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The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDARdependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.

γ-Aminobutyric acid type A (GABA A ) receptor interacting factor-1 (GRIF-1) was originally discovered as a result of studies aiming to find the elusive GABA A receptor clustering protein. It was identified as a GABA A receptor associated protein by virtue of its specific interaction with the GABA A receptor β2 subunit intracellular loop in a yeast two-hybrid screen of a rat brain cDNA library. Further work however, established that GRIF-1, now known as trafficking kinesin protein 2 (TRAK2), is a member of the TRAK family of kinesin adaptor proteins. A pivotal role for TRAK1 and TRAK2 in the transport of mitochondria is well recognized. Notwithstanding this progress, there is a body of evidence that still supports a role for TRAKs in the intracellular transport of GABA A receptors. This is critically reviewed in this article.

A new N-methyl D aspartate neurotransmitter receptor interacting protein has been identified by yeast two-hybrid screening of a mouse brain cDNA library. C-terminal binding protein 1 (CtBP1) was shown to associate with the intracellular C-terminal regions of the N-methyl D aspartate receptor subunits GluN2A and GluN2D but not with GluN1-1a cytoplasmic C-terminal region. In yeast mating assays using a series of GluN2A C-terminal truncations, it was demonstrated that the CtBP1 binding domain was localized to GluN2A 1157–1382. The GluN2A binding domain was identified to lie within the CtBP1 161–224 region. CtBP1 co-immunoprecipitated with assembled GluN1/GluN2A receptors expressed in mammalian cells and also, in detergent extracts of adult mouse brain. Co-expression of CtBP1 with GluN1/GluN2A resulted in a significant decrease in receptor cell surface expression. The family of C-terminal binding proteins function primarily as transcriptional co-repressors. However, they are also known to modulate intracellular membrane trafficking mechanisms. Thus the results reported herein describe a putative role for CtBP1 in the regulation of cell surface N-methyl D aspartate receptor expression.

The basis for differences in activity-dependent trafficking of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) remains unclear. Using single-molecule tracking, we found different lateral mobilities for AMPARs and NMDARs: changes in neuronal activity modified AMPAR but not NMDAR mobility, whereas protein kinase C activation modified both. Differences in mobility were mainly detected for extrasynaptic AMPARs, suggesting that receptor diffusion between synaptic and extrasynaptic domains is involved in plasticity processes.

Expression of a form of GFP-Miro in which the two EF-hand calcium binding domains were inactivated by mutation i.e., GFP-Miro1 ΔEF, did not change basal mitochondrial dendritic transport but importantly, glutamate-induced arrest did not occur. [...]rather than perfusing with glutamate, local stimulation of neurones with glutamate delivered via a patch pipette resulted in the halting of mitochondrial movement within a 15 µm region for 150 s demonstrating that synaptic activation results in the recruitment of passing mitochondria to synapses. [...]they showed that introduction of a kinesin function-blocking antibody into neurones resulted in a decrease in the percentage of mobile mitochondria proving the engagement of motor proteins. Co-immunoprecipitations and pull downs from extracts of rat brain ratified these in vitro findings. [...]it was concluded that the presence of Ca2+ inhibits the Miro1/kinesin protein– protein interaction hence the motor is dissociated from mitochondria yielding arrested movement.

γ-aminobutyric acid has become one of the most widely known neurotransmitter molecules in the brain over the last 50 years, recognised for its pivotal role in inhibiting neural excitability. It emerged from studies of crustacean muscle and neurons before its significance to the mammalian nervous system was appreciated. Now, after five decades of investigation, we know that most neurons are γ-aminobutyric-acid-sensitive, it is a cornerstone of neural physiology and dysfunction to γ-aminobutyric acid signalling is increasingly documented in a range of neurological diseases. In this review, we briefly chart the neurodevelopment of γ-aminobutyric acid and its two major receptor subtypes: the γ-aminobutyric acidA and γ-aminobutyric acidB receptors, starting from the humble invertebrate origins of being an ‘interesting molecule’ acting at a single γ-aminobutyric acid receptor type, to one of the brain’s most important neurochemical components and vital drug targets for major therapeutic classes of drugs. We document the period of molecular cloning and the explosive influence this had on the field of neuroscience and pharmacology up to the present day and the production of atomic γ-aminobutyric acidA and γ-aminobutyric acidB receptor structures. γ-Aminobutyric acid is no longer a humble molecule but the instigator of rich and powerful signalling processes that are absolutely vital for healthy brain function.

Eric Barnard was a protein biochemist who played a leading role in the delineation of the molecular components of neuromuscular transmission and the emergence of molecular neuroscience as a scientific discipline. He began his career at King's College London, moving to the State University of Buffalo, New York, in 1965 before returning to Imperial College, London, in 1975. In 1985 he became the Director of the Medical Research Council (MRC) Molecular Neurobiology Unit in Cambridge. Upon retirement from the MRC, he moved to the Royal Free Hospital in London where he continued as Director of Molecular Neurobiology, but in 1998 returned to the University of Cambridge (Department of Pharmacology) as Emeritus Professor. In 2014, at the age of 86, he finally retired from active research. Although Eric was elected FRS for his early pioneering work on the protein chemistry of enzymes and the nicotinic acetylcholine receptor, his seminal contribution, initiated during his time at Imperial, was the application of molecular biological methods to the study of many neurotransmitter receptors. With Ricardo Miledi FRS (and later David Brown FRS and colleagues), he developed the Xenopus oocyte system for the expression of receptors from total tissue mRNA. His was the first group to clone a neurotransmitter receptor subunit cDNA, the nicotinic acetylcholine receptor α subunit of Torpedo marmorata . This was followed by purification and subsequent cloning of inhibitory γ-aminobutyric acid (GABA) A receptor subunit cDNAs. This achievement, driven by Eric and aided by his collaborator Peter Seeburg, led to the discovery of the ligand-gated ion channel superfamily, the discovery of neurotransmitter receptor heterogeneity, and the development of concepts of receptor families and superfamilies. His pioneering work was pivotal for the foundation of modern central nervous system drug discovery.

Issue Title: Special Issue Dedicated to Richard W. Olsen γ-Aminobutyric acid type A (GABA^sub A^) receptor interacting factor-1 (GRIF-1) was originally discovered as a result of studies aiming to find the elusive GABA^sub A^ receptor clustering protein. It was identified as a GABA^sub A^ receptor associated protein by virtue of its specific interaction with the GABA^sub A^ receptor [beta]2 subunit intracellular loop in a yeast two-hybrid screen of a rat brain cDNA library. Further work however, established that GRIF-1, now known as trafficking kinesin protein 2 (TRAK2), is a member of the TRAK family of kinesin adaptor proteins. A pivotal role for TRAK1 and TRAK2 in the transport of mitochondria is well recognized. Notwithstanding this progress, there is a body of evidence that still supports a role for TRAKs in the intracellular transport of GABA^sub A^ receptors. This is critically reviewed in this article.[PUBLICATION ABSTRACT]

The potency of two novel glycine site antagonists, GV150,526A and GV196,771A, was assessed by their ability to inhibit the binding of [3H]‐MDL105,519 to cell homogenates prepared from mammalian cells transfected with either NR1‐1a, NR1‐2a, NR1‐1a/NR2A, NR1‐1a/NR2B, NR1‐1a/NR2C or NR1‐1a/NR2D NMDA receptor clones. The inhibition constants (Kis) for GV150,526A displacement of [3H]‐MDL105,519 binding to either NR1‐1a or NR1‐2a expressed alone were not significantly different and were best fit by a one‐site binding model. GV150,526A inhibition to NR1‐1a/NR2 combinations was best fit by a two‐site model with the NR1‐1a/NR2C having an approximate 2–4 fold lower affinity compared to other NR1‐1a/NR2 receptors. The Kis for GV196,771A displacement of [3H]‐MDL105,519 binding to NR1‐1a, NR1‐2a and all NR1‐1a/NR2 combinations was best fit by a two‐site binding model. There was no significant difference between the Kis for the binding to NR1‐1a and NR1‐2a; NR1‐1a/NR2A receptors had an approximate 4 fold lower affinity for GV196,771A compared to other NR1‐1a/NR2 combinations. The Kis for both GV150,526A and GV196,771A for the inhibition of [3H]‐MDL105,519 binding to membranes prepared from adult rat forebrain were determined and compared to the values obtained for binding to cloned NMDA receptors. The Kis for a series of glycine site ligands with diverse chemical structures were also determined for the inhibition of [3H]‐MDL105,519 binding to NR1‐1a/NR2A receptors. L689,560 displayed similar binding characteristics to GV150,526A. It is suggested that glycine site antagonists may be divided into two classes based on their ability to distinguish between NR1 and NR1/NR2 receptors with respect to binding curve characteristics. British Journal of Pharmacology (2000) 130, 65–72; doi:10.1038/sj.bjp.0703298