The basis for stronger association of PSD-95 with
GluN2BWT compared to GluN2B2A(CTR) could be due to different sequences immediately upstream of the conserved C-terminal PDZ ligand. We generated a chimeric variant of GluN2B in which the final 12 amino acids of its CTD have been replaced by those of GluN2A (three amino acid differences, GluN2B(2A-PDZ)). Coimmunoprecipitation studies revealed that GluN2B(2A-PDZ) had a similar affinity for PSD-95 as GluN2B (Figure S4C), indicating that immediate upstream sequence differences are not the basis for differential association of PSD-95 with the CTDs of GluN2B and GluN2A. Recently, additional PSD-95 interaction domains have been discovered on internal regions of CTD2B (1086–1157; see more Cousins et al., 2009), which could contribute to the overall affinity of the CTD for PSD-95. The role of these additional regions in neurons is not yet clear, but could act to stabilize the primary interaction with the C-terminal PDZ ligand, or even act independently. Deletion of this region (creating GluN2BΔ(1086–1157)) resulted in a small reduction in PSD-95 association (Figure 5G). Importantly, NMDA-induced death following overexpression of GluN2BΔ(1086–1157)
in primary rat hippocampal neurons (as per the assays used in Figure 1) was significantly lower than in neurons overexpressing GluN2BWT (Figure 5H), even though whole-cell NMDAR currents were found to be the same in GluN2BΔ(1086–1157) as wild-type GluN2BWT-expressing Selleckchem HA 1077 neurons (Figure 5I), implicating this region of the CTD in contributing to prodeath NMDAR signaling. We have demonstrated distinct roles for the CTDs of GluN2B and GluN2A in determining the dose response of NMDAR-mediated excitotoxicity. CTD2B promotes neuronal death more efficiently than CTD2A, an effect which is observed regardless
of whether the CTD is tethered to the channel portion of GluN2B almost or of GluN2A. Moreover, this difference is observed both in the context of acute chimeric subunit expression in wild-type neurons, as well as in a knockin mouse where the CTD is swapped at the genetic level. Using the latter approach, we demonstrated the influence of the GluN2 CTD subtype in controlling excitotoxic lesion volume in vivo. We also show that the GluN2 CTD subtype’s ability to influence excitotoxicity is overcome when strong excitotoxic insults are applied. These findings raise the question as to whether subunit composition (and CTD identity) underlies the known differential prodeath signaling from synaptic versus extrasynaptic NMDARs, or whether it represents an additional factor that influences excitotoxicity (Hardingham and Bading, 2010). Although some studies have reported that GluN2B is enriched at extrasynaptic sites (Groc et al., 2006, Martel et al.