Molecular aspects of glutamate receptors and sodium-calcium exchange carriers in mammalian brain: Implications for neuronal development and degeneration

N-Methyl-d-aspartate (NMDA) andl-glutamate activate membrane receptor that produce substantial permeation of Na⁺, K⁺ and Ca²⁺ through the neuronal membrane. These ionic fluxes are intimately linked to processes that regulate neuronal survival, growth and differentiation. Intracellular free Ca²⁺ concentrations are thought to be particularly important determinants of the vulnerability of neurons to excessive excitatory stimulation produced through activation of NMDA receptors. In order to understand the molecular events involved in both NMDA receptor activation and regulation of intracellular Ca²⁺ levels, we have purified and reconstituted the protein complexes that form the NMDA/glutamate receptors in rat brain synaptic membranes and those that constitute the Na⁺-Ca²⁺ antiporters in bovine brain synaptic membranes. The molecular properties of these protein complexes are described, and information from the most recent studies of exploration of the molecular structures of these receptors and transport carriers is summarized.Special issue dedicated to Dr. Frederick E. Samson     

Nicotine receptors in the mammalian brain

Nicotine is a drug of abuse that presumably exerts its psychoactive effect through its interactions with nicotine binding sites in the central nervous system. Among its potential sites of action are the neuronal nicotinic acetylcholine receptors and the neuronal alpha-bungarotoxin binding sites. In this review we focus on the neuronal nicotinic acetylcholine receptors, their diversity, distribution, and functions as nicotine receptors or as mediators of synaptic transmission in the mammalian brain. We find that the complexity characteristic of the gene family encoding the subunits of these receptors is reflected both in the pattern of expression of the genes and in the pharmacological diversity of the expressed receptors.     

Molecular biology of mammalian amino acid receptors

The amino acid receptor proteins are ubiquitous transducers of most excitatory and inhibitory synaptic transmission in the brain. In July 1987 two reports appeared describing the molecular cloning of a pair of subunits of the GABAA receptor (7) and one subunit of the glycine receptor (13). These papers sparked wide interest and led quickly to the concept of a ligand-gated receptor-ion channel superfamily that includes nicotinic acetylcholine receptors as well as certain amino acid receptors. The identification of additional subunits of each receptor followed; with the recent cloning of a kainate receptor subunit (14), only the NMDA receptor remains elusive. Several disciplines have been brought to bear on these receptor clones, including in situ hybridization and functional expression in Xenopus laevis oocytes and mammalian cell lines. In this review we compare cloning strategies that have been used for amino acid receptors and discuss structural similarities among the receptor subunits. Two findings that have arisen from molecular cloning and expression of these receptors receive special attention. First, the molecular heterogeneity of GABAA receptors is larger than expected from pharmacological studies of native receptors. Second, although the native receptors are thought to be heterooligomers, much like the model proposed for the nicotinic receptors, some individual amino acid receptor subunits can form functional receptor channels, presumably in a homomeric configuration. This review focuses, therefore, on what we have learned from cloning efforts about amino acid receptors and what might lie ahead in this field.     

谷氨酸受体分子的多样性及其分子机理

谷氨酸是中枢神经系统一种重要的兴奋性神经递质,它与相应受体分子相互作用,通过细胞膜对阳离子通透性的改变或与G蛋白和第二信使系统相偶联,从而引起一系列复杂的信号转导反应。近年有关谷氨酸受体分子及其基因的研究表明:由于多基因家族、选择性剪接、RNA编辑以及异聚体形成等分子机理,使谷氨酸受体分子的结构和功能具有多样性,这种多样性是生物多样性的分子基础,也在微观水平上证明了生物多样性的原理。这方面的深入探索必将为中枢神经系统该受体表达及调控以及相关神经精神疾病发病的分子机理和治疗性药物设计提供新的线索。     

Glial glutamate receptors: likely actors in brain signaling.

It has become clear that the neurotransmitter glutamate does not confine its excitatory effects to central nervous system neurons but interacts also with glial cells. Neurons and glia share the same types of ionotropic and metabotropic glutamate receptors except for the N-methyl-D-aspartate receptor, which is not found on glia. Applied on cultured glial cells, glutamate regulates the opening of receptor channels, activates second messengers, and causes the release of neuroactive compounds. Although glutamate and glutamate receptors confer on cultured glia the ability to receive and emit signals, it remains to be established whether glial signaling takes place in vivo. The chick Bergmann glial cells provide a unique experimental system with which to test the contribution of glial glutamate receptors to neuronal electrical activity. These cells are the exclusive carriers in the cerebellum of functional kainate receptors. The synaptic location of these receptors, their ion channel properties, and their regulation by phosphorylation reactions suggest that glial kainate receptors play a role in regulating synaptic efficacy and plasticity. If proved, this concept may require a modification of the anatomical and functional definition of a synapse to include a glial component as well.     

Cell-selective effects of ammonia on glutamate transporter and receptor function in the mammalian brain

Increased brain ammonia concentrations are a hallmark feature of several neurological disorders including congenital urea cycle disorders, Reye's syndrome and hepatic encephalopathy (HE) associated with liver failure. Over the last decade, increasing evidence suggests that hyperammonemia leads to alterations in the glutamatergic neurotransmitter system. Studies utilizing in vivo and in vitro models of hyperammonemia reveal significant changes in brain glutamate levels, glutamate uptake and glutamate receptor function. Extracellular brain glutamate levels are consistently increased in rat models of acute liver failure. Furthermore, glutamate transport studies in both cultured neurons and astrocytes demonstrate a significant suppression in the high affinity uptake of glutamate following exposure to ammonia. Reductions in NMDA and non-NMDA glutamate receptor sites in animal models of acute liver failure suggest a compensatory decrease in receptor levels in the wake of rising extracellular levels of glutamate. Ammonia exposure also has significant effects on metabotropic glutamate receptor activation with implications, although less clear, that may relate to the brain edema and seizures associated with clinical hyperammonemic pathologies. Therapeutic measures aimed at these targets could result in effective measures for the prevention of CNS consequences in hyperammonemic syndromes.     

RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors

In the central nervous system, synaptic signal transduction depends on the regulation of neurotransmitter receptors by interacting proteins. Here, we searched for proteins interacting with two metabotropic glutamate receptor type 8 isoforms (mGlu8a and mGlu8b) and identified RanBPM. RanBPM is expressed in several brain regions, including the retina. There, RanBPM is restricted to the inner plexiform layer where it co-localizes with the mGlu8b isoform and processes of cholinergic amacrine cells expressing mGlu2 receptors. RanBPM interacts with mGlu2 and other group II and group III receptors, except mGlu6. Our data suggest that RanBPM might be associated with mGlu receptors at synaptic sites.     

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.     

Structural characteristics of ionotropic glutamate receptors revealed by channel blockade

The topography of the channel binding site in glutamate receptors (AMPA and NMDA types of rat brain neurons, receptors of molluscan neurons and insect muscle), and in two subtypes of nicotinic cholinoreceptors (in frog muscle and cat sympathetic ganglion), has been investigated by comparison of the blocking effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The channels studied can be divided into two groups. The first one includes AMPA receptor and glutamate receptors of mollusc and insect, and is characterised by the absence of activity of monocationic drugs and the strong dependence of dicationic once on the internitrogen distance in the drug molecule. The second group includes NMDA receptor and both nicotinic cholinoreceptors. Contrary, here the blocking potency of monocations and dications are practically equal irrespective of molecule length. The data obtained suggest that hydrophobic and nucleophilic components of the binding site are located close to each other in the channels of the NMDA receptor type but are separated by approximately 10 A in the AMPA receptor channel.     

Morphofunctional characteristics of the mammalian brain during increased calcium metabolism

The electron microscopic studies of the rat brain neocortex and neurosecretory structures have revealed that after the intensification of calcium metabolism by parathormone injection the destructive changes mainly occur in the structures involved in the maintenance of intracellular calcium homeostasis (mitochondria and microvesicles). The morphofunctional signs of decreased axonal terminal activity have been also shown by their over-excitability and suppression of conductivity.     

Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors

The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.     

Metabotropic glutamate receptors

Metabotropic glutamate receptors (mGlus) are a family of G-protein-coupled receptors activated by the neurotransmitter glutamate. Molecular cloning has revealed eight different subtypes (mGlu1-8) with distinct molecular and pharmacological properties. Multiplicity in this receptor family is further generated through alternative splicing. mGlus activate a multitude of signalling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of neurotransmitter release. In this review, we summarize anatomical findings (from our work and that of other laboratories) describing their distribution in the central nervous system. Recent evidence regarding the localization of these receptors in peripheral tissues will also be examined. The distinct regional, cellular and subcellular distribution of mGlus in the brain will be discussed in view of their relationship to neurotransmitter release sites and of possible functional implications.This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (R.S.) and by the Austrian Science Fund FWF (grant no. P16720 to F.F.).     

Molecular characteristics of receptors for atrial natriuretic factor

Specific, high-affinity receptors for atrial natriuretic factor (ANF) have been identified on membranes from a variety of tissues and cultured cells. By affinity labeling procedures, radioactivity from 125I-labeled ANF was specifically incorporated into three different polypeptides of ca. 120,000, 70,000, and 60,000 daltons, which may represent the binding subunits of ANF receptors. These polypeptides were present in varying amounts in different target tissues. In rat adrenal membranes, the 120,000- and 70,000-dalton peptides were specifically labeled whereas in A10 rat smooth muscle cells, only the 60,000-dalton peptide was labeled. Membranes from rat kidney and rabbit aorta contain all three peptides. Gel filtration chromatography of solubilized receptors suggested that intact ANF receptors are large molecular complexes with apparent molecular masses in the range of 250,000-350,000 daltons. The differential labeling pattern observed with the various tissues suggested that there might be at least two different receptors composed of unique ANF-binding polypeptides.     

Ionotropic glutamate receptors

In the brain, most fast excitatory synaptic transmission is mediated through L-glutamate acting on postsynaptic ionotropic glutamate receptors. These receptors are of two kinds—the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) and theN-methyl-D-aspartate (NMDA) receptors, which are thought to be colocalized onto the same postsynaptic elements. This excitatory transmission can be modulated both upward and downward, long-term potentiation (LTP) and long-term depression (LTD), respectively. Whether the expression of LTP/LTD is pre-or postsynaptically located (or both) remains an enigma. This article will focus on what postsynaptic modifications of the ionotropic glutamate receptors may possibly underly long-term potentiation/depression. It will discuss the character of LTP/LTD with respect to the temporal characteristics and to the type of changes that appears in the non-NMDA and NMDA receptor-mediated synaptic currents, and what constraints these findings put on the possible expression mechanism(s) for LTP/LTD. It will be submitted that if a modification of the glutamate receptors does underly LTP/LTD, an increase/decrease in the number of functional receptors is the most plausible alternative. This change in receptor number will have to include a coordinated change of both the non-NMDA and the NMDA receptors.