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 (). Kal7KO
mice exhibit a significantly decreased NMDA/AMPA ratio, indicative of a decrease in overall NMDA receptor function (). 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) or field (F.L.-C. unpublished observations) LTP induction protocols.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 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 ( 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 (). 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 (; 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
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 (). 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 ( right). We examined the NR2B and NR2A subunits of the NMDA receptor as well as the GluR1 subunit of the AMPA receptor (). 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 (); 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 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 (). 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 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 ( left) (Genotype, Treatment and Interaction effects: all p>0.75 for 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 ( 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 ( 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.
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
) (). 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 (). 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 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 (), biochemical () and behavioral (&) 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 , 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 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 (), but not the AMPA receptor subunit GluR1 (). 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) (). 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 (); 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 (, KO Input) (Ma et al., 2008b
). NR2B immunoprecipitation from Kal7KO
synaptosomes revealed co-precipitation of Kal9 and Kal12 (, 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 (). In cells co-transfected with vectors encoding Kal7, NR1 and either NR2B or NR2A, Kal7 monoclonal antibody beads co-precipitated NR2B, but not NR2A (). Immunoprecipitation of NR2B (), but not NR2A (), 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 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 (). 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 (). 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 (). 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 ().
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 (). 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 (). Immunoprecipitation of ΔNR2B with antibody directed to its extracellular N-terminal domain co-precipitated Kal7 (). 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 (). As before, the TiamGEF domain and ΔNR2B did not co-precipitate ().
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 (). Five of the 19 residues in the M1→M2 loop differ while none of the 11 residues in the M2→M3 loop differ (; loop diagrams in ). Eight of the 17 residues that follow M4 in ΔNR2B (M4→End) differ (). 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 (). 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 (). 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 (). 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 ().
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 . 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 (). For each of the three mutants, co-precipitation of ΔNR2B with KalPH1-GFP was reduced to levels just above the IgG control (). 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 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 ...)