GLUTAMATE RECEPTORS

Glutamate receptors are numerous and highly complex; more than 20 glutamate receptors have been identified in the mammalian central nervous system. They fall into two main categories, ionotropic (voltage sensitive) and metabotropic (ligand sensitive). Each ionotropic or metabotropic receptor has three types, depending on binding specificity, ion permeability, conductance properties, and other factors. Each type has multiple subtypes (Table 2-1). Ionotropic receptors are fast acting and, once opened, can produce large changes in current flow even if the voltage difference across the membrane is small. After glutamate, as a ligand, binds to an ionotropic receptor, the receptor's channel undergoes a conformational change to allow an immediate influx of extracellular sodium among other ions and an efflux of potassium ions. This triggers membrane depolarization in the post-synaptic cell sufficient to induce signal transmission. One of the main glutamate ionotropic receptors, N-methyl D-aspartate (NMDA), is permeable to calcium ions in addition to sodium and potassium ions; calcium ions have both beneficial and toxic effects. The NMDA receptor is unusual because it is a coincidence detector; for the channel to open, glutamate must bind to the receptor and the post-synaptic cell must be depolarized because the channel is blocked by magnesium at physiological levels and only opens when the cell is depolarized.

TABLE 2-1

Glutamate Receptor Protein Subunit Composition and Properties.

Ionotropic receptor channels are formed by assemblies of heterotetrameric or homotetrameric protein subunits. The three types of ionotropic receptors are named after the ligand that expressly binds to one, but not to the other two: NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid. Once these ligands were discovered, many others, whether agonists or antagonists, were subsequently found (Lesage and Steckler, 2010). Although their properties differ somewhat, as do their anatomical distribution, glutamate receptors are best known for mediating glutamate's role in learning and memory through plasticity, or modification, of channel properties; enhanced glutamate neurotransmission; and gene expression (Barco et al., 2006). Not only are NMDA receptors highly expressed on neurons, but they are also expressed on astrocytes (Lee et al., 2010). The human brain's expansive capacity for plasticity, learning, memory, and recovery from injury is attributed to improvement in synaptic anatomy and physiology of NMDA signaling, most notably in the hippocampus and other regions of the mammalian CNS (Barco et al., 2006). The basic mechanisms underlying plasticity include neurogenesis, activity-dependent refinement of synaptic strength, and pruning of synapses.

Metabotropic glutamate receptors are slower acting; they exert their effects indirectly, typically through gene expression and protein synthesis. Those effects are often to enhance the excitability of glutamate cells, to regulate the degree of neurotransmission, and to contribute to synaptic plasticity (Lesage and Steckler, 2010). Once glutamate binds with a metabotropic receptor, the binding activates a post-synaptic membrane-bound G-protein, which, in turn, triggers a second messenger system that opens a membrane channel for signal transmission. The activation of the protein also triggers functional changes in the cytoplasm, culminating in gene expression and protein synthesis. There are three broad groups of glutamate metabotropic receptors, distinguished by their pharmacological and signal transduction properties. Altogether, a total of eight metabotropic glutamate receptor subtypes have been cloned thus far.

Group I metabotropic receptors are largely expressed on the postsynaptic membrane. They have been implicated in problems with learning and memory, addiction, motor regulation, and Fragile X syndrome (Niswender and Conn, 2010). Group II metabotropic receptors are situated not only on post-synaptic cells, but also on pre-synaptic cells, possibly to suppress glutamate transmission (Swanson et al., 2005). Their dual location may enable them to exert a greater degree of modulation of glutamate signaling (Lesage and Steckler, 2010). Dysfunction of group II metabotropic receptors have been implicated in anxiety, schizophrenia, and Alzheimer's disease. Group III metabotropic receptors, like group II, are pre-synaptic and inhibit neurotransmitter release. They are found within the hippocampus and hypothalamus and may play a role in Parkinson's disease and anxiety disorders (Swanson et al., 2005).

GLUTAMATE TRANSPORTERS

Tight regulation of extracellular glutamate concentrations at both the synapse and in extra-synpatic locations is critical for normal synaptic transmission and to prevent excitotoxicity. Glutamate transporters regulate glutamate concentrations and are situated on both pre- and post-synaptic neurons as well as on surrounding astrocytes, a type of glial cell (Kanai et al., 1994). Five excitatory amino acid transporters (EAATs), previously known as glutamate transporters, have been cloned: EAAT-1 to EAAT-5, with EAAT-2 expressed predominantly on cells in brain regions rich in glutamate (Eulenburg and Gomeza, 2010). It is widely accepted that glutamate transporters on glial cells are primarily responsible for maintaining extracellular glutamate concentrations. However, the presence of transporters on multiple cell types suggests a high level of cooperation (Eulenburg and Gomeza, 2010; Foran and Trotti, 2009; Tanaka, 2000).

Glial cells, most often astrocytes but also microglia and oligodendrocytes (Olive, 2009), perform a key role in modulating extracellular glutamate levels. Under normal conditions, glutamate is recycled continuously between neurons and glia in what is known as the glutamate–glutamine cycle. Excess glutamate in the synapse is taken up by glial cells via EAAT transporters, where it is converted to glutamine. Glutamine is then transported back into neurons, where it is reconverted to glutamate (Rothman et al., 2003). However, glial cells, under certain conditions, may also release glutamate by at least six mechanisms, one of which is reversal of uptake by glutamate transporters (Malarkey and Parpura, 2008). This kind of reverse transport may be involved in brain damage and stroke (Grewer et al., 2008). Finally, altered expression of EAAT-2 is found in amyotrophic lateral sclerosis. The disease is also marked by excess glutamate levels in the cerebral spinal fluid (Rothman et al., 2003). New therapies are being developed to interfere with this pathological process by targeting EAAT-2 on astrocytes (Rothstein et al., 1992).

Given the number of receptors and transporters, the range of cell types expressing them, the variety of regulatory controls, and the narrow concentration difference between normal synaptic function and excitotoxicity, many fundamental questions remain about how to choose potential pharmacological targets. One presenter at the workshop, Schoepp, raised a series of questions and concerns that addressed both biomarkers and choices of molecular target. The foremost concern was whether the biomarker could distinguish between normal physiology versus pathology. What kind of feedback mechanisms and crosstalk at the synaptic cleft must be considered, especially in light of the probable need for chronic dosing of any new medication? Long-term use of any medication might produce unexpected changes, with the potential for side effects or paradoxical effects. The greatest danger is the specter of any glutamate-related drug inducing excitotoxicity and its ramifications.