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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC Feb 29, 2012.
Published in final edited form as:
PMCID: PMC3172699
NIHMSID: NIHMS321975
Kalirin binds the NR2B subunit of the NMDA receptor, altering its synaptic localization and function
Drew D. Kiraly, Fouad Lemtiri-Chlieh,# Eric S. Levine, Richard E. Mains, and Betty A. Eipper
Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030
Corresponding Author: Betty Eipper, Address: 263 Farmington Avenue, Department of Neuroscience, Farmington, CT 06030, eipper/at/nso.uchc.edu
#Current address: The King Abdullah University of Science and Technology, Thuwal, 23955-6900, Kingdom of Saudi Arabia
The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor (GEF) localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins including PSD-95, DISC-1, AF-6 and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal LTP as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell-surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.
Kalirin-7 (Kal7), one of approximately a dozen Rho guanine nucleotide exchange factors (GEFs) localized to the PSD (Kiraly et al., 2010a), is one of many molecules important for dendritic spine formation and function (Carlisle and Kennedy, 2005; Bourne and Harris, 2007; Hung et al., 2008). Kal7 is critical for spine formation in cultured hippocampal and cortical neurons (Ma et al., 2003; Ma et al., 2008a) and in vivo (Ma et al., 2008b). Kal7 knockout (Kal7KO) mice exhibit decreased hippocampal spine density at baseline (Ma et al., 2008b), with aberrant spine plasticity in the nucleus accumbens after repeated cocaine injections (Kiraly et al., 2010b). In addition to morphological alterations, Kal7KO mice have decreased passive avoidance fear conditioning and decreased conditioned place preference for cocaine (Ma et al., 2008b; Kiraly et al., 2010b). However, Kal7KO mice are normal in object recognition, radial arm maze acquisition and acute locomotor response to cocaine. Decreased levels of the NR2B subunit of the NMDA receptor were observed in PSDs from Kal7KO mice (Ma et al., 2008b). These studies indicate that Kal7 has profound, specific effects on normal synaptic function.
NMDA receptors are ionotropic glutamate receptors critical for many forms of synaptic plasticity, including long-term potentiation (LTP) and depression (LTD), considered cellular correlates of learning and memory (Cull-Candy and Leszkiewicz, 2004; Kerchner and Nicoll, 2008). NMDA receptors are tetramers primarily composed of obligate NR1 subunits, binding co-agonist glycine, and NR2 subunits (mainly A and B in adult forebrain), which bind glutamate (Cull-Candy and Leszkiewicz, 2004). NR2A-containing receptors support higher peak currents and faster deactivation kinetics, while NR2B-containing receptors exhibit prolonged channel open times and greater overall Ca2+ current per event (Sobczyk et al., 2005; Yashiro and Philpot, 2008). Within the PSD, NMDA receptors are embedded in a macromolecular complex (Husi and Grant, 2001). The juxtamembrane intracellular domains of the NMDA receptor subunits bind proteins that affect channel function and the cytoskeleton. Calmodulin binding to the juxtamembrane region of NR1 leads to its inactivation (Ehlers et al., 1996). Spectrin and α-actinin bind to the C-terminal domains of NR1 and NR2B (Wyszynski et al., 1997; Wechsler and Teichberg, 1998). Different endocytic motifs in the juxtamembrane regions of NR1 and NR2B target receptors for recycling and degradation (Scott et al., 2004). The coordinated interactions of NMDA receptors with these binding partners are critical for proper synaptic function.
A growing body of evidence suggests that NR2B plays a critical role in many forms of learning and plasticity. Genetic elimination of NR2B in hippocampal neurons abolishes NMDA receptor-dependent LTP, reduces dendritic spine density and decreases the ratio of filamentous to globular actin (Akashi et al., 2009). NR2B localized with CamKII in perisynaptic regions is critical for LTP development in hippocampal slices (Barria and Malinow, 2005; Foster et al., 2010). In agreement with electrophysiological experiments, the contribution of NR2B subunits to channel function is important for conditioned place preference for cocaine and morphine and for normal fear conditioning (Rodrigues et al., 2001; Ma et al., 2006; Pascoli et al., 2011). Here we demonstrate that Kal7 interacts with the juxtamembrane region of NR2B, and that Kal7KO mice have decreased NR2B subunit-containing NMDA receptor currents and cell surface expression. Furthermore, behavioral differences between Wt and Kal7KO mice in cocaine place preference and passive avoidance are abrogated by blockade of NR2B subunit-containing receptors.
Electrophysiology
For electrophysiological recordings, Wt and Kal7KO animals (P28-40) were decapitated under isoflurane anesthesia and the brains were placed into ice-cold “cutting and incubating” (CI) solution composed of (in mM): 125 NaCl, 2.5 KCl,1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 4 MgCl2, 4 MgSO4, 4 lactic acid, 2 pyruvic acid, 20 glucose, and 0.4 ascorbic acid, carboxygenated with 95% O2 - 5% CO2 (pH 7.3, 310 ± 5 mmol·kg−1). Transverse cortical slices (350 μm) were cut using a vibratome and placed into an incubating chamber containing CI solution at 35°C for 30 minutes before being transferred to room temperature for at least 30 minutes prior to recording. During recordings, slices were continuously perfused at 2 ml/min with artificial cerebrospinal fluid (aCSF) consisting of (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, and 15 glucose (pH 7.3, 310 ± 5 mmol·kg−1); pH was equilibrated by continuous bubbling with 95% O2 - 5% CO2.
Whole cell voltage-clamp recordings (Vhold = -70 mV) were obtained from layer 2/3 cortical pyramidal neurons (PNs). The pipette solution contained (in mM): 117 CH3O3SCs, 8 CsCl, 10 HEPES, 2 EGTA, 0.2 CaCl2, 4 Mg2+-ATP, 0.3 Na+-GTP, and 5 QX-314 (pH 7.3, 295 ±5 mmol·kg−1). Upon breaking into whole-cell configuration, a brief series of voltage ramps (50 ms, 2 mV/ms) were applied to promote the activity-dependent block of the sodium conductance by QX-314. Electrical events were filtered at 2.9 kHz and digitized at ≥ 6 kHz. Series resistance was compensated 70% at 10–100 μs lag. Input resistance (Ri) was monitored with 5 mV/50 ms hyperpolarizing voltage steps. Electrically-evoked excitatory postsynaptic currents (EPSCs) were elicited with a bipolar tungsten electrode (resistance = 1 MΩ) positioned 60–120 μm lateral to the patched neuron in layer 2/3. Voltage measurements were not corrected for the calculated liquid junction potential of −10.2 mV. Stimulation consisted of a single square-wave current pulse (duration: 50–150 μs, amplitude: 50–200 μA at a frequency of 0.067 Hz) designed to evoke half maximal responses. Neurons were discarded from analyses if (i) Rs was >25 MΩ at the time of break-in or >10.5 MΩ after compensation, (ii) if Ri changed by more than 15% during the course of an experiment, or (iii) if Ri fell below 100 MΩ. For determination of NMDA/AMPA receptor current ratios the AMPA receptor current was quantified as the peak current recorded at Vh= −70 mV and the NMDA receptor current was quantified 40 ms after stimulation onset at Vh= +40 mV. For antagonist studies, NR2B subunit antagonists (Ifenprodil and Ro 25-6981; Tocris Bioscience) were dissolved in water.
BS3 Crosslinking
Methods for labeling cortical slices with membrane-impermeable BS3 crosslinker were adapted from published procedures (Boudreau and Wolf, 2005; Sears et al., 2010). Age matched Wt and Kal7KO mice were decapitated and their brains were removed into slush-cold Slicing ACSF (S-ACSF) composed of (in mM): 26 HEPES [pH 7.4], 250 Sucrose, 2.3 KCl, 2 CaCl2, 2 MgSO4, 1.26 KH2PO4, 10 Glucose. Coronal cortical Vibratome slices (400μM) were transferred from slush-cold S-ACSF to tubes containing ice-cold crosslinking ACSF (X-ACSF) composed of (in mM): 26 HEPES [pH 7.4], 125 NaCl, 2.3 KCl, 2 CaCl2, 2 MgSO4, 1.26 KH2PO4, 10 Glucose. To prevent any receptor trafficking, slices were kept ice-cold for all steps until tissue lysis. Slices were washed twice with X-ACSF, before addition of X-ACSF containing 2mM BS3 [Bis(Sulfosuccinimidyl) suberate; Pierce, Rockford, IL]. Slices were incubated with BS3 for 40 minutes with agitation to allow for full labeling of cell surface receptors. Unreacted BS3 was quenched by adding glycine to 10mM. This solution was aspirated and replaced with X-ACSF + 5mM glycine. The second wash solution was aspirated and slices were sonicated into 250μl of SDS lysis buffer (50mM Tris [pH=8.0], 2% SDS, 5mM EDTA, 50mM NaF, 1mM dithiothreitol, 1mM PMSF, and protease inhibitor cocktail (Xin et al., 2004). After incubation at 55°C for ten minutes, lysates were spun for 20 minutes at 25,000 × g at room temperature. Protein concentrations were determined by bicinchoninic acid assay (Pierce), and 20μg of protein was mixed with 1X Laemmli buffer and heated to 55°C for five minutes. Proteins separated on a 4–15% acrylamide gel were transferred to PVDF membranes and Western blotting was performed as described (Ma et al., 2008b). Quantification of discrete intracellular and diffuse surface bands was performed using GeneGnome software (Ma et al., 2008b). Surface receptor included signal associated with a higher molecular weight than the non-crosslinked receptor; background signal was subtracted out.
Behavioral experiments
Animals were group housed in the UCHC animal facility on a 12-hour light-dark cycle (lights on 7:00 AM, off 7:00 PM). All experiments were performed in accordance with UCHC Institutional Animal Care and Use Committee and National Institutes of Health procedures for animal care. Male Wt and Kal7KO (Ma et al., 2008b) littermate mice 2 to 6 months of age were used throughout. These mice have been backcrossed into the C57BL/6 background for ≥15 generations. Behavioral studies using ifenprodil to distinguish the role of NR2B-containing NMDA receptors have used doses ranging from 1–10mg/kg administered intraperitoneally (Rodrigues et al., 2001; Liu et al., 2006; Ma et al., 2006; Schumann and Yaka, 2009). To minimize any effect on NR2A-containing NMDA receptors (Williams, 1993), we chose a low dose (2mg/kg) that was reported to reduce conditioned place preference and passive avoidance (Rodrigues et al., 2001; Liu et al., 2006; Ma et al., 2011).
Conditioned place preference
Conditioned place preference methods were adapted from our previously published experiments (Kiraly et al., 2010b). To determine the role of NR2B-containing receptors in these behaviors, ifenprodil (2mg/kg in saline) was injected (i.p.) ten minutes before the injection of saline or cocaine (National Institute on Drug Abuse, Bethesda, MD). The timing of these injections was chosen based on a morphine place preference study in rats (Ma et al., 2006). On the first day of the experiment, each animal was given a 20 minute pre-test session in which they were allowed to explore all chambers of the place preference apparatus freely. The least preferred side became the side paired with drug injections. Animals that showed a <30 second difference in baseline preference were assigned to balance chamber side with treatment and genotype. During training there were two conditioning sessions each day; in the morning all animals received saline injections; in the afternoon animals received either ifenprodil (2mg/kg) or saline ten minutes before cocaine (10mg/kg) or saline. Five minutes after the cocaine or saline injection, animals were placed into the conditioning chamber for 15 minutes. This conditioning was repeated for four days. Twenty four hours after the final conditioning session, animals were again placed into the boxes and allowed to explore all chambers freely. Preference score was calculated as the time spent in the conditioned chamber on the final day minus the amount spent in the same chamber on the pretest day.
Locomotor Monitoring
Measurement of the effects of ifenprodil on locomotor activity with and without cocaine was performed using a Latin square design (Benavides et al., 2007; Kiraly et al., 2010b). After a single day of habituation to the locomotor chambers, animals were given i.p. injections of saline, ifenprodil (2mg/kg), cocaine (10mg/kg), or concomitant ifenprodil + cocaine in a randomized order over four days. Immediately after injection, animals were placed into the locomotor monitoring chambers (San Diego Instruments, San Diego, CA) and their activity was recorded for 45 minutes.
Passive Avoidance
Methods were adapted from our previous procedure (Ma et al., 2008b). A single injection of ifenprodil (2mg/kg, i.p.) or saline was administered 15 minutes before passive avoidance conditioning. For conditioning, animals were placed into one side of a shuttle box; after five seconds, the house light came on in that chamber only and the door between chambers was opened. When the animal crossed to the dark compartment, the door was closed and the animal received a single 0.3mA × 2sec scrambled footshock. Previous studies with these mice showed that this stimulus was sufficient to elicit robust conditioning and that there were no genotypic differences in shock sensitivity (Ma et al., 2008b). After the context-shock pairing, animals were returned to their home cages. Twenty-four hours after training, animals were returned to the same boxes and their latency to cross was measured. Increases in latency to cross were used as an index of the strength of conditioning.
Synaptosomes
For co-immunoprecipitation experiments, synaptosomes were prepared from adult rat or mouse brain (pooled cortex, striatum and hippocampus). Briefly, brains were homogenized in 20 volumes Buffer A (20mM Tris-HCl [pH 7.4], 320mM sucrose, 5mM EDTA, 50mM NaF, 2mM sodium orthovanadate, 1mM PMSF and protease inhibitor cocktail (Xin et al., 2004)) and centrifuged at 1000 × g for ten minutes. The supernatant was then centrifuged at 15,000 × g for 15 minutes, yielding a crude synaptosomal fraction (P2), which was resuspended in TE buffer (10mM Tris-HCl, pH7.4, 5mM EDTA, PMSF, protease inhibitor cocktail). Three solubilization protocols were used. In the first protocol, a mixture of Triton X-100 (TX-100; 1.0%) and SDS (0.1%) was used; samples were tumbled for one hour at 4°C and then centrifuged for 40 minutes at 20,000 × g. The TX/SDS supernatant was used for analysis. For protocol 2, 10% deoxycholate (DOC) in 500mM Tris (pH 9.0) was added to a concentration of 1% DOC, and samples were tumbled for 1 hour at 4°C. Triton X-100 (0.1 volume of 1.0% TX-100 in 500mM Tris.HCl [pH 9.0]) was added to DOC lysates, where were dialyzed overnight against binding buffer (50mM Tris.HCl [pH 7.4], 0.1% TX-100) (Dunah et al., 2000; Wyszynski et al., 2002). Dialyzed DOC lysates were centrifuged for 40 minutes at 40,000 × g and supernatants were used for immunoprecipitation. For protocol 3, 10% SDS was added to a final concentration of 1% and samples were tumbled for 30 minutes at room temperature; samples were then centrifuged and the supernatant was used for immunoprecipitation. Protocol 2 (DOC) was used for all co-immunoprecipitation experiments with the exception of a single methods comparison experiment.
Immunoprecipitation
Samples were pre-cleared by incubation with 15μl of Protein A/G beads (Thermo Scientific, Rockford, IL) for 30–45 minutes. An aliquot of each sample was saved as input; antibody (3–4 μg) and Protein A/G beads (15 μl) were added to each sample, which was tumbled at 4°C for 4 hours or overnight. For Kal7 immunoprecipitation, Kal7 monoclonal antibody 20D8 (Ma et al., 2008b) was enriched by ammonium sulfate precipitation (45% saturation) of ascites fluid and dialyzed into 10 mM Na phosphate, pH 7.4, 150 mM NaCl. The ammonium sulfate precipitate (0.4 mg protein) was linked to 0.2 ml AminoLinkR Plus Coupling Resin using sodium cyanoborohydride (Thermo Scientific, Rockford, IL). After incubation with antibody plus Protein A/G beads or Kal7 monoclonal antibody beads, unbound proteins were removed and beads were washed twice with TMT buffer (0.5 ml 20 mM NaTES, 10 mM mannitol, pH 7.4, 1.0% Triton X-100) and then twice with TM buffer (0.5 ml 20 mM NaTES, 10 mM mannitol, pH 7.4). Bound proteins were eluted by boiling into 1X Laemmli sample buffer. For each sample, incubation of equal amounts of protein with pre-immune IgG was used as a negative control for non-specific binding. IgG was covalently linked to AminoLinkR Plus Coupling Resin in the same manner as the Kal7 monoclonal antibody.
Antibodies
Kalirin antibodies were described previously (Ma et al., 2008b) and include rabbit polyclonal antibodies specific to the terminal 20 amino acids of Kal7 (JH2959) or to spectrin repeat regions 4 to 7 of Kalirin (JH2582) and mouse monoclonal Kal7 antibody 20D8. The myc monoclonal antibody (9E10) was described previously (Borjigin and Nathans, 1994; Caldwell et al., 1999). Commercially available antibodies used included: NR2B (clone 59/20; Neuromab), NR2A (clone A12W, #04-901; Millipore), NR1 (#556308; BD Biosciences), PSD-95 (clone K28/43; Neuromab), GluR1 (ab31232; Abcam), GFP (ab290; Abcam), Myc (ab9106; Abcam), HA (ab9110; Abcam), pan-MAGUK (clone K28/86; Neuromab).
Transfection of non-neuronal cells
pEAK Rapid cells (Edge Biosystems, Gaithersburg, MD) were maintained in DMEM:F12 medium containing 200 U/ml penicillin G, 20 μg/ml streptomycin sulfate, 25 mM HEPES, and 10% fetal bovine serum. Cells were fed with serum-free medium for 2 hours before transfection. Vectors encoding rat NMDA receptor subunits and Kal7 were transfected in a mass ratio of 1 NR1: 3 NR2: 1.5 Kal7. Expression vectors encoding rat NR1, NR2A and NR2B were generous gifts from Dr. Jon Johnson at the University of Pittsburgh (Qian et al., 2005; Clarke and Johnson, 2008), and the expression vector encoding ΔNR2B was a generous gift from Dr. Kelly Foster at Commonwealth Medical College (Foster et al., 2010). Modifications of the ΔNR2B vector were made to create the NR2B juxtamembrane mutants. For the “2B→2A” construct the terminal 17 residues of ΔNR2B were swapped for the corresponding residues from NR2A; for the “Stub” truncation construct the NR2A stop transfer sequence (KLR) followed by an AG spacer was inserted; for the “GAGA” construct the NR2A stop transfer sequence followed by a string of 14 alanine and glycines was inserted. Constructs were verified by DNA sequencing. The Kal7 expression vectors used were described previously: His-Myc-Kal7, His-Myc-ΔKal7, His-myc-KGEF1→7end (Xin et al., 2008), His-myc-I-10 (Ratovitski et al., 1999), His-myc-Kal7/ND/AA (Schiller et al., 2008), His-myc-Kal7ΔCT (Penzes et al., 2001b) and His-myc-KalGEF1 (Ferraro et al., 2007). The Tiam1 GEF vector (TiamGEF) included tandem C-terminal HA-tags (Ferraro et al., 2007). KalPH1-GFP was made by inserting EGFP into the N-terminal portion of the previously described Kalirin PH1 vector (Chakrabarti et al., 2005). Vectors were mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM (Life Technologies) and added to cells for 6 hours at 37°C. Cells were then fed with glutamate-free medium (MEM plus 200U/ml penicillin G, 20 μg/ml streptomycin sulfate, 25 mM HEPES, and Glutamax [Invitrogen]) to prevent NMDA receptor-induced excitotoxicity. After 24–48 hours, cells were scraped into spent medium, pelleted and solubilized in DOC as described above. Dialyzed lysates were then subjected to immunoprecipitation as described above.
Peptide pulldown
Synthetic rNR2B(845-861) preceded by a Gly-Ala spacer [H-GAGAQ845FRHCFMGVCSGKPG MV861NH2] (Biomatik, Wilmington, DE) was covalently linked to AffiGel-10 beads (Bio-Rad, Hercules, CA); this sequence follows transmembrane domain 4 and is referred to as NR2B juxtamembrane domain (2B-JM). Control AffiGel-10 beads were either blocked with ethanolamine in the absence of peptide or linked to β-endorphin, a peptide with a positive charge similar to that of 2B-JM. Readily soluble proteins (KalPH1-GFP and GFP) were solubilized in TE buffer + 0.02% TX-100. For less soluble proteins, samples were solubilized in DOC and dialyzed as described for immunoprecipitation; solutions were diluted to 0.02% TX-100 before binding. Lysates were mixed with 15μl peptide-coated or control beads. Binding was performed for 4 hours or overnight at 4°C. After binding, beads were pelleted and washed 4 times with binding buffer. Bound proteins were then eluted by boiling into Laemmli sample buffer for five minutes. Similar results were obtained using two separate preparations of KalPH1-GFP.
Statistical Analyses
For behavioral experiments, statistical analysis was performed using SigmaPlot. Two-way ANOVA tests were used to determine main effects of genotype, treatment and any interactions. For experiments in which there was a significant genotype × treatment interaction, Holm-Sidak post hoc tests were used to parse out specific effects. For analysis of NR2B subunit-containing NMDA receptor currents, an unpaired two-tailed t-Test was used to compare the NMDA/AMPA receptor current ratios as well as percentage change of evoked currents after ifenprodil or Ro 25-6981 application. Unpaired two-tailed t-Tests were also used to compare surface/intracellular receptor ratios for crosslinking experiments.
NR2B subunit-containing NMDA receptor currents are reduced in Kal7KO cortical neurons
One of the few changes observed in PSDs isolated from Kal7KO mice was a decrease in NR2B levels (Ma et al., 2008b). To examine changes in ionotropic glutamate receptor signaling in Kal7KO mice we quantified the ratio of NMDA to AMPA receptor currents in slices of somatosensory cortex from young adult (P28-40) Wt and Kal7KO mice. The ratio of peak EPSC at Vh +40mV 40ms after stimulation (NMDA receptor mediated current) over the peak current at Vh −70mV (AMPA receptor mediated current) was calculated (Fig. 1A,B). Kal7KO mice exhibit a significantly decreased NMDA/AMPA ratio, indicative of a decrease in overall NMDA receptor function (Fig. 1C). These findings are in line with our previous findings that Kal7KO mice do not form normal NMDA receptor-dependent LTP when looking at single cell (Ma et al., 2008b) or field (F.L.-C. unpublished observations) LTP induction protocols.
Figure 1
Figure 1
NMDA/AMPA receptor current ratio and NR2B subunit-containing NMDA receptor mediated currents are diminished in Kal7KO neurons. A–C. To measure NMDA/AMPA receptor current ratio in layer 2/3 pyramidal neurons, the AMPA receptor response was quantified (more ...)
Given these findings, we used pharmacological tools to compare NR2B subunit-containing NMDA receptor mediated signaling in Wt and Kal7KO cortical neurons. For these experiments NMDA receptor-mediated activity was isolated using the AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μM); and cells were voltage clamped at +50mV to remove the voltage-dependent blockade of NMDA receptors. Extracellular stimulation within layer 2/3 evoked NMDA receptor-mediated EPSCs in slices from both Wt and Kal7KO animals (Fig. 1D,E dark traces); these EPSCs were completely blocked by the NMDA receptor antagonist 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP - 3 μM; data not shown). To determine the NR2B subunit-mediated component of the NMDA receptor current, ifenprodil (3μM) (Tovar and Westbrook, 1999; Madara and Levine, 2008) was added to the bath solution. In slices from Wt animals, the ifenprodil-sensitive component represented 56 ± 4% of the peak current, whereas in slices from Kal7KO animals, the ifenprodil-sensitive component was only 39 ± 6% of the peak current (Fig. 1D-F). A different NR2B-specific antagonist, Ro 25-6981 (0.5 μM) (Liu et al., 2004; Haseneder et al., 2009), produced the same result. In slices from Wt animals, the Ro 25-6981-sensitive component was 48 ± 2% of the peak current; in slices from Kal7KO animals, it was only 30 ± 4% of the peak current, significantly less than in Wt tissue (Fig. 1F; traces not shown). These data indicate that the decrease in NR2B subunit levels observed in cortical PSDs is associated with a decrease in NR2B subunit-containing NMDA receptor functionality in Kal7KO animals.
Surface localization of NR2B is reduced in Kal7KO animals
To determine whether the decrease in ifenprodil-sensitive currents in the Kal7KO animals was due to changes in synaptic localization or receptor function, we used membrane-impermeable BS3 crosslinking to identify surface receptors (Boudreau and Wolf, 2005; Sears et al., 2010). Cortical slices from adult Wt and Kal7KO mice were chilled to 4°C before cross-linking to prevent receptor trafficking. High molecular weight bands representing crosslinked NMDA and AMPA receptors were seen only in crosslinked slices (Fig. 2A). Additionally, Western blot analysis of tubulin, an intracellular protein, revealed a single discrete band of the appropriate molecular weight, demonstrating that BS3 was unable to enter cells (Fig. 2A right). We examined the NR2B and NR2A subunits of the NMDA receptor as well as the GluR1 subunit of the AMPA receptor (Fig. 2B-D). Quantification of Surface (high molecular weight) to Intracellular ratios for these receptor subunits revealed a specific decrease in surface expression of the NR2B subunit in Kal7KO mice (Fig. 2E); surface levels of the NR2A subunit and the GluR1 subunit were unaltered. Surface biotinylation experiments of cortical slices kept at 4°C to block trafficking revealed the same patterns of surface expression for these three receptor subunits (data not shown).
Figure 2
Figure 2
Knockout of Kal7 specifically decreases surface localization of NR2B subunit. A. Exposure of cortical slices kept at 4°C to BS3 crosslinks surface receptors, producing high molecular weight bands that are not observed in non-crosslinked control (more ...)
Differences in Wt and Kal7KO mouse place preference for cocaine are eliminated by ifenprodil
Given the specific decreases in NR2B subunit localization and function in Kal7KO mice, we wanted to determine if any of the behavioral differences in Kal7KO mice might be attributable to a diminished contribution from NR2B-containing NMDA receptors. Kal7KO animals display a substantially reduced conditioned place preference for cocaine despite their normal place preference for food (Kiraly et al., 2010b). Previous studies from other labs demonstrated that NR2B function is critical for development of cocaine or morphine place preference (Ma et al., 2006; Pascoli et al., 2011). To test our hypothesis, we repeated the place preference experiment for cocaine, but tried to eliminate the contribution of NR2B subunit-containing NMDA receptor signaling by injecting ifenprodil before cocaine on each conditioning day (Fig. 3A). To optimize specificity for NR2B-containing receptors (Williams, 1993), we chose a dose of ifenprodil (2mg/kg) in the lower end of the range previously reported to have behavioral effects (Rodrigues et al., 2001). A higher dose of ifenprodil or Ro 25-6981 (10mg/kg for both) was used in previous studies of NR2B in drug preference (Ma et al., 2006; Pascoli et al., 2011).
Figure 3
Figure 3
Blockade of NR2B subunit-containing NMDA receptors eliminates differences in cocaine conditioned place preference response in Kal7KO and Wt animals. A. Timeline of daily injections. Cocaine injections (10mg/kg) were preceded by ifenprodil injections (2mg/kg (more ...)
As expected, animals receiving only saline injections showed no significant changes from baseline; ifenprodil on its own created no preference or aversion (Fig. 3B left) (Genotype, Treatment and Interaction effects: all p>0.75 for Fig. 3B left; two-way ANOVA). In animals receiving cocaine after either saline or ifenprodil, there were no main effects of genotype (p=0.19) or treatment (p=0.11), but there was a significant genotype × treatment interaction (F(1,32)=4.32, p=0.047; two-way ANOVA). In animals receiving saline prior to cocaine, there was a strong effect of genotype (Fig. 3B right) (p=0.02; Holm-Sidak post hoc test); as expected, Kal7KO mice showed a diminished place preference for cocaine (Kiraly et al., 2010b). When Wt animals were given ifenprodil before each injection of cocaine, they showed a significant decrease in preference (effect of ifenprodil within Wt: p=0.01); in contrast, ifenprodil did not alter the response of Kal7KO animals to cocaine (effect of ifenprodil within Kal7KO: p=0.77). Pre-treatment with ifenprodil abolished the genotypic difference between Wt and Kal7KO mice (Fig. 3B right; effect of genotype within ifenprodil: p=0.61). While blockade of NR2B subunit-containing NMDA receptors decreased preference in Wt mice, ifenprodil treatment did not eliminate the conditioned response entirely, an effect similar to published results in mice (Pascoli et al., 2011). Our data are consistent with the hypothesis that NR2B subunit-containing NMDA receptors play an important role in cocaine-induced place preference and that the absence of Kal7 diminishes the contribution of NR2B subunit-containing NMDA receptors to this behavior.
Kal7KO mice demonstrate a normal locomotor response to a wide range of acute cocaine doses (Kiraly et al., 2010b). To eliminate a potential confound due to an aberrant locomotor response to ifenprodil, or to the combination of ifenprodil and cocaine, we monitored the locomotor response of Wt and Kal7KO mice to an injection of ifenprodil, cocaine or both using a Latin-square design (Benavides et al., 2007; Kiraly et al., 2010b) (Fig. 3C). Animals were given each of the four treatments in a randomized order over the course of four days. While the expected significant effect of cocaine was observed (F(1,31)=50.44, p<0.001; 3-way ANOVA), there were no effects of genotype (p=0.79), ifenprodil (p=0.46) or any interactions (all p≥0.64). Thus the differences noted in cocaine place preference do not reflect an altered acute behavioral response of either genotype to ifenprodil.
Differences in Wt and Kal7KO mouse passive avoidance behaviors are abrogated by ifenprodil
We previously observed a deficit in the passive avoidance form of contextual fear conditioning in Kal7KO animals (Ma et al., 2008b). NR2B subunit-containing NMDA receptors are known to play an essential role in fear memory formation and maintenance (Rodrigues et al., 2001; Sotres-Bayon et al., 2007). Additionally, transgenic animals engineered to over-express NR2B showed enhanced fear conditioning and extinction behavior (Tang et al., 1999). To determine if the Kal7KO animals had an altered response to NR2B blockade, we injected animals with either saline or ifenprodil (2mg/kg) 15 minutes before training. There were no significant genotype or treatment differences in latency to cross on the training day (data not shown). The avoidance test was administered 24 hours after conditioning (Fig. 4). While there were no main effects of genotype (p=0.11) or treatment (p=0.89) there was a strong genotype × treatment interaction (F1,35 = 11.30, p=0.002; 2-way ANOVA). Post-hoc tests revealed a strong genotype effect within the saline (p=0.003, Holm-Sidak test) but not the ifenprodil treated animals. Interestingly, while the Wt animals showed a significant decrease in conditioning (effect of ifenprodil within Wt: p=0.01), the Kal7KO animals showed a significant enhancement of conditioning with ifenprodil pretreatment (effect of ifenprodil within Kal7KO: p=0.04). Saline treated Kal7KO mice showed a decrease in conditioning compared to Wt mice that was similar to that reported previously (Ma et al., 2008b). Thus, administration of ifenprodil abolished behavioral differences between Wt and Kal7KO animals for both passive avoidance conditioning and cocaine place preference.
Figure 4
Figure 4
Blockade of NR2B subunit-containing NMDA receptors abrogates genotypic differences in passive avoidance fear conditioning. Left. In animals receiving an injection of saline before passive avoidance conditioning, Kal7KO animals showed significantly decreased (more ...)
Endogenous Kal7 and NMDA receptor complexes interact
Based on the electrophysiological (Fig. 1), biochemical (Fig. 2) and behavioral (Fig. 3&4) findings that suggest a specific role for Kal7 in NR2B subunit-containing NMDA receptor localization and function, we investigated the possibility that Kal7 and NR2B interact directly. In order to explore this possibility, synaptosomes were solubilized using three different protocols. Given that the PSD is a complex and highly interconnected piece of machinery, the method used to solubilize PSD proteins is a critical determinant of the interactions that can be measured. As shown in Fig. 5A, the three detergent conditions tested solubilized different amounts of NR2B, Kal7, GluR1 and PSD-95. In the least stringent detergent protocol (1% TX-100/0.1%SDS; TX), very little NR2B or PSD-95 was solubilized while substantial amounts of Kal7 and GluR1 were recovered from the supernatant. Deoxycholate (DOC) solubilization increased the recovery of NR2B and PSD-95 from the soluble fraction, but had relatively little effect on the recovery of Kal7 or GluR1. Solubilization with 1% SDS (at room temperature), further increased the solubilization of NR2B and GluR1.
Figure 5
Figure 5
Kal7 associates with NMDA receptor complexes in vivo. A. Solubilization of rat forebrain synaptosomal proteins with different detergents yielded different amounts of soluble NR2B, Kal7, GluR1 and PSD-95. Samples were loaded as equal percentages of the (more ...)
Based on its ability to solubilize NR2B and Kal7 and the fact that it has been widely used for examining protein-protein interactions in the PSD (Wyszynski et al., 2002; Collins et al., 2006; Al-Hallaq et al., 2007) we chose the DOC protocol as our primary method of solubilization for co-immunoprecipitation experiments. Using this protocol, immunoprecipitation of Kal7 led to co-precipitation of a small but significant amount of NR2B, NR2A, and NR1 as well as PSD-95 (Fig. 5B), but not the AMPA receptor subunit GluR1 (Fig. 5B). Co-precipitation of both NR2B and NR2A from synaptosomes was expected as 30–40% of endogenous NMDA receptors are thought to be tri-heteromeric [NR12/NR2A/NR2B] (Al-Hallaq et al., 2007). To determine the stability of the Kal7-NMDAR interactions we performed one set of experiments using more stringent solubilization conditions (1% SDS) (Fig. 5C). Following SDS extraction, immunoprecipitation of Kal7 still co-precipitated NMDA receptor subunits NR2B and NR2A as well as PSD-95. Under these same conditions, immunoprecipitation of NR2B resulted in robust co-precipitation of NR2A, indicating that NMDA receptor complexes remained intact (data not shown). Maintenance of the Kal7-NMDAR interaction despite strong detergent application indicates that the interaction is a stable one.
To verify the Kal7-NMDAR interaction, we asked whether NR2B antibody co-precipitated Kal7 (Fig. 5D); using a pan-Kalirin antibody, co-precipitation of Kal7 was demonstrated. In addition, larger isoforms of Kalirin were detected. Kal7KO mice were engineered to lack Kal7 while still expressing the larger isoforms; as reported previously, levels of Kal8, Kal9 and Kal12 are increased in Kal7KO animals (Fig. 5D, KO Input) (Ma et al., 2008b). NR2B immunoprecipitation from Kal7KO synaptosomes revealed co-precipitation of Kal9 and Kal12 (Fig. 5D, IP). Unlike NR2B and Kal7, Kal9 and Kal12 lack a PDZ binding motif (Al-Hallaq et al., 2007). Our findings indicate that the Kalirin/NR2B subunit interaction is not solely dependent on PDZ domain binding.
The KalPH1 domain of Kal7 specifically interacts with a membrane-proximal region of NR2B
To determine whether Kalirin and NR2B interact directly and to map sites of interaction, we turned to non-neuronal cells. pEAK Rapid cells, a HEK-293 derivative, do not express PSD-95 or other MAGUK proteins (Fig. 6A). In cells co-transfected with vectors encoding Kal7, NR1 and either NR2B or NR2A, Kal7 monoclonal antibody beads co-precipitated NR2B, but not NR2A (Fig. 6B). Immunoprecipitation of NR2B (Fig. 6C), but not NR2A (Fig. 6D), from these same lysates led to co-precipitation of Kal7. The fact that co-immunoprecipitation of Kal7 and NR2B took place outside of the meshwork of the PSD and was specific for NR2B, suggests that Kal7 may interact directly with NR2B.
Figure 6
Figure 6
KalPH1 interacts with NR2B. A. pEAK Rapid cell (pE) and mouse brain (Br) lysate (10 or 20 μg protein) were blotted for PSD-95 and other MAGUK scaffolding proteins. B. In pEAK Rapid cells co-transfected with vectors encoding NR1, NR2B and Kal7 (more ...)
Kal7 is a large protein; to identify the region responsible for its interaction with NR2B, vectors encoding fragments of Kalirin were expressed along with NR1 and NR2B (Fig. 6E). A fragment which begins in spectrin repeat 3 and ends in spectrin repeat 9 (SR3-9) and has been shown to interact with iNOS (Ratovitski et al., 1999) and DISC-1 (Hayashi-Takagi et al., 2010), did not co-precipitate with the NR2B subunit. Kal7ΔCT, which lacks the 60 C-terminal residues of Kal7, including the PDZ binding motif, co-precipitated with NR2B, as did the GEF1 domain. GEF domains consist of a catalytic DH domain followed by a PH domain (Rossman et al., 2005). When expressed alone, a KalPH1-GFP fusion protein interacted with NR2B (Fig. 6E). To verify this, we co-expressed KalPH1-GFP and NR1/NR2B and used an antibody to GFP to look for an interaction; immunoprecipitation of KalPH1-GFP led to co-precipitation of NR2B (Fig. 6F). Interestingly, the PH2 domain of Kalirin, which is only present in the larger isoforms, also co-precipitated NR2B (data not shown). To determine whether this interaction were specific to Kalirin, we co-transfected NR1/NR2B and vector encoding the GEF domain of Tiam1 (TiamGEF), another Rac GEF localized to the PSD (Tolias et al., 2005). Immunoprecipitation of TiamGEF did not result in co-precipitation of the NR2B subunit (Fig. 6G).
We next mapped the region of the NR2B subunit that interacts with Kal-PH1; NR2B contains a large extracellular domain that begins with the N-terminal regulatory domain where ifenprodil and other allosteric modulators bind and is followed by the glutamate binding domain (Fig. 7A). Three transmembrane domains and a non-penetrant membrane domain form the ion channel; the ~80 kDa intracellular C-terminal domain of NR2B interacts with multiple cytosolic proteins including CaMKII (Barria and Malinow, 2005) and α-actinin (Wyszynski et al., 1997; Cull-Candy and Leszkiewicz, 2004) and terminates with a PDZ-binding motif known to interact with PSD-95 (Lau and Zukin, 2007). We first tested ΔNR2B, which terminates shortly after the final transmembrane domain (Foster et al., 2010). When Kal7, NR1 and ΔNR2B were co-expressed, the Kal7 antibody beads co-precipitated ΔNR2B (Fig. 7B). Immunoprecipitation of ΔNR2B with antibody directed to its extracellular N-terminal domain co-precipitated Kal7 (Fig. 7C). To ensure that this was the same interaction we were seeing before, we co-transfected KalPH1-GFP, NR1 and ΔNR2B; co-precipitation of KalPH1-GFP and ΔNR2B was observed (Fig. 7D). As before, the TiamGEF domain and ΔNR2B did not co-precipitate (Fig. 7E).
Figure 7
Figure 7
Interaction between KalPH1 and NR2B involves a membrane proximal intracellular segment of NR2B. A. Diagram of the NR2B subunit showing the C-terminus of ΔNR2B (residues 1-861 of NR2B) (Foster et al., 2010) and the antibody binding epitope. Sites (more ...)
KalPH1 interacts directly with the final juxtamembrane region of NR2B
The intracellular region of ΔNR2B is limited to three short sequences; since KalPH1-GFP interacts with NR2B, but not with NR2A, we compared their sequences in these three regions (Fig. 8A). Five of the 19 residues in the M1→M2 loop differ while none of the 11 residues in the M2→M3 loop differ (Fig. 8A; loop diagrams in Fig. 7A). Eight of the 17 residues that follow M4 in ΔNR2B (M4→End) differ (Fig. 8A). To test the M4→End region, an NR2B juxtamembrane peptide (2B-JM) was synthesized and linked to AffiGel beads. Lysates of pEAK Rapid cells expressing Kal7, KalPH1-GFP, SR3-9, or GFP only were incubated with 2B-JM beads or control (no peptide) beads. Kal7 and KalPH1-GFP bound to 2B-JM beads, but not to control beads (Fig. 8B). KalPH1-GFP lysates were also incubated with β-endorphin beads; no binding was seen, even with this positively charged peptide (data not shown). Neither SR3-9 nor GFP bound to the 2B-JM beads. Incubation of 2B-JM beads with lysates from cells expressing KalPH2 revealed a similar interaction with KalPH2 (not shown). To determine if binding to the 2B-JM beads saturated, we incubated increasing concentrations of KalPH1-GFP lysate with a fixed amount of beads. As the concentration of lysate was increased, we saw a plateau in binding (Fig. 8C). Finally to determine whether the juxtamembrane region of NR2B could interact with endogenous Kal7, we incubated 2B-JM beads with mouse brain synaptosomes solubilized with TX-100 or DOC. The 2B-JM beads bound Kal7 solubilized with either detergent but control beads did not (Fig. 8D). From these experiments we can conclude that there is a specific and stable interaction between the PH domains of Kalirin and the juxtamembrane region at the C-terminus of NR2B (Fig. 8E).
Figure 8
Figure 8
Interaction of KalPH1 with the juxtamembrane region of NR2B A. Clustal analysis comparing intracellular regions of NR2B and NR2A; M1, M2, M3 and the C-terminus of ΔNR2B (End) are as shown in Fig. 7A. B. Lysates from cells expressing the indicated (more ...)
To confirm that the 2B-JM region was in fact the region of ΔNR2B responsible for its interaction with KalPH1-GFP, we constructed three ΔNR2B mutants. The 2B-JM region of ΔNR2B was replaced by the corresponding region of NR2A to generate ΔNR2B→2A. ΔNR2B Stub stopped shortly after the final transmembrane domain of NR2B. In ΔNR2B GAGA, the 2B-JM region was replaced with a repeating sequence of Gly-Ala residues. pEAK Rapid cells were transfected with NR1, KalPH1-GFP and ΔNR2B or one of these three mutant constructs. As seen previously, immunoprecipitation of KalPH1-GFP strongly co-precipitated ΔNR2B (Fig. 9A). For each of the three mutants, co-precipitation of ΔNR2B with KalPH1-GFP was reduced to levels just above the IgG control (Fig. 9B-D). Importantly, each ΔNR2B mutant co-precipitated NR1 as effectively as ΔNR2B, indicating that they were capable of forming receptor complexes (data not shown). Thus the 2B-JM region of ΔNR2B is essential for the interaction of KalPH1 and ΔNR2B.
Figure 9
Figure 9
Mutation of the 2B-JM region decreases KalPH1-ΔNR2B co-precipitation. The sequences of the final juxtamembrane region of ΔNR2B (A), the NR2B→2A mutant (B), the Stub mutant (C) and the GAGA mutant (D) are shown (black text) with (more ...)
NR2B function is altered when Kal7 is absent
Purified PSDs from the cortices of Kal7KO animals contain slightly reduced levels of NR2B (Ma et al., 2008b). Here we demonstrate that this decrease results in altered synaptic physiology. Kal7KO animals have an overall decrease in the ratio of their NMDA/AMPA receptor currents, largely due to a decrease in current conducted via NR2B subunit-containing receptors (Fig. 1). Additionally, we demonstrate that this decrease is due to a decrease in cell surface localization of NR2B subunit-containing NMDA receptors (Fig. 2). Given that approximately 1/3 of NMDA receptors are thought to be tri-heteromeric, and surface levels of NR2A are unaltered in Kal7KO mice, NR1/NR2B di-heteromeric receptors may be especially sensitive to the absence of Kal7. Interestingly, ifenprodil more potently inhibits NR1/NR2B di-heteromeric receptors (Hatton and Paoletti, 2005). Localization of NR2B subunit-containing receptors to synapses is essential for induction of long-term potentiation, and neurons overexpressing NR2B show enhanced potentiation (Tang et al., 1999; Akashi et al., 2009). More recent studies have shown that NR2B is essential because of the many proteins recruited to the synapse via their interactions with its cytosolic tail (Foster et al., 2010). The ability of NR2B to localize CamKII and its other binding partners directly to the source of large calcium currents seems to be critical for LTP induction (Barria and Malinow, 2005). Decreased synaptic NR2B subunit-containing receptor levels and currents may explain why Kal7KO animals exhibit significantly impaired NMDA receptor-dependent LTP (Ma et al., 2008b)(Lemtiri-Chlieh et al., unpublished).
Smaller spines, and spines where presynaptic input has been blocked, have greater accumulations of NR2B-containing receptors than larger spines or those receiving stronger inputs (Sobczyk et al., 2005; Lee et al., 2010). Additionally, smaller spines are more easily potentiated and demonstrate more robust and pronounced increases in dendritic head size in response to stimulation (Matsuzaki et al., 2004; Bourne and Harris, 2007). Numerous studies have suggested that synapses expressing higher levels of NR2B have a lower threshold for potentiation (Lau and Zukin, 2007). Given this, it seems likely that localization of NR2B-containing receptors to newly developing synapses is a key to ensuring synapse stabilization and maturation. Kal7 has been implicated in the formation and stabilization of new dendritic spines both in vitro and in vivo (Ma et al., 2008a; Ma et al., 2008b). As shown here, Kal7KO mice are deficient in NR2B subunit-containing receptor signaling and surface localization (Figs. 1&2). If NR2B is an important component in stabilizing newly formed spines, this may partially explain how Kal7 expression leads to increases in spine density.
Behavioral differences between Wt and Kal7KO animals are abrogated by ifenprodil
One of the most striking deficits observed in Kal7KO mice is a decrease in conditioned place preference for cocaine (Kiraly et al., 2010b). The circuitry involved in this response is complex and includes the striatum, prefrontal cortex and ventral tegmental area, with roles for dopaminergic, glutamatergic, GABAergic and cholinergic transmission (Hyman et al., 2006; Kauer and Malenka, 2007; Williams and Adinoff, 2008; Kalivas, 2009). NR2B subunit-containing NMDA receptor function is known to be necessary for cocaine and morphine place conditioning (Ma et al., 2006; Pascoli et al., 2011). In Kal7KO animals, loss of the Kal7/NR2B interaction and subsequent reduction in NR2B-containing receptor mediated plasticity may underlie this behavioral deficit. We thus hypothesized that blocking NR2B–containing receptors would reproduce this deficit in Wt animals, but would have less effect in Kal7KO mice. Consistent with this prediction, ifenprodil administration eliminated the difference between Kal7KO and Wt mice, although mice of both genotypes continued to exhibit a preference for cocaine (Fig. 3). This is consistent with the fact that this behavioral response is not entirely dependent on NR2B-containing NMDA receptors.
Similar to the conditioned place preference effect, we had seen decreases in passive avoidance fear conditioning in Kal7KO animals (Ma et al., 2008b). When Wt and Kal7KO animals were given ifenprodil before conditioning, this genotypic difference was also abolished (Fig. 4). NR2B-containing NMDA receptor function has been shown to be crucial for consolidation of fear conditioning memories (Tang et al., 1999; Rodrigues et al., 2001). Interestingly, ifenprodil pretreatment had distinctly different effects in Wt and Kal7KO mice, with reduced fear conditioning in Wt mice and increased fear conditioning in Kal7KO mice. Region-specific responses to elimination of Kal7 may be a factor; for example, while spine density is reduced in Kal7KO vs Wt CA1 hippocampal pyramidal neurons under baseline conditions (Ma et al., 2008b), basal spine density is unaltered in the Kal7KO nucleus accumbens (Kiraly et al., 2010b).
Kal7KO mice may have compensatory developments that allow their function to rely more heavily on NR2A subunit-containing NMDA receptors, which are expressed at normal levels (Fig. 2), or other non-NMDA receptor dependent forms of plasticity. Mice lacking NR2A subunits have reduced LTP as well, indicating that NMDA receptor mediated LTP is not solely dependent on NR2B (Sakimura et al., 1995). Additionally, non-NMDA receptor dependent forms of LTP have been described (Cavus and Teyler, 1998). Interestingly, recent electrophysiological experiments on Kal7KO mice indicate that they exhibit impaired NMDA receptor-dependent LTP, but normal non-NMDA receptor mediated LTP (Lemtiri-Chlieh et al., unpublished). Increased reliance on these alternate pathways in Kal7KO mice may explain why inhibition of NR2B-containing receptors does not further accentuate their behavioral deficits.
Binding of Kal7 to NR2B-containing NMDA receptors is stable and specific
The PSD is an intricate complex of hundreds of proteins (Sheng and Hoogenraad, 2007); proper pairwise connections amongst specific proteins in this network are critical for synaptic function. Both Kal7 and NMDA receptors interact directly with multiple PSD constituents, including multiple PDZ domain containing proteins. The C-terminal PDZ binding motif of Kal7 interacts with PSD-95 in a way that decreases its catalytic activity (Penzes et al., 2001b), and with afadin (AF-6) in a way that helps anchor it in the PSD (Xie et al., 2008). Interactions of the C-terminus of NR2B with PSD-95 and SAP-97 are critical for stabilizing synaptic NMDA receptors (Niethammer et al., 1996; Roche et al., 2001). It is currently not clear whether Kal7 and NR2B can bind simultaneously to PSD-95, which has three PDZ domains. We show here that Kalirin and NR2B-containing NMDA receptors interact directly, in a way that is not dependent on PDZ binding. In addition to NR2B, the first PH domain of Kal7 binds to TrkA, the high affinity NGF receptor (Chakrabarti et al., 2005).
NMDA receptors exist as part of huge macromolecular complexes in the PSD, reflective of direct and indirect interactions with receptors, scaffolding proteins and enzymes (Husi and Grant, 2001). Interestingly, the interactions of Kalirin with the NR2B subunit involve its juxtamembrane region (Figs. 79), placing Kalirin in an excellent position to modulate channel function or respond to calcium influx through the receptor. A number of other proteins interact with the juxtamembrane regions of specific NMDA receptor subunits. Calmodulin binds the C-terminus of the NR1 subunit in a calcium dependent manner and causes channel inactivation (Ehlers et al., 1996). The interaction of calmodulin with the NR1 subunit seems to be directly competitive with the actin binding protein α-actinin, which may help crosslink these receptors to the actin cytoskeleton and stabilize them in the synapse (Wyszynski et al., 1997). Additionally, both the NR1 and NR2B subunits have AP-2 binding sites in their juxtamembrane C-terminal regions, and binding of AP-2 leads to dynamin dependent receptor internalization (Scott et al., 2004). The 2B-JM peptide used to pull down Kal7 and KalPH1-GFP (Fig. 8&9) contains part of this AP-2 binding motif and Kalirin may affect surface localization of NR2B-containing receptors by competing with AP-2. BLAST searches of the 2B-JM region of the NR2B subunit, which is fully conserved in mouse, rat, chimp and human, identify the corresponding region of NR2A as the closest match (9/17 residues). Replacing the 2B-JM region of ΔNR2B with the corresponding sequence from NR2A eliminated its interaction with KalPH1-GFP. In addition to the 2B-JM region, the full-length NR2B subunit may have additional interactions with Kal7.
Kalirin activates Rac1, a GTPase known to mediate actin rearrangement (Penzes et al., 2001a; Hotulainen and Hoogenraad, 2010). Interactions of NMDA receptors with the actin cytoskeleton are known to be essential for NMDA receptor function and localization (Rosenmund and Westbrook, 1993; Allison et al., 1998). Both spectrin and α-actinin bind to NMDA receptors, linking them to the actin cytoskeleton (Wyszynski et al., 1997; Wechsler and Teichberg, 1998). Kal7 is not the only Rho-GEF known to bind to NMDA receptors. The Rac-GEF Tiam-1 binds to the NR1 subunit and is critical for NMDA induced spine dynamics in neuronal cultures (Tolias et al., 2005). RasGRF1, a dual Ras-and Rac-GEF, binds directly to NR2B (Krapivinsky et al., 2003), and RasGRF1 knockout mice show decreases in cocaine place preference, as do Kal7KO mice (Fasano et al., 2009). The activity of both Tiam-1 and RasGRF1 is modulated by calcium and/or calcium-calmodulin dependent kinases (Fleming et al., 1999; Tian et al., 2004; Schmitt et al., 2005; Tolias et al., 2005; Tian and Feig, 2006). By being directly coupled to NMDA receptors, these GEFs are uniquely positioned to transduce calcium dependent signals. We have directly identified multiple CamKII sites in Kal7 (Kiraly et al., 2011); these sites are distinct from the putative CamKII site previously identified through indirect means but are consistent with a role for calcium in controlling Kal7 signaling. Given the rapid (seconds → minutes) time course over which NMDA receptor dependent changes in spine size occur (Murakoshi et al., 2011), GEF interactions with NMDA receptors may be critical for morphological plasticity.
Acknowledgments
These studies were supported by NIH grants DA-15464, DA-18274, DA-23082 and DA-26706. The authors would like to thank Darlene D’Amato Yanping Wang and Chris Mazzone for amazing technical support. We would also like to thank Jodi Eipper-Mains for the original version of the drawing used in Figure 7 and Dr. Marina Wolf for advice on crosslinking.
Footnotes
Conflict of Interest: None
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