The NMDA receptor complex assembly

1.9.2.1 From transcription to membrane insertion

The transcription of mRNA coding for the NMDA receptor subunits and their subsequent translocation to form functional NMDA receptors at the cell membrane

is a complex process that is as yet not defined. It has been shown that the same neurone can express a variety of NMDA receptor complexes that have distinct distribution patterns, thus a complex mechanism to include or exclude particular NR2 or NR3 subunits with N Rl and specific translocation mechanisms must exist (Tovar & Westbrook, 1999).

The N R l subunit appears to be constitutively formed and more than 50% is held within the endoplasmic reticulum (ER) before degradation (Hall & Soderling, 1997). In contrast more than 90% of the NR2B subunit is found at the cell surface (Hall & Soderling, 1997). N Rl subunit can be expressed on the cell surface in mammalian expression systems and has been demonstrated in Purkinje cells that do not express NR2 subunits (Petralia et al., 1994b). Different splice varients of N R l appeared to be preferentially trafficked to the cell surface, which appeared to be determined by the presence or absence of exon 21 in the C terminus. The splice variants containing exon 21 were held within the ER due to the presence of an ER retention signal, whilst the absence of exon 21 were trafficked to the surface membrane (Standley et a l, 2000; Xia et al., 2001). This retention signal could be overcome by the addition of a PDZ domain -binding motif contained in exon 22 (Standley et al., 2000; Xia et al., 2001). Despite this all N R l splice variants can form functional surface receptors in Xenopus oocytes and ER retention is not required for assembly (Okabe et al., 1999). Whilst it is generally assumed that assembly of NMDA subunits occurs in the cell body there is some evidence for dendritic assembly. Golgi associated proteins and N R l have been identified in the dendrites that could potentially allow for rapid local synaptic modification (Misra et al., 2000a). The half life of N Rl subunits varies considerably. Those held in the cytoplasmic pool are rapidly degraded with a half life of under 5 hours, whilst subunits assembled with NR2 at the surface had a half life of up to 34 hours (Huh & Wenthold, 1999). Post-transciptional maturation via addition of carbohydrate residues andphosphorylation lengthens the half-life and cell surface trafficking (Garcia-Gallo et al., 2001).

Whilst N R l subunits can be expressed on the cell surface the NR2 and NR3 subunits require co-expression with N R l to be expressed (Mcllhinney et al., 1996;

Das et al., 1998; Mcllhinney et al., 1998). The N' terminal residues of N R l are vital for oligomerization with NR2A and the subsequent transportation of NR2A to the cell surface (Mcllhinney et al., 1998; Meddows et al., 2001), where the half-life for NR2A or B appears to be approximately 20 hours (Huh & Wenthold, 1999). The C terminal end of the NR2A determines its trafficking into synaptosomes as a

truncated form was not targeted into post-synaptic densities (Steigerwald et al.,

2000). No selective incorporation of NR2A and B with N R l splice variants were demonstrated, with both NR2 subunits similarly enriched at the synapse (Al-Hallaq

et al.,2001).

Where the NMDA receptor assembly occurs, within the ER, cytosol or within the cell membrane (or combinations of all routes) is as yet unknown. In addition it is becoming apparent that the surface insertion of NMDA receptors alone does not yield an active synapse. The formation and structure of an active synapse is a new kind of signalling machine with multiple proteins involved: neurotransmitter receptors, cell-adhesion proteins, adaptor molecules, signalling enzymes and cytoskeleton proteins (Grant & O'Dell, 2001).

The spatial organisation of the NMDA receptor in relation to other receptors and secondary messenger / transducer systems depends on the array of scaffolding proteins such as PSD-95, and cytoskeletal proteins. These are arrayed in different layers on the intra-cellular side of the plasma membrane. Valtschanoff and Weinberg demonstrated that PSD-95 layer 2nm in from the plasma membrane, closely associated with NR2, C terminal, neuronal (n) NOS layer a further 6nm deep with other scaffolding proteins deeper between 10-20nm (Valtschanoff & Weinberg, 2001). Interaction between the NMDA receptor and PSD-95 occurs through the NR2 and certain N R l subunits containing a consensus motif of three amino acids at the end of the C terminal and PDZ domains (Koranu et al., 1995; Marsh gr a/., 2001).

All the PSD-95 / SAP-90 family of proteins have a similar structure with three tandem PDZ domains, a Src homology, and an inactive yeast guanylate kinase domain. PDZ domains, approximately 90 amino acids, mediate protein - protein

interactions. The PSD-95 proteins are highly enriched around post-synaptic densities, drive synaptic formation and co-expression with NR2 subunits induces clustering (Niethammer et ah, 1996; El-Husseini et al., 2000). In addition PSD-95 interacts with neuroligins, neuronal adhesion molecules, whose extra-cellular domain binds to the pre-synaptic p-neurexins, inducing tight adhesion between the two synaptic membranes (Irie et ah, 1997). The second domain of PSD-95 can bind to nNOS enabling a close association between NMDA and NO production, whilst the third PZD domain interacts with the mitogen-activated protein kinase pathway (O'Brien et ah, 1998). Linkage with the AMPA receptor appears to be via

cytoskeleton interactions of the PSD-95 protein and GRIP (glutamate receptor interacting proteins) (O'Brien et ah, 1998).

One group investigated the assembly of individual glutamatergic synapses. Results indicated that a functional pre-synaptic active zone preceeded PSD-95 accumulation and NMDA and AMPA receptors at the post-synaptic site, although an active synapse was formed within 1-2 hours of the initial contact (Friedman et ah, 2000). The synapses contained large multimolecular complexes which may be formed from pre-fabricated complexes, although there is some evidence that NR2B is shuttled to the synaptic site in discrete transport vesicles (Ziv & Gamer, 2001). The interaction with PSD-95 has functional consequences. Co-expression of NR1/NR2A with PSD- 95 reduced the receptor sensitivity of glutamate and enhanced expression of the NR2 subunits (Rutter & Stephenson, 2000).

1.9.2.2 Diheteromeric and triheteromeric assembly

The composition of the NMDA receptor alters throughout development and confers on the NMDA receptor changing properties. The complexity and diversity of the NMDA receptor is further increased with the expression of diheteromeric and triheteromeric synaptic receptors, combinations of N Rl with NR2 subunits and / or NR3 subunits (Sheng et ah, 1994; Buller & Monaghan, 1997; Chazot & Stephenson,

1997; Luo et ah, 1997; Das et ah, 1998; Tovar & Westbrook, 1999). Although most studies of in vivo NMDA receptors have identified either high or low conductances (NR2A or B or NR2C or D respectively) (Momiyama et ah, 1996; Cull-Candy et ah, 1998; Misra et ah, 2000b; Momiyama, 2000), co-expression of NR2A and

NR2D produced three channel conductances (Cheffings & Colquhoun, 2000). The novel channel was postulated to contain triheteromer of N R l, NR2A and D giving unique conductance (30, 40 and 50pS) and increased glycine sensitivity (Cheffings & Colquhoun, 2000). In addition a novel NMDA receptor current in neonatal dorsal horn has been described (Green & Gibb, 2001). Whilst in vivo the expression of triheteromeric assembles could explain the discrepancies in Mg^"^ sensitivity and EPSCs in maturing neurones, most work has been done ex vivo and the implications of synaptically active triheteromeric channels has not been demonstrated (Cathala et al., 2000). In addition ex vivo expression can not be directly extrapolated to the in vivo situation. Xenopus oocytes can express functional (albeit low conductance) NMDA receptor complexes comprised solely of N R l subunits, which whilst interesting requires in vivo confirmation.