Activity and Protein Kinase C Regulate Synaptic Accumulation of N-Methyl-d-aspartate (NMDA) Receptors Independently of GluN1 Splice Variant

Abstract

NMDA receptors are calcium-permeable ionotropic receptors that detect coincident glutamate binding and membrane depolarization and are essential for many forms of synaptic plasticity in the mammalian brain. The obligatory GluN1 subunit of NMDA receptors is alternatively spliced at multiple sites, generating forms that vary in N-terminal N1 and C-terminal C1, C2, and C2 cassettes. Based on expression of GluN1 constructs in heterologous cells and in wild type neurons, the prevalent view is that the C-terminal cassettes regulate synaptic accumulation and its modulation by homeostatic activity blockade and by protein kinase C (PKC). Here, we tested the role of GluN1 splicing in regulated synaptic accumulation of NMDA receptors by lentiviral expression of individual GluN1 splice variants in hippocampal neurons cultured from GluN1 (؊/؊) mice. High efficiency transduction of GluN1 at levels similar to endogenous was achieved. Under control conditions, the C2 cassette mediated enhanced synaptic accumulation relative to the alternate C2 cassette, whereas the presence or absence of N1 or C1 had no effect. Surprisingly all GluN1 splice variants showed >2-fold increased synaptic accumulation with chronic blockade of NMDA receptor activity. Furthermore, in this neuronal rescue system, all GluN1 splice variants were equally rapidly dispersed upon activation of PKC. These results indicate that the major mechanisms mediating homeostatic synaptic accumulation and PKC dispersal of NMDA receptors occur independently of GluN1 splice isoform. N-Methyl-D-aspartate (NMDA)3 type ionotropic glutamate receptors are present at excitatory synapses in the nervous system. These receptors play roles in neuronal development, learning and memory and neurological diseases (1-4). They require the simultaneous binding of glutamate and membrane depolarization for the channel to open, which makes them coincidence detectors of pre-and postsynaptic activity. Calcium flux through NMDA receptors triggers signal transduction cascades that are necessary for inducing many forms of synaptic plasticity. NMDA receptors are heteromeric cation channels composed of GluN1 and GluN2 subunits, and more rarely GluN3 subunits (5). The obligatory GluN1 subunit, which binds the co-agonist glycine, is encoded by a single gene which gives rise to eight GluN1 variants through alternative splicing of exons 5, 21, and 22 (6). The expression of GluN1 splice variants changes during development and across brain regions (7,8), and can be regulated by neuronal activity (9, 10). Therefore, the composition of NMDA receptor signaling complexes may be dynamically regulated through the splicing of GluN1.At the region encoding the N-terminal domain of GluN1, alternative splicing of exon 5 gives rise to splice variants containing or lacking the N1 cassette which regulates receptor deactivation kinetics as well as sensitivity to pH, Zn 2ϩ , and spermine (11-13). Alternative splicing of exons 21 and 22 generates four different splice variants at the intracellular C terminus of GluN1, var...

Requirement of phospholipase C and protein kinase C in cholecystokinin‐mediated facilitation of NMDA channel function and anxiety‐like behavior

“…Exogenous application of NMDA still enhances NMDA currents recorded from the dissociated granule cells thereby supporting that CCK targets the extrasynaptic NMDA receptors as well. There is compelling evidence indicating that activation of PKC enhances NMDA receptor function via activation of Src tyrosine kinase (Grosshans and Browning, 2001; Lu et al, 1999; Macdonald et al, 2005; MacDonald et al, 2001) or increases membrane trafficking of NMDA receptors (Carroll and Zukin, 2002; Ferreira et al, 2011; Fong et al, 2002; Lan et al, 2001a; Lan et al, 2001b; Lau et al, 2010). Further studies will determine the roles of Src and membrane trafficking in CCK-mediated augmentation of NMDA currents in dentate gyrus granule cells.…”

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-d-aspartate (NMDA) Receptors*

“…Thus, ligand binding controls delivery of this functionally important pool of NMDA receptors. NMDA receptor accumulation at synapses is also controlled by other features of GluN1 and GluN2 subunits (42,43) and multiple interacting proteins regulated by activity and kinases (27,44,45). …”

Alcohol and NMDA receptor: current research and future direction

“…NR1 exists as eight different splice variants due to the alternative splicing of exon 5 (NR1) and exon 21 and 22 (C and C2). Figure 5 shows that the eight splice variants exist as combinations of a two splice form involving the presence or absence of the NR1 cassette and a four splice form involving the combination of the C-terminal cassette (Bradley et al, 2006; Horak and Wenthold, 2009; Ferreira et al, 2011). These splice variants exhibit heterogeneity with respect to agonist and antagonist affinity, zinc modulation, and regional and developmental expression pattern.…”

“…Other major regulatory events influencing the activity of NMDARs are the post-translational modifications, especially the phosphorylation state of the receptor. NR2A, NR2B are phosphorylated on serine and tyrosine residues, and the alternatively spliced isoform of the NR1 subunits is phosphorylated on serine residues (Ferreira et al, 2011). Recently, evidence has emerged that the activity of several kinase (Fyn, Src, PKA, PKC, CaM kinase II, CDK5) and phosphatatese (PP1, PP2B) on NMDAR subunits' phosphorylation was shown to be affected by ethanol, leading to altered ethanol sensitivity and or functional activity of NMDARs (Nagy, 2003, 2008).…”