ReviewNMDA receptor subunits: diversity, development and disease
Introduction
N-methyl-d-aspartate receptors (NMDARs) have critical roles in excitatory synaptic transmission, plasticity and excitotoxicity in the CNS. The involvement of NMDARs in these diverse processes reflects their unique features, which include voltage-sensitive block by extracellular Mg2+, a high permeability to Ca2+ and unusually slow ‘activation/deactivation’ kinetics. NMDARs also display sensitivity to an array of endogenous ligands and modulators present in the vicinity of the synapse: the co-agonist glycine must bind before the receptors can be activated, whereas physiological levels of protons suppress NMDAR activation. Extracellular Zn2+ and polyamines also act on the receptor to modify its behaviour. Furthermore, NMDAR subunits interact with various intracellular scaffolding, anchoring and signalling molecules associated with the postsynaptic density.
Several distinct NMDAR subtypes have now been identified in central neurons, differing in their sensitivity to endogenous and exogenous ligands, permeation and block by divalent ions, kinetic properties, and interaction with intracellular proteins. Biophysical, pharmacological and molecular methods are all providing a clearer picture of the key features defined by particular subunits and are furnishing tools that can be used to determine the involvement of specific subunits in synaptic transmission. Appreciating the roles played by distinct NMDAR subtypes is essential in understanding normal transmission in the CNS, and should provide information about how NMDAR subunit multiplicity can be exploited for therapeutic advantage. Several recent publications have reviewed the assembly and targeting of NMDARs and their role in developmental plasticity, learning and memory 1., 2., 3., 4.. Our aim here is to consider recent advances in the functional and pharmacological identification of the various NMDAR subtypes—including the relationship between subunit composition and receptor properties— and to consider the implications of this receptor diversity in normal and disease states.
Section snippets
NMDAR subunits and splice variants
Over the past decade, a variety of NMDAR subunits have been identified: the ubiquitously expressed NR1 subunit; a family of four distinct NR2 subunits (A, B, C and D); and two NR3 subunits 3., 5., 6., 7.. NR1 occurs as eight distinct isoforms owing to the presence of three independent sites of alternative splicing [1]. Similarly, each of the NR2 and NR3 subunits (apart from NR2A) has several splice variants (see Fig. 1), although the functional relevance of the different splice forms remains
NMDAR functional properties are determined by subunit composition
Studies of recombinant receptors 8., 11., 12., 13. have provided an understanding of how receptor properties are defined by individual NMDAR subunits. The likely subunit composition of native receptors has been inferred by examining subunit mRNA or protein distribution 8., 9., 14., 15., 16., 17., by using animals with specific subunit genes deleted 7., 18., 19., 20., and bycomparing the functional properties of native and recombinant NMDARs 21., 22., 23., 24., 25., 26., 27.. Together, these
NR2 and NR3 subunits
During synaptic transmission, NMDAR activation generates a current with a slow rise and an exceptionally slow decay time, which exceeds that of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR)-mediated component by at least two orders of magnitude. NMDAR channels first open about 10ms after glutamate is released into the synaptic cleft, and continue to open and close for hundreds of milliseconds until glutamate unbinds from receptor 13., 28.. The time course of decay of
NR1 isoforms
Although the effects of NR2 subunits have received most attention, NR1 splice variants also strongly influence NMDAR properties. For example, the pH sensitivity of NMDARs is determined by the presence of exon 5 (in the amino terminus). At physiological pH, splice variants that include exon 5 are fully active, whereas those that lack exon 5 are partially blocked [37]. It has been suggested that the exon 5 cassette forms a surface loop, with structural similarities to polyamines, and acts as a
NMDAR subtypes differ in their pharmacology
One way in which the functions of the various NMDAR subunits may be assessed is through the use of subunit selective agonists and antagonists. A number of pharmacological agents have been shown to distinguish between certain NMDAR subtypes (see Table 1). Competitive antagonists such as AP5 (2-amino-5-phosphonpentanoic acid) and d-CPPene (3-[2-carboxypiperazine-4-yl]-propenyl-1-phosphonic acid), channel blockers such as MK-801 (dizocilpine), ketamine, phencyclidine, amantadine and memantine, and
Developmental changes in synaptic NMDAR subtypes
One indicator of the functional importance of NMDAR subunit diversity comes from examining the subunit mRNA changes seen during development. At embryonic stages, the NR2B subunit is found in most brain regions, whereas the NR2D subunit is present in the diencephalon and brainstem. Soon after birth, NR2A mRNA is found in most regions, whereas NR2C appears later and is prominent in the cerebellum (see 8., 9.). Functional studies have now examined the possible subunit composition of NMDARs in a
Expression of the NR2A subunit and its role in synaptic plasticity
A gradual replacement or supplementation of NR2B by NR2A during postnatal development has been implicated in the speeding of NMDAR-EPSC decay—a phenomenon often linked with the ability of neuronal circuits to exhibit experience-dependent synaptic plasticity [4]. For example, in visual cortex the NMDAR-EPSCs are sensitive to NR2B-selective antagonists when the NMDAR-EPSC decay is slow (postnatal day [P] 3–5), and this sensitivity is lost by P7 when the NMDAR-EPSC decays rapidly. For some time it
Subcellular variation in NMDAR subunit composition
Growing evidence indicates that in some cells extrasynaptic and synaptic NMDARs differ in their subunit composition. Whereas NMDAR-EPSCs in visual cortex lose their sensitivity to NR2B-selective antagonists by P7, the extrasynaptic receptors are still blocked at this stage, suggesting that NR1/NR2B-containing receptors are present but are no longer targeted to the synapse [58]. Differences have also been noted between synaptic and extrasynaptic NMDARs in young cerebellar granule cells [23]. In
The presence of NR2C subunits in synaptic NMDARs
As described above, NMDARs containing NR2C (or NR2D) exhibit a low sensitivity to Mg2+. The functional significance of this reduced Mg2+ sensitivity has not been examined in detail, but it would be expected to allow these NMDARs to operate at more negative membrane potentials than conventional NR2A/B-containing receptors. This difference may explain, in part, the ability of antagonists with moderate selectivity for NR2A/B- or NR2C/D-containing receptors to differentially block long-term
NR1/NR2D receptors have been identified extrasynaptically, but not at the synapse
Although there is good evidence that diheteromeric NR2A-, NR2B- and NR2C-containing NMDARs participate in synaptic transmission, there is no evidence for NR1/NR2D-containing receptors at any central synapse, despite the fact that the distinctive single-channel properties of NR1/NR2D receptors have enabled their identification in the extrasynaptic membrane of several cell types 22., 59.. Recombinant NR1/NR2D receptors exhibit a remarkably slow macroscopic deactivation 8., 11., 12.. Therefore, if
Evidence for functionally distinct triheteromeric NMDARs in neurons
Molecular biological and immunoprecipitation studies have provided compelling evidence that some native NMDARs contain more than one type of NR2 subunit in the same assembly 14., 60., 61.. As described above, there is also evidence from studies of recombinant receptors that the NR3 subunit co-assembles with NR1/NR2 receptors to produce a functionally distinct triheteromeric NMDAR 7., 10.. However, the issue of whether triheteromeric assemblies represent a sizeable fraction of the synaptic
NMDAR diversity and disease
Inappropriate activation of NMDARs has been implicated in the aetiology of several disease states. In particular, excessive calcium influx through NMDARs can cause excitotoxic neuronal death, and thus blockade of NMDARs is neuroprotective in animal models of both stroke and seizure [65]. Stroke was, therefore, the first clinical indication considered for NMDAR antagonists, but the usefulness of most drugs was limited by their actions on normal synaptic transmission or by additional side
Conclusions
It is now possible able to make use of the characteristic biophysical and pharmacological properties of NMDARs to establish the subunit composition of many native subtypes. Recent studies using such approaches have described the targeting of particular NMDAR subtypes to specific locations in single cells, and have identified developmental changes occurring in the subunit composition of synaptic and extrasynaptic NMDARs.
Until fairly recently, only four types of functionally distinct
Update
Experience-dependent changes in the subunit composition of synaptic NMDARs have been shown to modify the temporal summation of EPSCs in the visual cortex, although the effect of these changes on neuronal integration and/or Ca2+ influx remains unknown [78]. Importantly, however, a temporal dissociation between changes in the pharmacology (subunit composition) and the kinetic behaviour of NMDARs seen during a critical period of development at thalamocortical synapses casts doubt on the idea that
Acknowledgements
We are grateful to the Wellcome Trust for support, and to our colleagues for many helpful discussions that have contributed to this article.
References and recommended reading
Papers of particular interest, published within the annual period of review,have been highlighted as:
• of special interest
•• of outstanding interest
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