The expression of dominant-negative subunits selectively suppresses neuronal AMPA and kainate receptors

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Neuroscience Vol. 115, No. 4, pp. 1199^1210, 2002 A 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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THE EXPRESSION OF DOMINANT-NEGATIVE SUBUNITS SELECTIVELY SUPPRESSES NEURONAL AMPA AND KAINATE RECEPTORS ANTOINE ROBERT,a RHONDA HYDE,a1 THOMAS E. HUGHESb and JAMES R. HOWEa
a

Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA b

Department of Ophthalmology and Visual Sciences, Yale University School of Medicine, 330 Cedar Street, New Haven, CT 06520-8066, USA

Abstract8Glutamate-gated ion channels are widely expressed in neurons where they serve a host of cellular functions. An appealing, but yet unexplored, way to delineate the functions of particular glutamate receptor subtypes is to direct the expression of dominant-negative and gain-of-function mutant subunits. We tested the ability of two dominant-negative subunits, an K-amino-3-hydroxy-5-methyl-isoxazolproprionic acid receptor subunit and a kainate receptor subunit, to silence recombinant and neuronal glutamate receptors. Co-expression studies in non-neuronal cells indicated that the inclusion of a single mutant subunit was su⁄cient to silence the receptor. When expressed in cerebellar granule cells, the dominant-negative subunits silenced native channels in a subtype-speci¢c fashion. Immunocytochemical staining of control and transfected neurons, as well as studies with a gain-of-function glutamate receptor-1 mutant, indicated that the mutant subunits were expressed at levels roughly equal to the total abundance of related native subunits, and both dominant-negatives suppressed native channel expression 60^65% when tested 24 h post-transfection. If co-assembly of the mutant subunits with related native subunits is combinatorial, this level of suppression gives receptor half-lives of approximately 20 h. A 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: glutamate receptor, dominant negative, AMPA, kainate, cerebellar granule cells, receptor turnover.

tional channels, native kainate receptors often include subunits from each gene family (Herb et al., 1992; Ripellino et al., 1998). Di¡erences in subunit composition and stoichiometry are likely a primary source of GluR functional diversity (Geiger et al., 1995; Washburn et al., 1997; Brorson et al., 1999; Cui and Mayer, 1999; Smith et al., 2000), but the redundancy of GluR expression in individual cells and the lack of strict constraints on subunit assembly complicate genetic approaches to unraveling the roles of individual receptor subtypes. For example, while metabotropic GluRs can be disrupted quite easily to produce instructive phenotypes, single GluR subunit knockouts are likely to alter rather than silence function, and these alterations may di¡er among cell types that express di¡erent complements of related subunits. An alternative strategy would be to direct the expression of mutant subunits that suppress or enhance GluR function when they co-assemble with native subunits. Several such mutations have been identi¢ed. For example, point mutations that create dominant-negative subunits were ¢rst reported for AMPA-type subunits (Dingledine et al., 1992), and other dominant-negative AMPA- and NMDA-type subunits have been identi¢ed (Watase et al., 1997; Sekiguchi et al., 1994; Laube et al., 1998). Conversely, gain-of-function GluR point mutants include the lurcher mutation (Zuo et al., 1997; Taverna et al., 2000) and a leucine-to-tyrosine mutation in the S1 domain of AMPA-type subunits that suppresses desensitization (Stern-Bach et al., 1998). However, although

Ionotropic glutamate receptors (GluRs) fall into three categories: K-amino-3-hydroxy-5-methyl-isoxazolproprionic acid (AMPA) receptors, N-methyl-D-aspartate (NMDA) receptors and kainate receptors. Four genes, GluR1^4, encode AMPA receptor subunits (Boulter et al., 1990; Keinanen et al., 1990) and each subunit forms functional homomeric channels in recombinant expression systems. AMPA receptors are believed to be tetramers (Rosenmund et al., 1998; Chen et al., 1999; Smith and Howe, 2000), and the native receptors expressed in neurons are often heteromeric combinations of at least two di¡erent subunits (Geiger et al., 1995; Wenthold et al., 1996). In contrast, kainate receptors are composed of subunits from two di¡erent gene families, GluR5^7 and KA1 and 2. Although all homomeric and heteromeric combinations of GluR5^7 form func-

1 Present address: Program in Neuroscience, Harvard University, 220 Longwood Ave., Boston, MA 02115, USA. *Corresponding author. Tel. : +1-203-737-2398; fax: +1-203-7857670. E-mail address: [email protected] (J. R. Howe). Abbreviations : AMPA, K-amino-3-hydroxy-5-methyl-isoxazolpropionic acid ; con A, concanavalin A; DIV, days in vitro ; GFP, green £uorescent protein; GluR, glutamate receptor; GYKI 53655, 1(4-amino-phenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine; MEM, modi¢ed Eagle’s medium; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide; NMDA, N-methyl-D-aspartate; PBS, phosphate-bu¡ered saline.

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dominant-negative and gain-of-function mutants have been widely and successfully used to delineate intracellular signaling cascades, they have only rarely been applied to studies of membrane channels (Ribera, 1996; Blaine and Ribera, 1998; Raghib et al., 2001; Selyanko et al., 2002). We show here that point mutations in the pore-forming regions of GluR1£ip (M599 R) and GluR6 (M589 R) create subunits that are dominant-negative when coexpressed with wild-type subunits in non-neuronal cells. Directing the expression of these mutants in cultured neurons substantially reduced native channel function in a subtype-speci¢c fashion. The results show that the targeted expression of dominant-negative subunits o¡ers a viable alternative approach to pharmacological methods for silencing neuronal GluRs.

EXPERIMENTAL PROCEDURES

Cell culture HEK 293 cells were plated onto 12-mm glass coverslips that had been coated with poly-L-lysine (100 Wg/ml) and were maintained in humidi¢ed 95% O2 /5% CO2 . The culture medium was modi¢ed Eagle’s medium (MEM; Gibco) containing 10% fetal bovine serum. HEK 293 cells were transiently transfected using Lipofectamine 2000 (Life Technologies, Invitrogen, Carlsbad, CA, USA) with 0.2^1 Wg of total cDNA per coverslip. The solution used for transfection consisted of 100 Wl of Opti-MEM medium (Gibco, Invitrogen, Carlsbad, CA, USA), 3 Wl Lipofectamine 2000, 0.5 Wg of a reporter cDNA encoding the green £uorescent protein (GFP) in pCMVsport, and 0.1^1 Wg GluR plasmid in a mammalian expression vector. The GluR1flip and GluR6 cDNAs were kindly provided by Peter Seeburg (MPI for Medical Research, Heidelberg, Germany). The GluR6 cDNA encodes the fully edited version of GluR6. Plasmids encoding GluR4flip and the Q version of GluR2flip were kind gifts from Michael Tang (Yale) and Mark Mayer (NIH), respectively. The transfection solution was ¢rst incubated at room temperature for 15 min and 25 Wl of this solution was added to each culture well. Mutagenesis Wild-type GluR1flip and GluR1flip (L497 Y) constructs were generously provided by Derek Bowie (Emory). Both constructs (in pRK5, a CMV-driven eukaryotic expression plasmid) were previously modi¢ed by Mark Mayer and Kathy Partin (NIH) to enhance expression in mammalian cells. To create the M599 R mutation in GluR1, the BglII fragment of the coding region was moved to pCR-Script. This was used as template in a QuickChange mutagenesis reaction (Stratagene, La Jolla CA, USA) with the primers: 5P-CCTGTGGTTCTCCCTGGGCGCCTTCAGGCAGCAAGGATGTGACATTTCCCCC-3P and 5P-ACATCCTTGCTGCCTGAAGGCGCCCAGGGAGAACCACAGGCTGTTGAATA-3P. These primers introduced both the M599 R mutation and a silent change that created a novel HaeI site. The HaeI site was used to identify the clones that carried the M599 R mutation, and the entire coding region was sequenced to ensure that the QuickChange reaction had not introduced unwanted mutations. The correct BglII fragment was then re-inserted into full-length GluR1flip in pRK5. This same fragment was inserted into GluR1flip (L497 Y) to create the double M/R, L/Y mutant. An analogous strategy was used to introduce the M589 R mutation into a BamHI fragment of GluR6 (with primers: 5P-GGAGCTCTCAGGCGGCAAGGTTCT-3P and 5P-CAGAACCTTGCCGCTGAGAGC-3P) that was subsequently moved into full-length GluR6 in pcDNA3 (Invitrogen).

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Cerebellar culture The cerebella of Sprague^Dawley rat pups (aged 5^7 days) were removed and minced with a razor blade. Cells were dissociated into the culture medium with a ¢re-polished Pasteur pipette in the absence of enzymes. The resulting solution was then ¢ltered through a nylon mesh and the cells were plated onto acid-washed coverslips coated with poly-L-lysine (400 Wg/ ml for 1 h). The culture medium was Dulbecco’s modi¢ed Eagle’s medium containing 25 mM KCl and 10% fetal bovine serum. Cells were transfected as described above using 150 ng of total DNA per coverslip. Electrophysiology Whole-cell patch-clamp recordings were made with an EPC9 ampli¢er (HEKA, Lambrecht, Germany). Patch electrodes were pulled from thin-walled borosilicate glass with inner ¢lament (Warner) to an open resistance of 1^3 M6 and 5^10 M6 for recordings from HEK 293 and cerebellar granule cells, respectively. Whole-cell currents were analog low-pass ¢ltered at 2.9 kHz (4-pole Bessel-type, 33 dB) and were written directly to the hard-drive of the computer at a sampling rate of 30 kHz. The cells were constantly superfused with normal external solution at a rate of 1 ml/min. All recordings were performed at room temperature (22^24‡C). The external solution was (in mM): 150 NaCl, 3 KCl, 2 CaCl2 , 1 MgCl2 , 5 glucose, 0.002 glycine and 10 HEPES (pH adjusted to 7.4 with NaOH). Tetrodotoxin (0.2 WM, Chemicon, Temecula, CA, USA) was added to the external solution for the experiments made on neurons. Patch pipettes were ¢lled with a solution containing (in mM): 120 CsF, 33 KOH, 2 MgCl2 , 1 CaCl2 , 0.1 spermine, 10 HEPES, and 11 EGTA (pH adjusted to 7.4 with CsOH). Frozen stock solutions of glutamate, kainate, NMDA, GYKI 53655 (1(4-amino-phenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine), and NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide, disodium salt) were diluted in external solution on the day of the experiment. Cyclothiazide was prepared as a 20-mM stock solution in DMSO and diluted to 100 WM in external solution. Concanavalin A (con A) was prepared in external solution. GYKI 53655 was a kind gift from David Leander (Eli Lilly, Indianapolis, IN, USA). Other chemicals were purchased from Sigma. Fast agonist applications Drugs were applied with a rapid superfusion system made from a pulled theta capillary. The tip of the theta glass was cleanly broken to a tip diameter of approximately 300 Wm and its septum snapped back so that the £ow of solution leaving the tip from either side of the theta capillary overlapped. Four capillaries (outer diameter 450 Wm; Polymicro Technologies, Phoenix, AZ, USA) were introduced into each barrel at the back of the theta glass and secured with Sylgard (Dow Corning, Midland, MI, USA). Each capillary was connected to plastic tubing, branched to a solenoid valve (The Lee Company, Westbrook, CT, USA). The valves were operated by a digital-analog interface (TIB 14, HEKA) that was controlled by the acquisition software of the EPC9. The open tip responses obtained with this system had 10^90% rise times of 150^300 Ws. Data analysis The digitized records were transferred to IGOR software (Wavemetrics, Oswego, OR, US). The amplitude of steadystate AMPA-, kainate-, and NMDA-receptor currents were measured and expressed as current density after dividing the amplitude by the whole-cell capacitance (determined from electronically cancelling the transients at the onset and o¡set of a 10-mV voltage pulse). For experiments in which wild-type GluR1flip and the GluR1flip (L497 Y) mutant subunits were compared, the decays of glutamate-evoked currents were ¢tted with functions consisting of the mixture of multiple exponential com-

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ponents and a steady-state plateau current. The segment of the decay to be ¢tted was de¢ned by placing cursors on the data trace, where the ¢rst cursor was placed near the peak of the current and the other cursor about 700 ms after the peak. Zero time was de¢ned as the time at which the peak inward current occurred. Fits were performed by sequentially including from one to three exponential components. At each stage, the quality of the ¢t was evaluated by visual inspection of the residual current (obtained by subtracting the ¢t from the data). If the residual current was not £at, an additional exponential component was added. Functions containing three exponential components were su⁄cient to obtain a £at residual current in all cases and inclusion of a fourth component did not improve the ¢ts signi¢cantly [see Robert et al. (2001)]. Sample means were compared with Student’s t-tests, and P values less than 0.05 were taken as statistically signi¢cant. Unless otherwise noted, results are given as mean V S.E.M. Immunocytochemistry Coverslips of cultured cerebellar cells were ¢xed in 4% paraformaldehyde for 5 min. The cells were then washed once with phosphate-bu¡ered saline (PBS) containing Triton X-100 (0.05%; Kodak). After incubation at room temperature for 1 h in PBS/Triton with 10% goat serum (Jackson ImmunoResearch, West Grove, PA, USA), the cells were exposed overnight at 4‡C to anti-GluR1 antisera (Upstate Biotechnology; 1:200 dilution in PBS/Triton and 10% goat serum). The following day the cells were rinsed three times with PBS and were then incubated with Texas Red-conjugated goat anti-rabbit antisera (Jackson ImmunoResearch; diluted 1:300 with PBS). The cells were then washed with PBS and mounted on slides with PBS-bu¡ered glycerol. Images of the cells were collected with a Zeiss microscope ¢tted with a 25U immersion lens and a Princeton Instruments (Roper Scienti¢c, Trenton, NJ, USA) cooled CCD camera. Epi£uorescence ¢lter sets were used to isolate and independently image the GFP and Texas Red signals (Chroma, Brattleboro, VT, USA). Identical coverslips of cells were processed without primary antibody as a control for non-speci¢c labeling caused by auto£uorescence and the secondary antisera. These control coverslips were used to adjust the collection times for the imaging. Cells transfected with only GFP served as a control for spurious labeling that could be due to the process of transfection and heterologous expression. The images were analyzed with IPlabs (Scanalytics, Fairfax, VA, USA) and IGOR software. To quantify GluR1 immunoreactivity, mean signal intensities were measured from the soma of control and transfected granule cells. Data from di¡erent experiments were pooled after scaling the image ¢les so that the mean intensity of untransfected cells was approximately 1000 (full scale 4096). Figure 6 was created with Adobe Photoshop 6 (Adobe, Mountain View, CA, USA). Image manipulations included changing the brightness/contrast of the ¢gure, pseudo-coloring and merging image sets, and the addition of scale bars and lettering.

RESULTS

AMPA- and kainate-type dominant-negative subunits in HEK 293 cells Previous work has shown that a methionine-to-arginine mutation near the Q/R editing site in GluR3 (M605 R) creates a mutant subunit that is non-conducting when expressed alone and that acts as a dominant-negative when co-expressed with wild-type GluR3 or GluR1 in Xenopus oocytes (Dingledine et al., 1992). Our ¢rst question was whether the analogous mutation in GluR1flip [GluR1flip (M599 R)] and GluR6 [GluR6 (M589 R)] would also produce dominant-negative ele-

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ments. To test this, the mutant subunits were coexpressed with the corresponding wild-type subunits in HEK 293 cells. Parallel sets of cells were transiently transfected with plasmid encoding the wild-type subunit alone or the same amount of wild-type plasmid was cotransfected with plasmid encoding the mutant subunit at a 1:1 cDNA ratio. Plasmid encoding GFP was included in the transfection mixture in all experiments and transfected cells were identi¢ed with epi£uorescence optics. The currents activated by saturating concentrations of glutamate (5 mM for GluR1flip ; 1 mM for GluR6) were measured (at 390 mV) in at least seven cells per group for each experiment, and the results were expressed as current densities (pA/pF). In the GluR1 experiments, AMPA receptor desensitization was reduced by including cyclothiazide (100 WM) in the external solutions. In the GluR6 experiments, kainate receptor desensitization was minimized by pre-exposure of the cells to con A (10 WM for 15 min). Figure 1A shows typical currents elicited by 5 mM glutamate in a cell expressing wild-type GluR1flip (left trace) and in a cell co-expressing GluR1flip and GluR1flip (M599 R) (right trace). Typical results obtained for GluR6 channels with and without co-expression of GluR6 (M589 R) are illustrated in Fig. 1B (currents elicited by 1 mM glutamate). For both GluR1 and GluR6, co-expression of the corresponding mutant subunit resulted in a substantial reduction in the expression of functional membrane receptors. Mean current densities obtained in single experiments for GluR1 and GluR6 are presented in Fig. 1C. In three separate experiments, the current densities in cells co-expressing both wild-type and mutant GluR1 were 4.9%, 7.6% and 4.6% (n = 27 cells) of the values obtained for the sister cultures transfected with GluR1 alone (n = 23 cells). The GluR1flip (M599 R) mutant also produced greater than 80% suppression of currents in separate co-transfection experiments with the flip versions of GluR4 and GluR2(Q). Similarly, cells coexpressing the wild-type and mutant GluR6 displayed current densities that were 4.6%, 6.7% and 2.6% (n = 38 cells) of the corresponding densities determined in parallel for cells expressing wild-type GluR6 alone (n = 25 cells). If the mutant and wild-type subunits are equally abundant (1:1 cDNA ratio) and they co-assemble indiscriminately, then homomeric wild-type channels will represent 6.25% of the total population of channels (assuming the channels are tetramers). In our experiments the amount of wild-type plasmid was kept constant, so the total amount of cDNA in the coexpression experiments was twice that in the control experiments. Thus the number of homomeric wild-type channels in the co-expression experiments should be 12.5% of the control expression. Our observation that each mutant subunit silences roughly 95% of the channel population is therefore consistent with the conclusion that a single mutant subunit is su⁄cient to render channel assemblies non-conducting. The results demonstrate that both GluR1flip (M599 R) and GluR6 (M589 R) are dominant-negative GluRs when expressed in heterologous systems.

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Fig. 1. The GluR1 and GluR6 point mutants are dominant-negative subunits in HEK 293 cells. (A) Currents elicited by 5 mM glutamate (bars) in the presence of 100 WM cyclothiazide in HEK 293 cells transiently transfected with wild-type GluR1flip (left) or wild-type GluR1flip and GluR1flip (M599 R) (+DN) at a cDNA ratio of 1:1 (right). (B) Currents elicited by 1 mM glutamate (bars) in HEK 293 cells transiently transfected with wild-type GluR6 (left) or wild-type GluR6 and GluR6 (M589 R) (+DN) at a cDNA ratio of 1:1 (right). The cells were exposed to con A (10 WM) for 15 min prior to recording to reduce kainate-receptor desensitization. (C) Mean AMPA- and kainate-receptor current densities obtained from control cells and from cells co-transfected with the corresponding mutant subunit. Error bars indicate S.E.M. Left: Data from nine cells transiently expressing GluR1flip and 11 cells from a sister culture co-transfected with GluR1flip and GluR1flip (M599 R) (+DN). The mean control density was 480 V 57 pA/pF (n = 9), whereas the mean current density in cells coexpressing wild-type GluR1flip and GluR1flip (M599 R) was 22 V 5 pA/pF (n = 11). Right: Data from nine cells transiently expressing wild-type GluR6 and 12 cells co-transfected with wild-type GluR6 and GluR6 (M589 R) (+DN). The mean control density was 39.5 V 5.2 pA/pF (n = 9) and the corresponding value for the cells co-expressing GluR6 (M589 R) was 1.9 V 0.6 pA/pF (n = 12).

Transient expression of cDNAs in cerebellar granule cells To determine whether the dominant-negative subunits would co-assemble with and silence native AMPA- and kainate-type channels, we sought to express them transiently in cultured neurons. The protocol used for HEK 293 cells gave transfection e⁄ciencies of 0.5^1% in primary cultures of cerebellar granule cells, and the GFP signal made it possible to record from the transfected neurons. To ensure that the transfection procedure alone, or the expression of GFP, did not in£uence native GluRs, we compared GluR currents in control and GFPtransfected cerebellar granule cells. To measure AMPA-, kainate-, and NMDA-receptor expression levels, whole-cell currents were evoked by sequential applications of 200 WM kainate, 10 WM kai-

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nate in the presence of the AMPA-receptor antagonist GYKI 53655 (50 WM), and 100 WM NMDA. The cultures were pre-incubated with 10 WM con A to reduce kainate-receptor desensitization. Currents through AMPA- and kainate-type channels were measured at 390 mV, whereas NMDA-type currents were measured at 330 mV (to minimize Mg2þ block). The EC50 value for kainate activation of kainate receptors in cultured granule cells is 2^4 WM (Pemberton et al., 1998), whereas the corresponding value for AMPA receptors is 50^100 WM (Pemberton et al., 1998; Traynelis and Cull-Candy, 1991). Thus both AMPA and kainate receptors contributed to the currents activated by 200 WM kainate and the amplitude of the AMPA-receptor component was taken as the current evoked by 200 WM kainate minus the amplitude of the current evoked in the same cell by 10 WM kainate in GYKI 53655. Typical results obtained from a control granule cell using the above protocol are shown in Fig. 2A. In addition to their di¡erential sensitivity to GYKI 53655, the AMPA- and kainate-receptor currents showed di¡erent apparent deactivation rates. Currents evoked by 10 WM kainate in the presence of AMPA-receptor blockade decayed slowly upon termination of the kainate application (Fig. 2A, middle trace, arrowhead). The currents evoked by 200 WM kainate decayed bi-exponentially, with a major fast component and a much slower tail that displayed an amplitude and kinetics similar to those of the current evoked in the same cell by 10 WM kainate in GYKI 53655 (Fig. 2A, right trace, arrowhead). These observations are consistent with the presence of both AMPA- and kainate-receptor components in the currents evoked by 200 WM kainate and support the validity of the subtraction procedure used to obtain the amplitude of the AMPA-receptor component. The mean current densities obtained from one experiment for AMPA-, kainate- and NMDA-type currents are plotted in Fig. 2B. Cerebellar cultures were transfected with 100^150 ng of GFP plasmid per coverslip and GluR current densities of control (no detectable GFP signal) and GFP-expressing granule cells were compared 24 h post-transfection. The results shown in Fig. 2B represent data from one culture that was maintained in vitro for 6 days (DIV6 neurons). The AMPA-receptor densities in 13 control and 11 GFP-expressing cells were 41.2 V 3.8 and 38.8 V 4.5 pA/pF. Control kainate- and NMDA-type current densities were 7.5 V 1.1 pA/pF and 16.8 V 1.8 pA/ pF compared with values of 7.9 V 2.4 pA/pF and 18.0 V 2.3 pA/pF in GFP-expressing cells. Similar current densities were obtained from cells in untransfected sister cultures. Under the conditions of our experiments ( 9 150 ng plasmid per coverslip), we found no e¡ect of transfection on GluR expression or any obvious signs of toxicity. Figure 2C shows a photomicrograph of a DIV6 granule cell transiently expressing GFP. The GFPexpressing neurons displayed the typical morphological characteristics of granule cells: a small round cell body, short stubby dendrites, and in most cases a long axonal-like process that separated into two principal branches.

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GluR1flip (M599 R) silences AMPA receptors in granule cells To test the ability of GluR1flip (M599 R) to co-assemble with AMPA-type subunits in neurons, DIV5 granule

Fig. 3. GluR1flip (M599 R) selectively suppresses native AMPAreceptor currents. (A,B) Currents elicited in DIV6 granule cells by sequential applications of 200 WM kainate (left traces), 10 WM kainate in the presence of GYKI 53655 (middle traces), and 100 WM NMDA (right traces). The records in A are from a control neuron, whereas the currents in B are from a cell on the same coverslip co-transfected with GFP and GluR1flip (M599 R) 24 h before (GluR1 DN). Calibrations bars in B also apply to the corresponding column in panel A. (C) Mean current densities in eight untransfected granule cells (¢lled bars) and 10 granule cells in the same culture that were co-transfected with GFP and GluR1flip (M599 R) (open bars) 24 h previously. The results are from a single culture transfected at DIV5. The AMPA-receptor current densities (left axis) represent the di¡erence between the values obtained with 200 WM and 10 WM kainate and were signi¢cantly smaller in the transfected neurons (P 6 0.0001). The kainate- and NMDAtype current densities (right axis) were similar in control and transfected neurons (P = 0.67). Error bars indicate S.E.M. (D) Individual AMPA-receptor current densities obtained in two separate experiments. In total, AMPA-receptor current densities were a measured in 17 control (b,R) and 19 transfected neurons ( ,O). The data are from DIV6 cultures transfected 24 h earlier. Fig. 2. Transfection of GFP does not alter AMPA-, kainate- or NMDA-receptor-mediated currents in cerebellar granule cells. (A) Currents elicited by sequential applications of 200 WM kainate (left trace), 10 WM kainate in the presence of GYKI 53655 (middle trace), and 100 WM NMDA (right trace) in a granule cell maintained DIV7. In this and all subsequent ¢gures, the current elicited by NMDA was recorded at a holding potential of 330 mV. All other currents were recorded at 390 mV. The arrowheads point to the slow component of the decay of the current activated by kainate. (B) Mean AMPA-receptor current densities (left axis) and mean densities for kainate- and NMDA-type currents (right axis). The results are from 13 DIV6 control granule cells (¢lled bars) and 11 DIV6 granule cells transiently expressing GFP (open bars). In each cell, currents were evoked by sequential applications of 200 WM kainate, 10 WM kainate in GYKI 53655, and 10 WM NMDA (as in panel A). The AMPA-receptor current was taken as the di¡erence between the current evoked by 200 WM and 10 WM kainate. Error bars indicate S.E.M. The mean densities in control and GFP-expressing neurons did not di¡er signi¢cantly (P = 0.68). (C) Photomicrograph showing a DIV6 granule cell transiently expressing GFP.

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cells were co-transfected with GluR1flip (M599 R) and GFP (100 and 50 ng plasmid DNA, respectively). Twenty-four hours post-transfection, AMPA-, kainate-, and NMDA-receptor-mediated currents were recorded and the expression of each GluR subtype in the transfected cells was determined. Because the results in the previous section indicate that neither the transfection procedure nor expression of the GFP altered GluR current densities, the current densities in transfected neurons were compared with the corresponding densities of untransfected neurons (no detectable GFP signal) on the same coverslips. Typical currents recorded from control and transfected neurons are illustrated in panels A and B of Fig. 3. Expression of GluR1flip (M599 R) signi¢cantly reduced AMPA-receptor current densities. Figure 3C shows results from one experiment where the mean

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to the mean control density in sister control neurons (P 6 0.0001, n = 17). GluR6 (M589 R) silences kainate receptors in granule cells

Fig. 4. GluR6 (M589 R) selectively suppresses native kainate-receptor currents. (A,B) Currents evoked in a DIV6 granule cell by 200 WM kainate (right traces), 10 WM kainate (with GYKI 53655, middle traces), and 100 WM NMDA (right traces). The currents in A are from a control neuron, whereas the currents in B are from a granule cell on the same coverslip that was co-transfected with GFP and GluR6 (M589 R) 24 h earlier (GluR6 DN). Calibration bars in B also apply to the corresponding column in panel A. (C) Mean AMPA-receptor current densities (left axis, di¡erence between responses to 200 WM and 10 WM kainate) and the mean densities for kainate-, and NMDA-receptor currents (right axis) from a single experiment. The results from seven control neurons (¢lled bars) gave a mean ( V S.E.M.) kainate-receptor density of 7.4 V 0.64 pA/pF. The corresponding value from nine transfected neurons (open bars) was 2.4 V 0.5 pA/pF (P 6 0.0001). The mean densities of AMPA- and NMDA-type currents in control and transfected cells were not signi¢cantly di¡erent (40.0 V 2.9 vs. 36.0 V 3.7 pA/pF and 6.8 V 0.5 vs. 5.9 V 0.6 pA/pF, respectively; P = 0.29). Error bars indicate S.E.M. (D) Individual current densities obtained from kainate receptor-mediated responses in 17 a control (b,R) and 21 transfected cells ( ,O) from two separate cultures.

currents densities were 58.4 V 5.5 pA/pF in eight control cells vs. 18.1 V 3.3 pA/pF in 10 transfected neurons (P 6 0.0001). Importantly, expression of GluR1flip (M599 R) did not signi¢cantly alter kainate- and NMDA-receptor expression. The same transfected neurons gave kainate- and NMDA-receptor current densities (pA/pF) of 7.2 V 0.6 and 6.4 V 1.0, whereas the corresponding values in the control neurons were 7.5 V 0.9 and 7.0 V 0.9 (Fig. 3C). In Fig. 3D we have plotted the individual AMPAreceptor results from two separate experiments. Although the current densities for the control and transfected neurons showed considerable variation, on average the AMPA-receptor current density in the transfected neurons was reduced 59.7 V 6.6% relative

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To determine whether a similar strategy would work for kainate receptors, we tested whether GluR6 (M589 R) would selectively silence native kainate channels in DIV5 granule cells. Typical results from control neurons and neurons transfected with the GluR6 dominant-negative mutant are illustrated in Fig. 4 (panels A and B, respectively). The mean current densities for each receptor subtype that were obtained in one experiment are presented in Fig. 4C. In each of two experiments with DIV5 cultures, expression of GluR6 (M589 R) signi¢cantly suppressed kainate-type currents without altering the expression of functional AMPA and NMDA receptors. Individual kainate-receptor densities from the two experiments are shown in Fig. 4D. When the densities in the transfected neurons were expressed as a percentage of the mean density in their sister control neurons, the pooled results gave a mean percentage reduction of 63.5 V 5.9% (P 6 0.0001, n = 16). Similar results were obtained in another experiment with DIV7 cells. Kainate-receptor densities in DIV7 transfected neurons (expressed relative to the mean control density) were reduced 61.1 V 6.1% (P 6 0.0001, n = 7) 24 h post-transfection, whereas AMPA- and NMDA-receptor current densities were not signi¢cantly altered. The GluR1 dominant-negative is more e¡ective in early cultures Although the suppression of GluR currents obtained with both mutant subunits was clearly statistically significant, there was substantial cell-to-cell variation. In part, this may re£ect di¡erences in the level of native channel expression between individual granule cells. In contrast to most CNS neurons, cerebellar granule cell development takes place principally in postnatal life. Most granule cells undergo di¡erentiation between postnatal days 5 and 15 (Altman, 1972a,b) and retain their developmental program in culture (Powell et al., 1997). In situ, granule cells begin to express AMPA receptors about the time they di¡erentiate and the level of expression rises during migration and synaptogenesis (Smith et al., 1999, 2000). When granule cells from rats 5^7 days old are isolated and put in culture, most cells initially show small AMPA-receptor currents and the mean AMPA-receptor current density increases about six-fold from DIV2 to its steady-state level at DIV7 (data not shown). In contrast, kainate-receptor current densities are relatively constant during granule cell maturation (Pemberton et al., 1998; Smith et al., 1999). The developmental changes in AMPA-receptor expression allow a direct test of the e¡ect of variation in native channel expression. If the silencing obtained with the dominant-negative subunits varies with the abundance of native channels, then the GluR1 mutant should be more e¡ective at early stages in culture when AMPAreceptor expression is uniformly low. To test the e¡ect

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(A) and in a transfected neuron on the same coverslip (B). The mean current densities for each receptor subtype are shown in Fig. 5C and the AMPA-receptor results from individual neurons are plotted in Fig. 5D. The mean AMPA-receptor density was suppressed nearly 90% in the transfected neurons (2.6 V 0.5 pA/pF vs. 23.7 V 3.9 pA/pF in the control neurons; n = 10 and 9 cells, respectively, P 6 0.0001). Compared with the results in older neurons, the percentage reductions (relative to the mean control value) were more uniform (88.3 V 2.2%). This suggests that the large variation in the amount of suppression seen in older cells arose in part from neuron-to-neuron di¡erences in the level of native channel expression at the time of transfection. The di¡erences in control expression are to be expected given the substantial developmental heterogeneity of the granule cell population in 5^7-day-old rats. As in the earlier experiments, kainate- and NMDA-receptor current densities did not di¡er in the control and transfected neurons. Estimating the relative expression of wild-type and dominant-negative GluR1

Fig. 5. GluR1flip (M599 R) suppresses AMPA-receptor currents more completely in immature granule cells. (A, B) Currents elicited in granule cells by sequential applications of 200 WM kainate (left traces), 10 WM kainate (with GYKI 53655, middle traces) and 100 WM NMDA (right traces). The currents in (A) are from a control neuron maintained in vitro for 4 days and the currents in (B) are from a granule cell on the same coverslip that was cotransfected with GFP and GluR1flip (M599 R) 24 h earlier (GluR1 DN). (C) Mean AMPA-receptor current densities (left axis, di¡erence between responses to 200 WM and 10 WM kainate) and mean densities for kainate- and NMDA-type currents (right axis) from 9 control granule cells (¢lled bars) and 10 granule cells in the same cultures that were co-transfected with GFP and GluR1flip (M599 R) 24 h earlier (open bars). The mean AMPA-receptor current density in transfected neurons (2.6 V 0.5 pA/pF) was signi¢cantly less than the corresponding value in control neurons (23.7 V 3.9 pA/pF, P 6 0.0001). Kainate- and NMDA-receptor densities in control and transfected neurons did not di¡er signi¢cantly (4.9 V 0.7 pA/pF vs. 5.0 V 0.6 pA/pF and 6.4 V 1.1 pA/pF vs. 5.3 V 0.8 pA/pF, respectively; P = 0.66). Error bars indicate S.E.M. (D) Individual AMPA-receptor current densities that gave the results in panel C.

of GluR1flip (M599 R) in immature granule cells, neurons were harvested from 6-day-old pups and were maintained in culture for 3 days. At this time, AMPA-receptor expression is relatively low and most of the cells have only recently di¡erentiated. The DIV3 cultures were transiently transfected with GluR1flip (M599 R) and GFP, and whole-cell AMPA-, kainate-, and NMDAtype currents were compared in transfected and untransfected granule cells 24 h later. Figure 5 shows typical currents elicited by sequential applications of 200 WM kainate, 10 WM kainate (in GYKI 53655), and 100 WM NMDA in a control neuron

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The results above indicate that the expression of dominant-negative subunits can suppress the expression of native glutamate-receptor channels. While the subtype selectivity of the e¡ect argues against it resulting simply from overexpression of recombinant protein, it was of interest to compare the expression and distribution of recombinant and native subunits. The low transfection e⁄ciency prohibited biochemical estimates of the relative abundance of recombinant and native subunits, so as a ¢rst approach we turned to immunocytochemical labeling. This also allowed us to compare directly the expression of GluR1 in cells that did and did not show detectable GFP signals. Cerebellar cultures were processed with antisera directed against the C-terminus of the GluR1 protein that should recognize native GluR1, wild-type GluR1flip , and GluR1flip (M599 R). Transfected neurons were identi¢ed with antisera directed against GFP. These experiments allowed us to compare the expression of recombinant subunit in di¡erent cellular compartments (e.g. soma vs. processes) and enabled an assessment of the neuron-to-neuron variation in expression. The background labeling of the cultures was ¢rst established by ¢nding the exposure times necessary to image cells processed without primary antibody. A comparison of these images with those collected from cultures processed with primary antibody revealed a small, but signi¢cant, increase in labeling that would be consistent with either non-speci¢c labeling from the primary antisera or a low level of GluR1 expression throughout the culture. Granule cells transfected only with the plasmid encoding GFP continued to £uoresce strongly even after the immunohistochemical processing, but the anti-GluR1 staining seen in these GFP-expressing cells was similar to that in untransfected cultures or in surrounding cells devoid of GFP. In contrast, granule cells co-transfected

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Fig. 6. Transiently expressed GluR1 subunits are localized to the soma and dendrites of transfected granule cells. Primary cultures of cerebellum were co-transfected with plasmids encoding GFP and wild-type GluR1flip (A1^3) or GFP and GluR1flip (M599 R) (B1^3). Panels A1 and B1 are merged Nomarski and epi£uorescence images of GFP (£uorescent neurons appear larger). The low level of GluR1 immunoreactivity in untransfected neurons (A3 and B3) is consistent with a number of previous studies showing that GluR1 expression is low or absent in granule cells in situ (Ripellino et al., 1998 ; and references therein). In neurons expressing the recombinant subunits, the GluR1 staining was primarily limited to somatodendritic compartments, as expected from the known subcellular distribution of native GluR1 subunits (Ruberti and Dotti, 2000). Arrowheads in A2 and B2 point to long axon-like processes that show little GluR1 immunoreactivity (A3 and B3). Neurons transfected with wild-type GluR1flip or GluR1flip (M599 R) subunits showed similar levels of GluR1 staining, which is consistent with the conclusion that the mutation does not a¡ect expression.

with GFP and either GluR1flip or GluR1flip (M599 R) showed strong anti-GluR1 signals. About 95% of the GFP-positive cells gave mean signal intensities clearly above those measured for control neurons, and comparisons of the levels of GluR1 immunostaining in cultures transfected with wild-type or mutant GluR1 did not reveal any discernible di¡erences. With the gains and collection times used (see Experimental procedures), the anti-GluR1 signals in GFP-positive neurons were on average three-fold greater than the corresponding signals of untransfected neurons in the same images. The variability of the anti-GluR1 signals in transfected neurons was only slightly greater than the variability of the sig-

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nals in control neurons (coe⁄cient of variation: 0.29 vs. 0.21, respectively). Because some of the observed variation re£ects the intrinsic variability of immunocytochemical labeling, this latter result indicates that the expression of recombinant GluR1 protein obtained in transfected neurons was relatively uniform. Examples of transfected granule cells are shown in Fig. 6. The cultures in Fig. 6A were transfected with wild-type GluR1flip and those in Fig. 6B were transfected with GluR1flip (M599 R). In panels A1 and B1, the majority of the cells in the ¢eld are granule cells. The GFPexpressing granule cells (panels A2 and B2) show strong GluR1 immunoreactivity (panels A3 and B3). Neurons

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expressing either the GluR1flip or GluR1flip (M599 R) subunits appeared normal. Thick tapering processes that appeared to be dendrites extended the same distances and branched in similar patterns as seen for granule cells transfected with GFP alone. The GluR1 immunostaining, however, was largely absent from the long thin processes that appeared to be axons (compare arrowheads in Fig. 6A2^A3 and B2^B3). The strong signals produced by the antisera, and the localization of the staining in the processes, are consistent with the interpretation that the heterologously expressed subunits are abundant within the cell and processed correctly. The normal appearance of the cells 24 h after transfection indicates that short-term expression of the recombinant subunits does not detectably alter the viability of the neurons or the growth and branching of their processes. Estimating the incorporation of recombinant GluR1 into plasmalemmal receptors The immunocytochemical results show that the transfection protocol results in GluR1 expression that is signi¢cantly above native levels. However, because GluR2 and GluR4 are the predominant subunits expressed in granule cells (Monyer et al., 1991; Gallo et al., 1992; Mosbacher et al., 1994), these results provide little information about the relative abundance of recombinant and native subunits. In addition, because the anti-GluR1 antisera recognizes the intracellular C-terminus of GluR1 subunits, the immunostaining was necessarily done on permeabilized cells and therefore re£ects both plasmalemmal receptors and receptors in intracellular pools. To obtain an estimate of the relative incorporation of GluR1 mutant subunits into cell surface GluRs, we transfected granule cells with a GluR1 point mutant that is largely non-desensitizing [GluR1flip (L497 Y); Stern-Bach et al., 1998]. We have shown previously that co-transfection of this gain-of-function mutant with wild-type subunits results in three easily discernible desensitization phenotypes that depend on the stoichiometry of wild-type and mutant subunits (Robert et al., 2001). Thus the relative amplitudes of the kinetically distinct components present in the decay of glutamateevoked currents can be used to estimate the relative abundance of wild-type and mutant subunits. The GluR1flip (L497 Y) expression plasmid used for these experiments is identical to the GluR1flip (M599 R) plasmid except at residues 497 and 599. To prevent activation of the non-desensitizing recombinant channels by glutamate in the culture medium, NBQX (1 WM) was included in the medium in these experiments. The addition of NBQX had no signi¢cant e¡ect on the GluR current densities recorded from control cultures, and the untransfected neurons in transfected cultures gave control densities similar to those of untransfected neurons in cultures not exposed to NBQX. The ligand binding domain of GluR2 crystallizes as a dimer. The leucine corresponding to residue 497 in GluR1 appears to make contact with residues in the adjacent monomer (two symmetrical contacts per

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dimer), and mutation of this leucine to tyrosine strongly promotes dimerization (Armstrong and Gouaux, 2000; Sun et al., 2002). To determine whether the L/Y mutation alters subunit assembly, we introduced the M599 R mutation into GluR1flip (L497 Y) and compared the dominant-negative suppression obtained with the single and double mutants in co-expression experiments with wildtype GluR1 in HEK293 cells. When co-expressed at a wt:mutant plasmid ratio of 1:1, the double M/R, L/Y GluR1 mutant reduced GluR1-mediated current densities by 92.1 V 3.2% when compared with the cells in sister cultures transfected with wild-type GluR1 alone (n = 9 and 11 cells; mean control current density 392 V 37 pA/pF). In the same experiment, co-expression of the single M/R mutant suppressed functional GluR1 expression 93.7 V 2.9% (n = 5 cells). These results demonstrate that the L/Y mutation does not appreciably alter the incorporation of full-length GluR1 subunits into AMPA receptor assemblies. In control granule cells, sustained applications of 2 mM glutamate invariably elicited currents that decayed rapidly to a small plateau current (Fig. 7A). In contrast, granule cells transfected 24 h earlier with GluR1flip (L497 Y) gave much larger sustained currents (Fig. 7B). On average, the steady-state currents were 6 V 2 pA/pF in control neurons (n = 37 cells) and 348 V 35 pA/pF in neurons transfected with GluR1flip (L497 Y) (range: 194^478 pA/pF, n = 9 cells). The large increase in non-desensitizing phenotype demonstrates that the mutant GluR1 subunits were rapidly and robustly expressed in GFP-positive neurons. However, because the solution exchanges obtained in whole-cell recordings are too slow to accurately measure wild-type desensitization (Robert et al., 2001), these data cannot be used to estimate the relative abundance of native and mutant subunits. We therefore also recorded glutamate-activated currents in outside^out patches from granule cells where solution exchange is much faster. Figure 7C shows the current activated by 2 mM glutamate in a patch pulled from a DIV8 neuron transfected 24 h earlier with GluR1flip (L497 Y). In each of ¢ve such patches, three exponential components were required to obtain adequate ¢ts to the decays of the glutamate-activated currents. The individual exponential components ¢tted to the decay of the current in Fig. 7C are shown as dotted lines in Fig. 7D. The mean time constants for the three components were 1.65 V 0.43 ms (fast component), 25.4 V 3.3 ms (intermediate component) and 180 V 24 ms (slow component). The relative amplitudes of the fast, intermediate and slow components were 0.25 V 0.04, 0.33 V 0.08 and 0.42 V 0.11 (n = 5). The mean time constants of the three decay components present in patches from granule cells transfected with GluR1flip (L497 Y) are virtually identical to the time constants of the three similar components we detected in co-expression experiments with wild-type GluR1 (Robert et al., 2001). Very similar components are also evident in co-expression experiments with GluR2(Q) and GluR4 (A.R. and J.R.H., unpublished results). Our previous analysis indicated that the fast, intermediate, and slow components arise from channels

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Fig. 7. The non-desensitizing AMPA-receptor subunit GluR1flip (L497 Y) is expressed e⁄ciently in transfected neurons. (A, B) Whole-cell currents elicited by 2 mM glutamate in a control DIV7 granule cell (A) and in a DIV8 neuron transfected 24 h before with GFP and GluR1flip (L497 Y) (GluR1 GOF). Expression of the non-desensitizing mutant results in a large sustained current. (C) Current elicited by 2 mM glutamate in an outside^out patch from a DIV8 granule cell neuron transiently expressing GluR1flip (L497 Y) for 24 h. The smooth curve superposed on the data is a triple-exponential ¢t to the decay of the current. (D) Same record as in (C) on an expanded time scale. The three individual exponential components (f, fast; i, intermediate ; s, slow) are shown (dotted lines). Although kainate receptors were not blocked, glutamate-activated AMPA-type currents are 10- to 50-fold larger than kainate-type currents at this stage in culture and the deactivation kinetics of the currents measured in patches revealed no evidence of kainate-receptor activation

with 0 or one, two, and three or four GluR1flip (L497 Y) subunits, respectively. Our observation that one-third of the functional channels present in transfected cells show intermediate decay kinetics (two native and two mutant subunits) suggests that mutant and native subunits are expressed at roughly equal abundance. If all the channels are assumed to re£ect assemblies made after expression of the mutant subunits reached steady-state, then the relative amplitudes of the three components are those expected for a mutant/native subunit ratio of about 1.2:1. If the fast component is assumed to arise in part from channels present prior to transfection, then the relative amplitudes of the intermediate and slow components predict a mutant/native subunit ratio of about 1.5:1. These estimates assume that all subunits co-assemble indiscriminately and that all channel assemblies have an equal likelihood of reaching the cell surface.

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One ¢nding of the present work is that the methionineto-arginine mutation shown to produce a dominant-negative GluR3 subunit (Dingledine et al., 1992) likewise creates dominant-negative subunits in GluR1 and GluR6. Our co-expression studies with wild-type subunits show that the inclusion of a single mutant subunit in channel assemblies renders the channels non-conducting. This result agrees with many studies indicating that the pore-forming regions of AMPA- and kainate-type subunits are structurally similar (Dingledine et al., 1999). Introduction of the leucine-to-tyrosine mutation described by Stern-Bach et al., (1998) did not result in any obvious change in the incorporation of the dominant-negative GluR1 subunit into heteromeric assemblies with wild-type subunits, although this mutation does promote dimerization of the recombinant S1S2 ligand binding domain (Sun et al., 2002). This result is consistent with evidence that the assembly of full-length AMPA-receptor dimers is largely speci¢ed by sequence elements upstream of the S1 region (Ayalon and SternBach, 2001). Analytical ultracentrifugation studies with the amino terminal domain (ATD) of GluR2 indicate that the a⁄nity of ATD^ATD dimer formation is much higher than the corresponding value for the S1S2 species, with or without the L/Y mutation (R. Jin, R. Olson and E. Gouaux, personal communication). These results, and our ¢ndings, support the view that dimer assembly is dominated by ATD interactions and that mutations at the S1S2 dimer interface which reduce desensitization are unlikely to have large e¡ects on subunit assembly. Although it is not surprising that the mutant GluR subunits we have tested are dominant negative in heterologous expression systems, the extent to which they would silence native channels in neurons was less clear. At least three requirements must be met for the dominant-negative strategy to be successful in neurons. Firstly, the mutant subunits must co-assemble with native subunits. Our results with the dominant-negative subunits, as well as our ¢ndings with the non-desensitizing GluR1 mutant, demonstrate that this is the case. Secondly, the mutant subunits must to be expressed at levels su⁄cient to ensure incorporation into a substantial fraction of plasmalemmal receptors, and these levels of expression must not be generally deleterious to cell function or result in assembly with subunits from related families. One major result of our experiments is that expression levels su⁄cient to produce substantial silencing of the targeted receptor subtypes had no detectable e¡ect on the expression of other ionotropic GluRs. Thirdly, the silencing of some assemblies must not trigger compensatory up-regulation of native channel expression that overrides the initial dominant-negative e¡ect. The transient expression protocol used here did not really allow us to address this issue and we largely restricted our analysis to 24 h post-transfection. In a few experiments we found that the dominant-negative e¡ect persisted at 48 h. However, some transfected neurons began to show signs of toxicity at times longer than 72 h. This

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was initially manifest as swelling of the soma and processes and was also seen in neurons transfected with GFP alone. We therefore did not follow the cultures for times longer than 48 h. The nearly 90% suppression of AMPA-receptor current densities we obtained when the transfection was performed at times when native channel expression was initially low suggests that the GluR1 dominant negative was incorporated into all potential heteromeric assemblies. It is di⁄cult to evaluate the relative abundance of mutant and native subunits in the experiments on DIV3 neurons because native GluR expression increases rapidly between 3 and 6 days in culture. Although individual DIV6 neurons displayed variable levels of native channel expression, on average the GluR1 and GluR6 mutants caused a 60^65% reduction of native AMPAand kainate-receptor currents 24 h post-transfection. Our immunocytochemical results indicate that the level of mutant subunit expression was relatively uniform in transfected cells and was about three times greater than the expression of native GluR1. Many studies have shown that GluR2 and GluR4 are the predominant AMPA-type subunits expressed in granule cells. Thus the three-fold increase in GluR1 expression is likely to substantially overestimate the ratio of mutant and native subunits in the transfected neurons. This conclusion is consistent with our GluR1flip (L497 Y) results that show mutant subunits were incorporated into plasmalemmal assemblies at stoichiometries expected if mutant and native subunits were expressed at approximately equal levels. If this is true and the mutant subunits behave as dominant negatives in co-assemblies with all native subunits, then about 95% of the channels synthesized posttransfection would contain at least one mutant subunit and be non-conducting. Thus, in DIV6 neurons, the functional channels present at 24 h would largely be channels that were assembled prior to expression of the mutant, and the fraction of residual channels (30^35%) would give a receptor half-life of about 20 h, a value that agrees closely with previous estimates (Huh and Wenthold, 1999; Howe et al., 1995). This interpretation of the results also assumes, however, that receptor assembly is combinatorial and that both mutant and native subunits co-assemble indiscriminately. Many studies in heterologous systems have shown that all AMPA- and kainate-receptor subunits

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can co-assemble with subunits from the same subtype family (see for instance Mosbacher et al., 1994; Cui and Mayer, 1999), although recent work suggests that GluR1 and GluR2 preferentially form heteromeric dimers (Mansour et al., 2001). In neurons, some studies suggest that native channel assembly is also largely combinatorial (Geiger et al., 1995; Washburn et al., 1997), whereas other results indicate that some native subunit combinations are preferred over others (Wenthold et al., 1996). We wish to emphasize that our results do not exclude the possibility that some of the residual channels present 24 h post-transfection were newly synthesized receptors from which the mutant subunits were selectively excluded. Indeed, a novel way to determine whether certain subunit combinations are preferred over others might be to compare the ability of di¡erent dominant-negative subunits to silence native receptors. Much of what we know about the role of particular GluR subtypes has been gleaned from studies with subtype-selective agonists and antagonists. This approach has been especially useful for studies on single cells, but for other studies the utility of this approach is limited by the expression of GluRs in virtually every CNS cell type. For example, many pharmacological studies point to the involvement of GluRs in various developmental events, but it is di⁄cult to know whether the observed e¡ects are directly on the cell type in question or result from complex interactions involving multiple GluR-expressing cells and circuits. Likewise, the interpretation of the e¡ects of genetically disrupting individual GluR subunits is complicated by the oligomeric nature of the receptors and the redundancy of GluR expression at the level of individual neurons. One of the most powerful approaches to circumvent this latter problem has been the use of dominant-negative elements (Herskowitz and Marsh, 1987). We show here that such an approach can be used to selectively suppress the function of individual GluR subtypes in cultured neurons. In combination with genetic targeting strategies or the use of viral vectors, the dominant-negative approach o¡ers a promising way to elucidate the function of GluR subtypes in the intact CNS.

Acknowledgements4We thank Eric Gouaux for sharing unpublished results. This work was supported by NS 37904.

REFERENCES

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(Accepted 19 July 2002)

NSC 5871 12-11-02

Cyaan Magenta Geel Zwart