The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.The N-methyl-D-aspartate (NMDA) 2 receptor subtype of the glutamate receptor family are hetero-oligomeric proteins composed of three classes of receptor subunits: NR1, NR2, and NR3. The NR1 subunit is encoded by a single gene, which undergoes extensive splicing to generate eight different splice variants that differ in regional distribution and functional properties (1). The NR2 subunit class consists of four different subtypes, NR2A-NR2D, encoded by four separate but closely related genes (reviewed in Ref. 1). The NR3 subunit class consists of two different subtypes, NR3A and NR3B (2-4). A number of studies of mammalian cell lines either permanently or transiently transfected with the NMDA receptor subunits have indicated that the NR1 subunit alone does not form glycine-glutamate responsive channels and requires the presence of NR2 (5-7).Other studies have shown that the NR1 and NR2 subunits contribute differently to the binding sites of a functional NMDA receptor. The NR1 subunit forms the glycine binding site (8, 9), and the NR2 subunit provides part of the glutamate binding site (10,11). Similar studies on the NR3 subunit family suggested that it could act as a dominant negative subunit that reduced channel conductances when associated with NR1 and NR2 (4, 12). However, several recent studies have suggested that NR3 can combine with NR1 alone to form an excitatory glycine channel in both transfected cells and the central nervous system (13-15). Thus, different combinations of the NMDA receptor subunits must coassemble to form functionally distinct ion channels.Current evidence indicates that the glutamate-activated ion channels are tetrameric. Biochemical and biophysical evidence from studies on ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors strongly support such a structure (16). These studies also i...

Plasma membrane Ca2ϩ ATPase 2 (PMCA2) is a fast, highly effective mechanism to control resting cytosolic Ca 2ϩ and Ca 2ϩ excursions in neurons and other excitable cells. The strong expression of PMCA2 in the cerebellum and the cerebellar behavioral deficits presented by PMCA2Ϫ/Ϫ knock-out mice all point to its importance for cerebellar circuit dynamics. Here, we provide direct functional evidence for the influence of presynaptic PMCA2-mediated Ca 2ϩ extrusion for short-term plasticity at cerebellar parallel fiber to Purkinje neuron synapses. Dramatic structural alterations to the Purkinje neurons in the absence of PMCA2 also suggest a strong influence of this fast PMCA2 isoform for development and maintenance of cerebellar function.

SUMMARYThe plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events (Strehler et al, 2007), its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre-and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull down approaches confirmed that PMCA2 interacts with the postsynaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its Cterminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre-and post-synaptic signalling complexes.

The cerebellum expresses one of the highest levels of the plasma membrane Ca 2+ ATPase, isoform 2 in the mammalian brain. This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex; i.e. the Purkinje neurons (PNs). Here we review recent evidence, including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2 (PMCA2) knockout mouse, to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour. These studies have also revealed that deletion of PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development, they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs. THE CEREBELLUM, THE PURKINJE NEURON AND THE IMPORTANCE OF CALCIUM DYNAMICS AT CEREBELLAR SYNAPSESThe cerebellum is a major centre for the integration of sensory and motor information in the brain and plays a central role in our ability to learn and refine motor tasks; the specialised function of the cerebellum allows us to execute motor tasks in a finely controlled but, at the same time, "unaware" manner that can still be improved by learning. For a detailed review of cerebellar function TOPIC HIGHLIGHTWorld J Biol Chem 2010 May 26; 1(5): 95-102 ISSN 1949-8454 (online)