MAGUKs are proteins that act as key scaffolds in surface complexes containing receptors, adhesion proteins, and various signaling molecules. These complexes evolved prior to the appearance of multicellular animals and play key roles in cell-cell intercommunication. A major example of this is the neuronal synapse, which contains several presynaptic and postsynaptic MAGUKs including PSD-95, SAP102, SAP97, PSD-93, CASK, and MAGIs. Here, they play roles in both synaptic development and in later synaptic plasticity events. During development, MAGUKs help to organize the postsynaptic density via associations with other scaffolding proteins, such as Shank, and the actin cytoskeleton. They affect the clustering of glutamate receptors and other receptors, and these associations change with development. MAGUKs are involved in long-term potentiation and depression (e.g., via their phosphorylation by kinases and phosphorylation of other proteins associated with MAGUKs). Importantly, synapse development and function are dependent on the kind of MAGUK present. For example, SAP102 shows high mobility and is present in early synaptic development. Later, much of SAP102 is replaced by PSD-95, a more stable synaptic MAGUK; this is associated with changes in glutamate receptor types that are characteristic of synaptic maturation.

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission and synaptic plasticity as well as excitotoxicity. They have several binding sites for allosteric modulators, including neurosteroids, endogenous compounds synthesized by the nervous tissue and expected to act locally. Whole-cell patch-clamp recording from human embryonic kidney 293 cells expressing NR1-1a/NR2B receptors revealed that neurosteroid pregnenolone sulfate (PS) (300 μm), when applied to resting NMDA receptors, potentiates the amplitude of subsequent responses to 1 mmglutamate fivefold and slows their deactivation twofold. The same concentration of PS, when applied during NMDA receptor activation by 1 mmglutamate, has only a small effect. The association and dissociation rate constants of PS binding and unbinding from resting NMDA receptors are estimated to be 3.3 ± 2.0 mm^-1sec^-1and 0.12 ± 0.02 sec^-1, respectively, corresponding to an apparent affinityK_dof 37 μm. The results of experiments indicate that the molecular mechanism of PS potentiation of NMDA receptor responses is attributable to an increase in the peak channel open probability (P_o). Responses to glutamate recorded in the continuous presence of PS exhibit marked time-dependent decline. Our results indicate that the decline is induced by a change of the NMDA receptor affinity for PS after receptor activation.These results suggest that the PS is a modulator of NMDA receptorP_o, the effectiveness of which is lowered by glutamate binding. This modulation may have important consequences for the neuronal excitability.

Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids (“empty” or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both “empty” vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. There are three distinct subtypes of ionotropic glutamate receptors that have been identified including 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptors (AMPARs), N-methyl-D-aspartate receptors (NMDARs) and kainate receptors. The most common glutamate receptors in mature synapses are AMPARs that mediate the fast excitatory neurotransmission and NMDARs that mediate the slow excitatory neurotransmission. There have been large numbers of recent reports studying how a single neuron regulates synaptic numbers and types of AMPARs and NMDARs. Our current research is centered primarily on NMDARs and, therefore, we will focus in this review on recent knowledge of molecular mechanisms occurring 1) early in the biosynthetic pathway of NMDARs, 2) in the transport of NMDARs after their release from the endoplasmic reticulum, and 3) at the plasma membrane including excitatory synapses. Because a growing body of evidence also indicates that abnormalities in NMDAR functioning are associated with a number of human psychiatric and neurological diseases, this review together with other chapters in this issue may help to enhance research and to gain further knowledge of normal synaptic physiology as well as of the etiology of many human brain diseases.

BACKGROUND AND PURPOSE: NMDA receptors are glutamatergic ionotropic receptors involved in excitatory neurotransmission, synaptic plasticity and excitotoxic cell death. Many allosteric modulators can influence the activity of these receptors positively or negatively, with behavioural consequences. 20‐Oxo‐5β‐pregnan‐3α‐yl sulphate (pregnanolone sulphate; PA‐6) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective. We tested the hypothesis that the interaction of PA‐6 with the plasma membrane is critical for its inhibitory effect at NMDA receptors.EXPERIMENTAL APPROACH: Electrophysiological recordings and live microscopy were performed on heterologous HEK293 cells expressing GluN1/GluN2B receptors and cultured rat hippocampal neurons.KEY RESULTS: Our experiments showed that the kinetics of the steroid inhibition were slow and not typical of drug‐receptor interaction in an aqueous solution. In addition, the recovery from steroid inhibition was accelerated by β‐ and γ‐cyclodextrin. Values of IC₅₀assessed for novel synthetic C3 analogues of PA‐6 differed by more than 30‐fold and were positively correlated with the lipophilicity of the PA‐6 analogues. Finally, the onset of inhibition induced by C3 analogues of PA‐6 ranged from use‐dependent to use‐independent. The onset and offset of cell staining by fluorescent analogues of PA‐6 were slower than those of steroid‐induced inhibition of current responses mediated by NMDA receptors.CONCLUSION AND IMPLICATIONS: We conclude that steroid accumulation in the plasma membrane is the route by which it accesses a binding site on the NMDA receptor. Thus, our results provide a possible structural framework for pharmacologically targeting the transmembrane domains of the receptor.