Raymond Dingledine

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.

Identification of a site in glutamate receptor subunits that controls calcium permeability

The neurotransmitter glutamate mediates excitatory synaptic transmission throughout the brain. A family of genes encoding subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor has been cloned. Some combinations of these subunits assemble into receptors with a substantial permeability to calcium, whereas others do not. To investigate the structural features that control ion permeation through these ligand-gated channels, mutant receptor subunits with single-amino acid changes were constructed. Mutation of a certain amino acid that results in a net charge change (from glutamine to arginine or vice versa) alters both the current-voltage relation and the calcium permeability of non-NMDA receptors. A site has thus been identified that regulates the permeation properties of these glutamate receptors.

Excitatory amino acid receptors in epilepsy

Excitatory amino acid transmitters participate in normal synaptic transmission throughout the CNS (see Headley and Grillner, May TiPS), so it comes as no surprise that such excitatory pathways are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using NMDA receptors, although more recent evidence indicates potential roles for the AMPA receptors as well. In this article--the first of two to focus on the neurological dangers inherent in excitatory amino acid pathways--Raymond Dingledine, Chris McBain and James McNamara consider their involvement in epilepsy; next months article will cover brain damage following ischemia and hypoxia.

Two different responses of hippocampal pyramidal cells to application of gamma-amino butyric acid.

1. Extra‐ and intracellular recordings were made from CA1 cells in hippocampal slices in vitro. The effects of ionophoretically applied GABA on somatic and dendritic regions were studied. 2. Ionophoresis of GABA at dendritic sites gave a reciprocal effect by inhibiting the effect of excitatory synapses close to the dendritic application, while facilitating those lying further away. For example, GABA delivered to the mid‐radiatum dendritic region reduced the population spike generated by a radiatum volley, while facilitating the population spike evoked by oriens fibre stimulation. Similarly, when single cells were recorded from, mid‐apical dendritic delivery of GABA abolished the synaptically driven discharges evoked by fibres terminating at this part of the dendritic tree, but facilitated the responses to input from fibres terminating on the basal dendrites of the same cell. 3. With intracellular recording two effects were observed. Applied near the soma, GABA induced a hyperpolarization associated with an increased membrane conductance. When applied to dendrites, GABA caused a depolarization also associated with an increased membrane conductance. Both types of GABA applications could inhibit cell discharges, although in some cases the depolarizing response could facilitate other excitatory influences or cause cell firing by itself. 4. Both the hyperpolarizing and depolarizing GABA responses persisted after blockade of synaptic transmission by applying a low calcium high magnesium solution, indicating mediation via a direct effect upon the cell membrane. 5. The reversal potential for the hyperpolarizing GABA effect was similar to the equilibrium potential for the i.p.s.p. evoked from alveus or orthodromically, and was 10‐12 mV more negative than the resting potential. The size of the depolarizing response was also dependent upon the membrane potential. By extrapolation an estimated equilibrium potential was calculated as about ‐40 mV. 6. Our results support the idea that the hyperpolarizing basket cell inhibition at the soma is mediated by the release of GABA. This hyperpolarizing response causes a general inhibition of firing. The dendritic effects of GABA, however, seem to represent another type of inhibition, which by shunting synaptic currents makes possible a selective inhibitory influence on afferents synapsing locally while facilitating more remotely placed excitatory synapses. We propose the term discriminative inhibition for this postulated new type of control of pyramidal cell discharges.

Transcriptional repression by REST: recruitment of Sin3A and histonedeacetylase to neuronal genes

Many genes whose expression is restricted to neurons in the brain contain a silencer element (RE1/NRSE) that limits transcription in nonneuronal cells by binding the transcription factor REST (also named NRSF or XBR). Although two independent domains of REST are known to confer repression, the mechanisms of transcriptional repression by REST remain obscure. We provide multiple lines of evidence that the N-terminal domain of REST represses transcription of the GluR2 and type II sodium-channel genes by recruiting the corepressor Sin3A and histone deacetylase (HDAC) to the promoter region in nonneuronal cells. These results identify a general mechanism for controlling the neuronal expression pattern of a specific set of genes via the RE1 silencer element.

Naloxone as a GABA antagonist: Evidence from iontophoretic, receptor binding and convulsant studies

From the following three lines of evidence, it is proposed that at least part of the convulsant activity of naloxone is a result of GABA receptor blockade. Firstly, iontophoretic naloxone reversibly antagonized GABA-evoked depression of firing rate in 21 of 27 neurons tested in the rat olfactory tubercle-nucleus accumbens region, without blocking inhibition evoked in the same cells by glycine (15 cells) or morphine (6 cells). Secondly, i.p. naloxone in high doses caused convulsions in mice, and potentiated the convulsant activity of bicuculline, but not that of strychnine. Diazepam, which protected mice against convulsions elicited by bicuculline, but not by strychnine, also protected mice against naloxone. Thirdly, naloxone, morphine, levorphanol and its non-analgesic enantiomer dextrorphan displaced 3H-GABA from GABA receptor sites in homogenates of human cerebellum, all with comparable low potencies (IC50 = 250--400 micron). There was no correlation with affinities at the stereospecific receptor sites that mediate opiate-induced analgesia, since the potent opiates etorphine and diprenorphine were relatively inactive (IC50 greater than 3 mM). In addition naloxone displaced 3H-GABA from receptor sites in rate forebrain and cerebellum, with similar low potency.

Involvement of N-methyl-D-aspartate receptors in epileptiform bursting in the rat hippocampal slice.

The effects of the N‐methyl‐D‐aspartate (NMDA) receptor antagonist, D‐2‐amino‐5‐phosphonovaleric acid (D‐APV), and other excitatory amino acid antagonists, were studied on CA1 pyramidal neurones treated with picrotoxin or bicuculline to reduce synaptic inhibition mediated by gamma‐aminobutyric acid (GABA). Under these conditions epileptiform burst firing is readily produced by orthodromic stimulation of the pyramidal cell population. D‐APV reduced the plateau amplitude and duration of the depolarization underlying evoked and spontaneous bursts without affecting membrane potential, input resistance or the ability of the cell to fire a Ca2+ spike or a short train of Na+ spikes. A late component of the subthreshold excitatory post‐synaptic potential (e.p.s.p.) was voltage dependent, being reduced in amplitude on membrane hyperpolarization. D‐APV selectively removed this component of the e.p.s.p. in disinhibited slices. In contrast, in the absence of GABA antagonists, D‐APV had no noticeable effect on the e.p.s.p. as studied with field potential recordings. The concentration‐response relationship of the inhibitory effect of D‐APV and L‐APV on population spike bursts was studied. The action of APV was highly stereoselective; the EC50 of D‐APV was approximately 700 nM, whereas a similar inhibition required 540 microM‐L‐APV. A number of other excitatory amino acid antagonists were tested at a fixed concentration (100 microM). Among them, the quisqualate antagonist gamma‐D‐glutamylaminomethyl sulphonic acid was ineffective against epileptiform bursts. In the low nanomolar concentration range both D‐ and L‐APV potentiated bursting. These results suggest that in the absence of GABAergic inhibition, a significant component of the slow depolarization underlying burst firing is voltage dependent, synaptic in origin and mediated by NMDA receptors. We propose that, under normal (non‐epileptic) physiological conditions, the balance between synaptic inhibition mediated by GABA receptors and synaptic excitation mediated by NMDA receptors may modulate the excitability of pyramidal cell dendrites.

Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic diet

The full anticonvulsant effect of the ketogenic diet (KD) can require weeks to develop in rats, suggesting that altered gene expression is involved. The KD typically is used in pediatric epilepsies, but is effective also in adolescents and adults. Our goal was to use microarray and complementary technologies in adolescent rats to understand its anticonvulsant effect.

Differential Dependence on GluR2 Expression of Three Characteristic Features of AMPA Receptors

The GluR2 subunit controls three key features of ion flux through the AMPA subtype of glutamate receptors—calcium permeability, inward rectification, and channel block by external polyamines, but whether each of these features is equally sensitive to GluR2 abundance is unknown. The relations among these properties were compared in native AMPA receptors expressed by acutely isolated hippocampal interneurons and in recombinant receptors expressed by Xenopusoocytes. The shape of current–voltage (I–V) relations between −100 and +50 mV for either recombinant or native AMPA receptors was well described by a Woodhull block model in which the affinity for internal polyamine varied over a 1000-fold range in different cells. In oocytes injected with mixtures of GluR2:non-GluR2 mRNA, the relative abundance of GluR2 required to reduce the log of internal blocker affinity by 50% was two- to fourfold higher than that needed to half-maximally reduce divalent permeability or channel block by external polyamines. Likewise, in interneurons the affinity of externally applied argiotoxin for its blocking site was a steep function of internal blocker affinity. These results indicate that the number of GluR2 subunits in AMPA receptors is variable in both oocytes and interneurons. More GluR2 subunits in an AMPA receptor are required to maximally reduce internal blocker affinity than to abolish calcium permeability or external polyamine channel block. Accordingly, single-cell RT-PCR showed that approximately one-half of the physiologically characterized interneurons exhibiting inwardly rectifying AMPA receptors expressed detectable levels of edited GluR2. The physiological effects of a moderate change in GluR2 relative abundance, such as occurs after ischemia or seizures or after chronic exposure to morphine, thus will be dependent on the ambient GluR2 level in a cell-specific manner.

Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model.

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.