A Primer on GABA and Glutamate

A <strong>Primer</strong> on GABA and GlutamateKenny J. Simansky, Ph.D.Objectives:1. Name the immediate precursor and enzyme responsible for the synthesis of GABAand describe the functional biochemical significance of the GABA shunt;2. Name the classes of GABAergic receptors and how they can be differentiatedpharmacologically;3. Describe in detail the GABA A supramolecular receptor complex, including theactions of GABA, benzodiazepines, barbiturates and picrotoxin on chloride conductance;4. Name at least four therapeutic actions of drugs that are believed to rely uponGABAergic mechanisms for their clinical effects;5. Describe the NMDA receptor for glutamate, including its neurotransmitter ligands,modulators, and the effects of receptor activation on ionic conductance; and6. Name at least two physiological functions or pathophysiological conditions forwhich glutamate receptors are targets for developing novel therapeutic agents.GABAGABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the mammalianCNS. It is widely distributed with concentrations of GABA 1,000-fold higher than those ofmonoamine neurotransmitters. Some of the numerous pathways in which GABA is believed toserve a role as an inhibitory transmitter include:• Long-axon tracts connecting regions of the basal ganglia, including a pathway to thesubstantia nigra; and• Interneurons within the cortex, within various limbic areas including hippocampus,amygdala and septum, within the basal ganglia and cerebellum, within the raphe nuclei and withinthe medulla and spinal cord.GABA is also colocalized within neurons with other classical neurotransmitters and peptides,such as with 5-HT in neurons of the dorsal raphe and with cholecystokinin in the cortex.I. Metabolism and Synaptic Regulation of GABASynthesis and degradation. GABA is synthesized, degraded and replenished by a series ofintegrated metabolic pathways that form a closed loop called the GABA shunt. Ultimately,glucose is the precursor source for almost all synthesis of GABA although the conversion ofglutamate by the enzyme glutamic acid decarboxylase (GAD) is the primary immediate pathwayfor forming GABA. The steps in the GABA shunt are:• α-Ketoglutarate, which is formed from glucose metabolism in the Krebs cycle, isconverted by GABA-T (α-oxoglutarate transaminase) into glutamic acid;• Glutamic acid is decarboxylated by GAD into GABA;• GABA is metabolized by GABA-T to form succinic semialdehyde; and

• Succinic semialdehyde is oxidized by succinic semialdehyde dehydrogenase (SSADH) intosuccinic acid which reenters the Krebs cycle.Synaptic activity. Depolarization of GABAergic neurons releases the neurotransmitter fromvesicles into the synaptic cleft. GABA acts at postsynaptic receptors and its action is terminated byreuptake into the presynaptic nerve terminal and also into proximal glial cells.• The GABA that is taken back up into GABAergic nerve terminals via the GABAtransporter can be reused as a neurotransmitter if it is not degraded• The GABA that is taken up via active transport into glia cannot be reconverted to GABAbecause glia do not contain GAD. However, the GABA can ultimately be restored toneuronal GABA through the glutamine loop.The glutamine loop. In this loop, glutamate that is formed as GABA is transaminated is convertedenzymatically to glutamine. The glutamine is returned to the GABAergic terminal where it isreconverted to glutamate. The glutamate is then decarboxylated by neuronal GAD to GABA.II.GABA ReceptorsThree classes of GABA receptors have been identified, GABA A , GABA B and GABA C . TheA and B classes appear to be the major classes in mammals and are certainly the best characterized.GABA A and GABA B receptors differ in their pharmacological profiles (i.e., agonists andantagonists), their structures, the transduction mechanisms for their cellular effects and theirrelevance as targets for therapeutic agents.The GABA A receptor is actually one binding site on a large complex containing multiple subunitswith a chloride channel at the core. Besides the chloride channel and the GABA A binding site forthe neurotransmitter, this large GABA A supramolecular receptor complex contains distinct bindingdomains for benzodiazepines, barbiturates, picrotoxin and neurosteroids. The complex isconsidered a ligand-gated, ionotropic receptor because the binding of GABA (the ligand) to itssite alters the conformation of the channel (i.e., opens the gate) thereby allowing chloride ions toflow down the electrochemical gradient. Especially in the adult brain, GABA stimulatespostsynaptic GABA A receptors to enhance chloride flux into the postsynaptic cell with the resultbeing a hyperpolarization of the neuron. In some cells of the spinal cord, however, chloridemoves out of the postsynaptic cell with the result being a depolarization of the neuron. Thisdepolarization inhibits the propogation of action potentials along the axon because of local changesin membrane conductance and shunting of excitatory currents by a process called primary afferentdepolarization. This process probably involves inactivation of Na + channels.GABA A receptors are assembled from five subunits and are therefore pentameric complexes.There are multiple families of these subunits (α, β, γ, δ) which are each encoded by separate genes.Thus, hundreds of subtypes of GABA A receptors are possible. The β subunits contain binding sitesfor GABA itself. The α subunits contain binding sites for benzodiazepines (BZDs) and binding ofBZDs to these sites is influenced by the presence of certain γ subunits, which are required forhighest affinity binding of these drugs. Thus, the minimum requirement for a GABA A receptorcomplex that is modulated maximally by BZDs is a combination containing at least an α, β and γ

subunit. The particular types of subunits (e.g., γ-2) to optimize this interaction are known, but thematerial will not be covered in this depth in this course.Important features of the GABA A complex are illustrated in the figure. Note the multiplesubunits and the different binding sites for GABA, BZDs, barbiturates, neurosteroids, anddihydropicrotoxinin. The binding of GABA to its site opens the channel for chloride. This bindingcan be mimicked by the directly-acting agonist, muscimol, and blocked competitively by theantagonist, bicuculline. Picrotoxin and related compounds directly block the channel and thereforenoncompetitively antagonize the actions of GABA. The BZD and other sites act primarily in anallosteric fashion to modulate the actions of GABA. Two benzodiazepine receptors have beenidentified (omega-1 and omega-2). For the purposes of this discussion, we will assume theneurochemistry of these two subtypes (i.e., effects on GABA-A conductance) is identical.Allosteric modulation of GABA A function by benzodiazepines. Benzodiazepines, such asdiazepam and alprazolam, do not directly open the chloride channel when they bind to the BZDsite. Instead, the binding of BZD agonists produces an allosteric modification in theconformation of the complex which enhances the ability of GABA to bind to its receptor site andpotentiates the ability of GABA to open the chloride channel. BZD agonists actually increase thefrequency at which the channel opens when GABA binds. The actions and interactions ofGABA and BZD agonists are depicted in the figure below, which shows the impediment to chloridemovement in the absence of GABA binding to its site (A), the opening of the channel when GABAdoes bind (B), the failure of BZD to open the channel when it binds in the absence of binding byGABA (C), and the synergistic interaction between BZD and GABA binding (D). Note that BZDbinding increases binding of GABA to GABA receptors and, conversely, GABA binding enhancesthe binding of BZD to its receptors (D).The GABA A supramolecular receptor complex

Allosteric modulation of GABA A function by benzodiazepinesBenzodiazepine positive modulators, antagonists and negative modulators. BZDs thatare used for treating anxiety positively modulate the actions of GABA. Other compounds havebeen either synthesized or isolated from brain tissue that negatively modulate the actions of GABAby reducing the ability of GABA to promote chloride movement via the GABA A complex.Antagonists, such as flumazenil, block the effects of both positive and negative modulators but donot allosterically modulate actions of GABA. A peptide, called diazepam binding inhibitor(DBI), that displaces diazepam from the BZD binding site, has been isolated from the brains ofrodents and humans. DBI, or a peptide fragment of DBI, acts as a negative modulator. In animalmodels, DBI produces an anxiogenic effect. Other related peptides and nonpeptide compoundshave been proposed to modulate GABA A function. It is interesting that the benzodiazepineantagonist, flumazenil, reverses neurological symptoms associated with hepatic encephalopathy inmany patients. Flumazenil is used more commonly for reversing overdosage from BZDs and forshortening the time until patients awaken after various procedures by antagonizing BZDs used asadjunctive agents in anesthesia (e.g., midazolam).

The identification of an endogenous peptide that purportedly modulates GABA Afunction is analogous to the isolation of opioid peptides that interact with receptors formorphine. The demonstration of receptors for exogenous substances always suggests,but does not prove, that endogenous ligands exist for those sites. The isolation ofligands for BZD receptors suggests that GABA function is normally modulatedallosterically in a physiological system that regulates responses to behavioral challenges.Other modulators of the GABA A complex. A number of structural classes of compoundsother than BZDs modulate GABA A function. These include barbiturates, the convulsant picrotoxinand certain steroids including neurosteroids and congeners of progesterone and corticosterone.andneurosteroids (see table that follows). Barbiturates are no longer considered appropriate foranxiolytic therapy because they are less efficacious, less anxioselective and less safe thanbenzodiazepines for that purpose. Zolpidem (Ambien®) and zaleplon are two nonbenzodiazepineagonists that bind to benzodiazepine receptors and are marketed for their hypnotic actions.See Table on next page for ligand interactions with GABA A receptors.

LIGANDS AND THEIR ACTIONS AT THE GABA A COMPLEXSITEAGONISTLIGAND/DRUG 1CELLULARACTION(S) 2ASSOCIATEDFUNCTION(S)GABA A receptorGABAMuscimolOpen chloride channelInhibit activity ortransmitter release ofneuron containing theGABA A receptorBenzodiazepineDiazepamFlunitrazepamIncrease the frequencyof channel openingsproduced by GABAAnxiolyticSedative/HypnoticAnticonvulsantBarbiturateLow to moderateligand concentrationsHigh concentrationsPhenobarbitalPentobarbitalProlong the duration ofchannel openingsproduced by GABASedative/HypnoticPossibly generalanestheticMay contribute toanticonvulsant actionsbut not primarymechanismDirectly open chloridechannelSteroidLow to moderateligand concenconcentrationsHigh concentrationsAlphaxalone(synthetic)VariousendogenoussteroidsProlong the durationand increase thefrequency of channelopenings produced byGABASedative/HypnoticGeneral anestheticAnticonvulsantPossibly anxiolyticPossibly analgesicDirectly open chloridechannelPicrotoxinPicrotoxinDihydropicrotoxininBlocks chloridechannel (reducesduration of channelopenings produced byGABA)Convulsant.See note, next page.

1 These are examples of ligands, other agonist ligands exist. Additionally, inverse agonists existfor the benzodiazepine receptor and probably the barbiturate and steroid sites. Antagonist toolsare available for GABA A (bicuculline, a convulsant) and benzodiazepine receptors (e.g.,flumazenil, see lecture on anxiolytics for clinical uses).2 Benzodiazepines, barbiturates and steroids also allosterically modulate each others' binding andcellular actions.GABA A vs. GABA B RECEPTORSRECEPTORNATURE OFTRANSDUCTIONAGONISTS ANTAGONISTS ROLE(S)GABA Aligand-gated ionchannelGABAmuscimolGABA B G-protein linked GABAbaclofenbicucullinephaclofensaclofen↑ Cl - conductance↑K + conductance↓Ca ++ conductanceNote: Other agonists exist for each site and a series of antagonists have defined GABA Bsubtypes. GABA A subtypes exist based upon different combinations of the subunits of the ionchannel/receptor complex. GABA B has been linked via G-protein to the inhibition of adenylylcyclase.Other GABA receptors. GABA B receptors are also responsible for inhibitory functions butthey are considered metabotropic because they are linked via a G-protein to second messengersto modulate ion conductances rather than linking directly to an ion channel. Activatingpresynaptic GABA B receptors decreases release of neurotransmitter, probably by decreasingCa ++ conductance. Activating postsynaptic GABA B receptors produces inhibitory postsynapticpotentials, probably by increasing K + conductance. The GABA B agonist, baclofen, is usedtherapeutically as an antispastic agent with a presumed locus of action in the spinal cord (seelecture on centrally acting muscle relaxants). Evidence suggests that baclofen may also reducecraving in human drug addicts.GABA C receptors are very simple ion pores for chloride conductance that havebeen found in a variety of vertebrates, including several mammalian species. These receptors aredefinitely located in the retina; other regions have not yet been explored in detail. GABA Creceptors are not modulated by benzodiazepines nor are they inhibited by bicuculline; agonistshave been identified but the therapeutic relevance of these receptors remains unknown.

GLYCINEGlycine was first described as an important inhibitory neurotransmitter in the mammalianspinal cord. Glycine also gates chloride channels but with a very different pharmacology fromGABA. The convulsant strychnine is a selective and potent antagonist at inhibitory glycinereceptors. Glycine also serves an important role as a coactivator of an excitatory amino acidreceptor for glutamate, termed the NMDA receptor.GLUTAMATEGlutamate and aspartate are the major excitatory neurotransmitters in the central nervoussystem. They serve functions other than their neurotransmitter actions and, not surprisingly, aredistributed fairly ubiquitously throughout the CNS. Some important pathways include a massivecorticostriatal tract that interacts with the nigrostriatal dopaminergic pathway in the striatum,corticothalamic innervation, connections of the pyramidal cells of the hippocampus to the limbicforebrain and probably corticospinal projections. The granule cells of the cerebellum and the dorsalhorn of the spinal cord (probably due to glutamate in primary afferent neurons) are amongnumerous nontelencephalic regions containing glutamate. Glutamate and aspartate are nonessentialamino acids that are synthesized from glucose and other precursors. Neurons and glia eachparticipate in synthesizing these excitatory amino acid (EAA) neurotransmitters. Glutamate that isreleased from nerve terminals is taken up by glial cells and converted to glutamine which is thencycled back to the neuronal terminal to be reconverted to glutamate. Recall that this process alsosupplies glutamate as the precursor for GABA within cells that produce GAD (see above).I. Glutamatergic ReceptorsBy convention, EAA receptors for glutamate and aspartate are referred to as glutamatereceptors. Two families of glutamate receptors exist. The ligand-gated channels contain threeclasses that are named the AMPA, NMDA (N-methyl-D-aspartate) and KA (kainate) receptors. Theligand-gated channels for glutamate are classified separately from the ligand-gated channels of thenicotinic superfamily (cholinergic nicotinic, serotonergic 5-HT 3 , gabaergic GABA A andglycinergic). Multiple other classes—not ligand gated channels—contain metabotropic receptors(mGluR's) because they are linked via G-proteins to their intracellular, cytoplasmic effectors.Considerable attention has been directed at developing experimental tools and therapeutic agents atglutamateric receptors, especially the channels.Activating AMPA and NMDA receptors increases cation flow. Opening of the channel inthese complexes increases permeability to the monovalent cations of sodium and potassium andalso permits the movement of calcium into the cell. The activated NMDA receptor channel ishighly permeable to calcium. Opening of the NMDA channel involves the movement ofmagnesium ions out of the channel. This ionic movement occurs secondary to local changes in themembrane potential. AMPA receptors are less permeant to calcium than NMDA receptors. Theelevated intracellular stores of Ca ++ activate numerous calcium-dependent enzymes.

The NMDA receptor. Probably the greatest interest focuses on the NMDA receptor, whichis highly regulated and unusual because two neurotransmitters (glutamate and glycine) serve asrequisite cotransmitters for receptor activation. The NMDA receptor is assembled from a family ofsubunits to form a complex with binding sites:• for glutamate, aspartate and N-methyl-D-aspartate• for glycine that is insensitive to blockade by strychnine but is antagonizedby a new series of antagonists;• within the cation channel that binds the psychotomimetic drug phencyclidine (PCP),the dissociative anesthetic ketamine and the experimental drug dizocilpine(MK-801), each of which blocks the channel in the open state;• for polyamines such as spermine and spermidine that are not essential for activatingNMDA receptors but which positively modulate the ability of glutamate and glycineto open the channel (they increase the frequency of openings); and• associated with the channel separately for Zn ++ , H + and Mg ++ ; for example, magnesiumbinds within the channel and changes in the resting membrane potential of the neuron canreduce the binding of Mg ++ thus permitting cation influxCa ++ Na +GlutamateGlycineMg ++The NMDA channel is a multimeric complexwith separate binding sites for two neurotransmittersand a cation pore that isblocked by magnesium ions.

Ca ++ Na +GlutamateMg ++Glycine++++Changes in the resting membrane potentialpermit Mg to “pop out” and concurrent glu+ gly stimulation increases cation influxCa ++ Na +GlutamateGlycineDizocilpine (MK-801), ketamine andphencyclidine (PCP) block noncompetitvelythe open state NMDA channel

Ca ++ Na +GlutamateGlycineDifferent competitive antagonists exist forthe two co-transmitter sites on the NMDAcomplexII.Clinical Relevance of the Actions of Glutamate and Other EAA NeurotransmittersNo existing therapeutic agents were developed on the basis of their interactions withglutamate or other EAA targets. Some evidence exists that the anticonvulsant barbiturates, such asphenobarbital, act partly by inhibiting glutamatergic mechanisms. The anticonvulsant felbamate(withdrawn due to toxicity) may have acted as an antagonist at the glycine receptor on the NMDAcomplex. Certainly, it is possible that other agents interact with EAA transmitter function but thatthese actions remain to be identified. Currently, however, EAA mechanisms are the focus of drugdiscovery programs. These programs are directed, for example, at creating novel therapies for:• Limiting or preventing ischemic cell damage in the CNS after stroke or cardiac arrestHypoxia depletes neuronal and glial energy stores leading to acidosis andrelease of highly reactive free radicals. The impaired cellular metabolismultimately alters membrane potentials leading to depolarization and therelease of glutamate. This release of glutamate occurs for a prolonged timeand at high levels. The glutamate activates postsynaptic AMPA and NMDAreceptors with the excessive increase in intracellular calcium leading toneurotoxicity. In models of stroke, a glutamate receptor antagonist(NMDA antagonist) has been reported to protect the hippocampus and striatum.

• Antiepileptic therapyThe activation of EAA receptors has been implicated in initiating and augmentingepileptic seizures. Seizures are thought to begin with excessive stimulation ofAMPA receptors and recruitment of NMDA receptors enhances their intensityand duration. AMPA antagonists prevent initiation and NMDA antagonistsreduce the magnitude and time interval of convulsive activity.• Controlling neurodegenerative diseasesDysregulated EAA activity has been suggested to mediate degenerative processesin certain progressive neurologic disorders. EAA antagonists are being exploredfor treating such neuropathologies.• Enhancement of learning and memoryModels testing the effects of drugs and neurotransmitters on learning and memoryhave implicated EAA receptors—especially NMDA receptors—in these functions,perhaps via a process identical or similar to long-term potentiation. Thus, drugsthat positively modulate AMPA or NMDA receptors may be developed ascognitive enhancers.