial synaptic targeting of AMPARs, a different pathway involving the association of GluR2 with ABP/ GRIP may be required for receptor stabilization. A number of issues remain unanswered. The site(s) on the AMPARs and stargazin responsible for binding has not been identi®ed and may involve the transmembrane domains. It is unlikely that the interaction between stargazin and AMPARs occurs within the intracellular C-terminal domain of AMPARs, as very little homology of this domain exists among GluR1, -2 and -4. How stargazin controls surface expression of AMPARs in granule cells is unknown. Stargazin might act as a chaperone or might mask a retention signal on the AMPARs. However, because surface expression of AMPARs can be detected in many cells including oocytes, HEK293 cells and so on, it would seem that either all these

Figure 7 StargazinDC downregulates hippocampal excitatory synapses. a, In situ hybridization shows discrete neuronal localizations of stargazin (g-2) and g-3 in brain. In cerebellum (cb) stargazin occurs in granule cells (G), in Purkinje neurons (P) and in stellate cells of the molecular layer (M). By contrast, g-3 occurs in Golgi cells of granule cell layer and in some Purkinje neurons, but is absent from granule cells and molecular layer. In hippocampus (hipp), stargazin and g-3 are co-expressed in similar neuronal populations. b, Stargazin±GFP, when transfected into hippocampal neurons, exhibits a punctate

distribution, whereas stargazinDC±GFP shows a diffuse pattern. Scale bar, 10 mm. c, Stargazin±GFP colocalizes with GluR1 in 12-DIV cultures. Scale bar, 10 mm. d, GAD 65 staining does not overlap with stargazin±GFP. Scale bar, 10 mm. e, StargazinDC transfection downregulates AMPAR mEPSC amplitude (P , 1 x 10-6) and frequency (P , 0.05) in hippocampal neurons. For the analysis of mEPSC amplitude, a 6-pA detection threshold (dashed line) was used: cells with no detectable mEPSCs were assigned a value of 6 pA.

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articles cell types possess a stargazin-like protein or that high GluR expression can bypass this regulation. Finally, although NMDAR function is independent of stargazin, kainate receptors, which are structurally similar to AMPARs, might require stargazin for normal traf®cking. We have identi®ed an essential role for stargazin in the synaptic targeting of AMPARs. We raise the intriguing possibility that stargazin-like proteins may participate in rapid activity-dependent traf®cking of AMPARs in synaptic plasticity, a proposal supported by the known impairment of stargazer mouse in motor learning23.M

Methods Antibodies The following antibodies were used: rabbit polyclonal antibodies to PSD-95, SAP-97, Kv1.4 (ref. 17), GluR1, GluR2 and GluR4 (Chemicon); monoclonal antibodies to PSD-95 (Af®nity Bioreagents), NR1 (Pharmingen), GFP (Quantum, Clontech), haemagglutinin A (HA) epitope tag (BABCO) and Myc epitope tag (Santa Cruz); mouse monoclonal antibodies to synaptophysin (Sigma) and GAD 65 (a gift from S. Baeskkoskov). The antibody to stargazin was generated by injecting rabbits with a fusion protein of the stargazin C-terminal 120 amino acids. The primary antibodies for the electron microscopy study (GluR2/3, GluR4, NR1 polyclonal and monoclonal) have been characterized24±27.

Plasmid constructs Stargazin cDNA (generously provided by V. Letts) was PCR ampli®ed and subcloned into eukaryotic expression vectors pcDNA3 (Stratagene) and GW1-CMV (British Biotechnology). The HA epitope was inserted into Stargazin at the unique XhoI site (between residues 105 and 106). EGFP was inserted into Stargazin at the BglII site (between residues 269±270). GluR4 was tagged with GFP by inserting GFP between amino acids 3 and 4 of the mature protein.

Tissue culture and immunolabelling Transfection (Lipofectamine Plus) and immunolabelling of COS cells were done as described28. For analysis of surface GluR, COS-7 cells transfected with GluR4±GFP were washed in ice-cold DMEM and then incubated with DMEM containing anti-GFP antibody (Clontech) for 30 min at 4 8C before ®xation in 2% paraformaldehyde. For immunoprecipitation, transfected COS cells were washed with ice-cold PBS and resuspended in 0.5 ml of lysis buffer containing TEE (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA), 150 mM NaCl, 1% Triton X100, PMSF, aprotinin and leupeptin. Cells were solubilized by brief sonication followed by extraction for 30 min at 4 8C. Insoluble material was removed by centrifugation at 10,000g for 10 min. Samples were then incubated with GFP antibodies for 1 h at 4 8C. After addition of 20 ml of Protein A/Sepharose beads (Pharmacia), samples were incubated for 1 h at 4 8C. Immunoprecipitates were washed three times with buffer containing TEE, 150 mM NaCl and 1% Triton X-100, boiled in SDS±PAGE sample buffer with 1 mM dithiothreitol for 2 min and analysed by SDS±PAGE. Input was 5% of the co-immunoprecipitation. Hippocampal primary cultures were prepared from newborn Sprague-Dawley rats as described29, except that B27-supplement (Life Technologies) was added to the culture media. Stargazer breeding pairs were from Jackson Laboratory. Stargazer cerebellar granule cell cultures were prepared from 5±7-day-old mouse pups as described14. Cells were in minimum essential medium (5.3 mM K+; Gibco), supplemented with glucose, transferrin, insulin, glutamine, cytosine arabinoside and 10% heat-inactivated fetal calf serum (Gibco). Tail samples from individual pups were used for genotyping with described primers11. Neuronal cultures were transfected using DOTAP (Roche) or Effectene (Qiagen). Immunocytochemistry was performed on 8±9-DIV granule cell cultures or 12±14-DIV hippocampal cultures. After ®xation with methanol (15 min, -20 8C) or 2% paraformaldehyde (15 min, room temperature), cells were washed with PBS containing 0.3% Triton X100 (PBST) before being incubated with primary antibody and secondary antibodies. Imaging used either epi¯uorescence with a ´ 63 oil-immersion objective or confocal microscopy with a ´60 oil-immersion objective.

Subcellular fractionation Subcellular fractions of rat brains were prepared by differential centrifugation as described30.

Electrophysiology Whole-cell patch-clamp recordings were made at room temperature using 3±7-MQ patch pipettes ®lled with an internal solution containing (in mM) 140 CsCl, 2 MgCl2, 5 EGTA, 10 HEPES, 0.3 Na3-GTP, 4 Na2-ATP pH 7.35. Cultures were continuously superfused with external solution containing (in mM) 119 NaCl, 26 NaHCO3, 2.5 KCl, 10 glucose, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 0.1 picrotoxin. Tetrodotoxin (TTX; 1 mM) was added for agonist application experiments and miniature EPSC recordings. During spontaneous and miniature EPSC recordings, cells were held at -60 mV (unless otherwise stated). For most experiments, neighbouring transfected and untransfected cells were selected for recording in an attempt to minimize any differences that may occur across cover slips and platings, such as the degree of connectivity. Fast agonist application was achieved by gravity feeding as described31. Calcium current recordings were obtained from 2-DIV granule cell cultures. External solution contains (in mM) 110 NaCl, 20 TEA-Cl, 26 NaHCO3, 2.5 KCl, 10 glucose, 3.0 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 0.1 picrotoxin and 1 mM

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TTX. The patch-pipette solution contains (in mM) 130 CsCl, 20 TEA-Cl, 2 MgCl2, 5 EGTA, 10 HEPES, 0.3 Na3-GTP, 4 Na2-ATP. Current records were ®ltered at 2 kHz and digitized at 5 kHz.

Postembedding immunogold labelling The postembedding immunogold method has been described32±35 and is modi®ed from an earlier method36. Parasagittal sections of the dorsorostral quadrant of the cerebellum were cryoprotected and frozen in a Leica EM CPS, then placed in a Leica AFS, in®ltrated with Lowicryl HM 20, and polymerized with ultraviolet light. Thin sections were immunolabelled using 10-nm immunogold (Amersham). Glomerular zones of the granular layer were selected and photographed from a wide area of the section. All synapses in the micrographs were counted. For both stg/stg and +/stg, most of these synapses showed ultrastructural features typical of granule cell dendrite synapses37. All immunogold particles from the postsynaptic density and cleft were counted, as described32±35. Received 17 July; accepted 31 October 2000. 1. Luscher, C., Nicoll, R. A., Malenka, R. C. & Muller, D. Synaptic plasticity and dynamic modulation of the postsynaptic membrane. Nature Neurosci. 3, 545±550 (2000). 2. Malenka, R. C. & Nicoll, R. A. Long-term potentiationÐa decade of progress? Science 285, 1870±1874 (1999). 3. Malinow, R., Mainen, Z. F. & Hayashi, Y. LTP mechanisms: from silence to four-lane traf®c. Curr. Opin. Neurobiol. 10, 352±357. 4. Liu, S. Q. & Cull-Candy, S. G. Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype. Nature 405, 454±458 (2000). 5. Luthi, A. et al. Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF±GluR2 interaction. Neuron 24, 389±399 (1999). 6. Man, Y. H. et al. Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization. Neuron 25, 649±662 (2000). 7. Song, I. et al. Interaction of the N-ethylmaleimide-sensitive factor with AMPA receptors. Neuron 21, 393±400 (1998). 8. P. Osten, S. et al. The AMPA Receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and SNAPs. Neuron 21, 99±110 (1998). 9. Chen, L., Bao, S., Qiao, X. & Thompson, R. F. Impaired cerebellar synapse maturation in waggler, a mutant mouse with a disrupted neuronal calcium channel gamma subunit. Proc. Natl Acad. Sci. USA 96, 12132±12137 (1999). 10. Hashimoto, K. et al. Impairment of AMPA receptor function in cerebellar granule cells of ataxic mutant mouse stargazer. J. Neurosci. 19, 6027±6036 (1999). 11. Letts, V. A. et al. The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit. Nature Genet. 19, 340±347 (1998). 12. Burgess, D. L., Davis, C. F., Gefrides, L. A. & Noebels, J. L. Identi®cation of three novel Ca2+ channel gamma subunit genes reveals molecular diversi®cation by tandem and chromosome duplication. Genome Res. 9, 1204±1213 (1999). 13. Klugbauer, N. et al. A family of gamma-like calcium channel subunits. FEBS Lett. 470, 189±197 (2000). 14. Randall, A. & Tsien, R. W. Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons. J. Neurosci. 15, 2995±3012 (1995). 15. Hsueh, Y. P. et al. Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses. J. Cell Biol. 142, 139±151 (1998). 16. Torres, R. et al. PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21, 1453±1463 (1998). 17. Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N. & Sheng, M. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378, 85±88 (1995). 18. Monyer, H., Burnashev, N., Laurie, D. J., Sakmann, B. & Seeburg, P. H. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529±540 (1994). 19. Sheng, M. & Pak, D. T. Glutamate receptor anchoring proteins and the molecular organization of excitatory synapses. Ann. N. Y. Acad. Sci. 868, 483±493 (1999). 20. Garner, C. C., Nash, J. & Huganir, R. L. PDZ domains in synapse assembly and signalling. Trends Cell Biol. 10, 274±280 (2000). 21. Braithwaite, S. P., Meyer, G. & Henley, J. M. Interactions between AMPA receptors and intracellular proteins. Neuropharmacology. 39, 919±930 (2000). 22. Osten, P. et al. Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor. Neuron 27, 313±325 (2000). 23. Bao, S., Chen, L., Qiao, X., Knusel, B. & Thompson, R. F. Impaired eye-blink conditioning in waggler, a mutant mouse with cerebellar BDNF de®ciency. Learn. Mem. 5, 355±364 (1998). 24. Wenthold, R. J., Yokotani, N., Doi, K. & Wada, K. Immunochemical characterization of the nonNMDA glutamate receptor using subunit-speci®c antibodies. Evidence for a hetero-oligomeric structure in rat brain. J. Biol. Chem. 267, 501±507 (1992). 25. Petralia, R. S. & Wenthold, R. J. Light and electron immunocytochemical localization of AMPAselective glutamate receptors in the rat brain. J. Comp. Neurol. 318, 329±354 (1992). 26. Petralia, R. S., Yokotani, N. & Wenthold, R. J. Light and electron microscope distribution of the NMDA receptor subunit NMDAR1 in the rat nervous system using a selective anti-peptide antibody. J. Neurosci. 14, 667±696 (1994). 27. Luo, J., Wang, Y., Yasuda, R. P., Dunah, A. W. & Wolfe, B. B. The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/ NR2B). Mol. Pharmacol. 51, 79±86 (1997). 28. El-Husseini, A. E. et al. Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering. J. Cell Biol. 148, 159±172 (2000). 29. Lester, R. A. J., Quarum, M. L., Parker, J. D., Weber, E. & Jahr, C. E. Interaction of 6-cyano-7nitroquinoxaline-2, 3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site. Mol. Pharmacol. 35, 565±570 (1989). 30. Jo, K., Derin, R., Li, M. & Bredt, D. S. Characterization of MALS/Velis-1,-2, and-3: a family of mammalian LIN-7 homologs enriched at brain synapses in association with the postsynaptic density95/NMDA receptor postsynaptic complex. J. Neurosci. 19, 4189±4199 (1999).

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articles 31. Lester, R. A. J. & Jahr, C. E. NMDA channel behavior depends on agonist af®nity. J. Neurosci. 12, 635± 643 (1992). 32. Wang, Y. -X., Wenthold, R. J., Ottersen, O. P. & Petralia, R. S. Endbulb synapses in the anteroventral cochlear nucleus express a speci®c subset of AMPA-type glutamate receptor subunits. J. Neurosci. 18, 1148±1160 (1998). 33. Petralia, R. S., Zhao, H. M., Wang, Y. X. & Wenthold, R. J. Variations in the tangential distribution of postsynaptic glutamate receptors in Purkinje cell parallel and climbing ®bre synapses during development. Neuropharmacology 37, 1321±1334 (1998). 34. Petralia, R. S. et al. Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses. Nature Neurosci. 2, 31±36 (1999). 35. Zhao, H. M., Wenthold, R. J. & Petralia, R. S. Glutamate receptor targeting to synaptic populations on Purkinje cells is developmentally regulated. J. Neurosci. 18, 5517±5528 (1998). 36. Matsubara, A., Laake, J. H., Davanger, S., Usami, S. & Ottersen, O. P. Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti. J. Neuroscience 16, 4457±4467 (1996). 37. Palay, S. L. & Chan-Palay, V. Cerebellar Cortex. Cytology and Organization (Springer, New York, 1974).

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Acknowledgements We thank R. F. Thompson and X. Qiao for providing the initial stargazer breeding pairs; K. P. Campbell for providing a stargazin antibody used in preliminary experiments; B. B. Wolfe for providing the NR1 monoclonal antibody used in the electron microscopy study; S. Tomita for subcloning Stargazin-3; E. Schnell for assisting in hippocampal culture transfection; Q. Zhou for assisting in confocal microscopy; and Y.-X. Wang for assisting in immunogold labelling. We also thank M. Frerking, L. Jan and D. Julius for their comments on the manuscript. D.S.B is supported by grants from NIH and HHMI. R.A.N. is supported by grants from the NIH and Bristol-Myers Squibb. D.S.B. is an established investigator of the American Heart Association, D.M.C. is a postdoctoral fellow of the HHMI. R.A.N. is a member of the Keck Center for Integrative Neuroscience and the Silvio Conte Center for Neuroscience Research. Correspondence and requests for materials should be addressed to R.A.N. (e-mail: [email protected]).

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