Regulation of γ-Aminobutyric Acid Synthesis in the Brain

Abstract: γ-Aminobutyric acid (GABA) is synthesized in brain in at least two compartments, commonly called the transmitter and metabolic compartments, and because reglatory processes must serve the physiologic function of each compartment, the regulation of GABA synthesis presents a complex problem. Brain contains at least two molecular forms of glutamate decarboxylase (GAD), the principal synthetic enzyme for GABA. Two forms, termed GAD₆₅ and GAD₆₇, are the products of two genes and differ in sequence, molecular weight, interaction with the cofactor, pyridoxal 5′-phosphate (pyridoxal-P), and level of expression among brain regions. GAD₆₅ appears to be localized in nerve terminals to a greater degree than GAD₆₇, which appears to be more uniformly distributed throughout the cell. The interaction of GAD with pyridoxal-P is a major factor in the short-term regulation of GAD activity. At least 50% of GAD is present in brain as apoenzyme (GAD without bound cofactor; apoGAD), which serves as a reservoir of inactive GAD that can be drawn on when additional GABA synthesis is needed. A substantial majority of apoGAD in brain is accounted for by GAD₆₅, but GAD₆₇ also contributes to the pool of apoGAD. The apparent localization of GAD₆₅ in nerve terminals and the large reserve of apo-GAD₆₅ suggest that GAD₆₅ is specialized to respond to short-term changes in demand for transmitter GABA. The levels of apoGAD and the holoenzyme of GAD (holoGAD) are controlled by a cycle of reactions that is regulated by physiologically relevant concentrations of ATP and other polyanions and by inorganic phosphate, and it appears possible that GAD activity is linked to neuronal activity through energy metabolism. GAD is not saturated by glutamate in synaptosomes or cortical slices, but there is no evidence that GABA synthesis in vivo is regulated physiologically by the availability of glutamate. GABA competitively inhibits GAD and converts holo- to apoGAD, but it is not clear if intracellular GABA levels are high enough to regulate GAD. There is no evidence of short-term regulation by second messengers. The syntheses of GAD₆₅ and GAD₆₇ proteins are regulated separately. GAD₆₇ regulation is complex; it not only is present as apoGAD₆₇, but the expression of GAD₆₇ protein is regulated by two mechanisms: (a) by control of mRNA levels and (b) at the level of translation or protein stability. The latter mechanism appears to be mediated by intracellular GABA levels.     

Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase

The interactions between glutamate decarboxylase (GAD) and its cofactor pyridoxal phosphate (PLP) play a key role in the regulation of GAD activity. The enzyme has two isoforms, GAD65 and GAD67. A comparison of binding constants, rate constants, and kinetic profiles for the formation of holoenzyme (holoGAD65 and holoGAD67) revealed that the two isoforms interact distinctively with the cofactor. GAD67 exhibits a higher binding constant for PLP binding, making it more difficult to dissociate PLP from holoGAD67 than holoGAD65. Meanwhile, PLP binding occurs at a much slower rate for GAD67 than GAD65, as evidenced by lower rate constants and a slower initial rate of the holoenzyme formation. Job's plots revealed a stoichiometry of 1:1 for PLP binding to GAD65 before and after the saturation level of PLP, while 1:2 for PLP binding to GAD67 prior to the saturation of PLP and 1:1 at the saturation level of PLP. These results suggested that the two binding sites of GAD65 exhibit similar affinities for PLP. In contrast, one binding site of GAD67 exhibits a significantly higher affinity for PLP than the other binding site. Based on these findings, it was proposed that a slower PLP binding to GAD67 than GAD65 and a less ease to dissociate PLP from holoGAD67 than holoGAD65 are important underlying factors. This attributes to GAD67 being more highly saturated by PLP and GAD65 being less saturated by PLP. A larger conformation change constant for GAD67 than GAD65 supported a significant conformational change induced by the initial PLP binding to GAD67, which affects the other binding site affinity of GAD67. The present studies provided valuable insights into distinctive properties between the two isoforms of GAD.     

ATP's impact on the conformation and holoenzyme formation in relation to the regulation of brain glutamate decarboxylase

To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed.     

Cofactor and tryptophan accessibility and unfolding of brain glutamate decarboxylase

Cofactor and tryptophan accessibility of the 65-kDa form of rat brain glutamate decarboxylase (GAD) was investigated by fluorescence quenching measurements using acrylamide, I-, and Cs+ as the quenchers. Trp residues were partially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than the apoenzyme (apoGAD). The fraction of exposed Trp residues were in the range of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs+ was less able to quench the fluorescence of pyridoxal-P in holoGAD. The cofactor was present in a positively charged microenvironment, making it accessible for interactions with anions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp residues upon pyridoxal-P binding strongly supported a significant conformational change in GAD. Fluorescence intensity measurements were also carried out to investigate the unfolding of GAD using guanidine hydrochloride (GdnHCl) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.     

Cofactor interactions and the regulation of glutamate decarboxylase activity

More than 50% of glutamate decarboxylase (GAD) in brain is present as apoenzyme. Recent work has opened the possibility that apoGAD can be studied in brain by labeling with radioactive cofactor. Such studies would be aided by a compound that inhibits specific binding. One possibility is 4-deoxy-pyridoxine 5-phosphate, a close structural analog of the cofactor pyridoxal 5-phosphate. The effects of deoxypyridoxine-P on the cyclic series of reactions that interconverts apo- and holoGAD was investigated and found to be consistent with simple competitive inhibition of the activation of apoGAD by pyridoxal-P. As expected from the cycle GAD was inactivated when incubated with glutamate and deoxypyridoxine-P even though cofactor was present, but no inactivation was observed with deoxypyridoxine-P in the absence of glutamate. Deoxypyridoxine-P also stabilized apoGAD against heat denaturation. These effects were quantitatively accounted for by a kinetic model of the apo-holoGAD cycle. Deoxypyridoxine-P inhibited the labeling by [³²P]pyridoxal-P of GAD isolated from rat brain. Hippocampal extracts were labeled with [³²P]pyridoxal-P and analyzed by SDS-polyacrylamide gel electrophoresis. Remarkably few bands were strongly labeled. The major labeled band (at 63 kDa) corresponded to one of the forms of GAD. Other strongly-labeled bands were observed at 65 kDa (corresponding to the higher molecular weight form of GAD) and at 69–72 kDa. Labeling of the 63- and 65-kDa bands was inhibited by deoxypyridoxine-P, but the 69–72 kDa bands were unaffected, suggesting that the latter were non-specifically labeled. The results suggest that the 63-kDa form of GAD makes up the majority of apoGAD in hippocampus.Special issue dedicated to Dr. Eugene Roberts.     

Two Forms of the γ-Aminobutyric Acid Synthetic Enzyme Glutamate Decarboxylase Have Distinct Intraneuronal Distributions and Cofactor Interactions

Glutamate decarboxylase (GAD) catalyzes the production of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter. The mammalian brain contains two forms of GAD, with Mrs of 67,000 and 65,000 (GAD67 and GAD65). Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals. In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate. In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme. We suggest that the relative levels of apo-GAD65 and holo-GAD65 in synaptic terminals may couple GABA production to neuronal activity.     

Regulatory properties of brain glutamate decarboxylase

1. Glutamate decarboxylase is a focal point for controlling gamma-aminobutyric acid (GABA) synthesis in brain. Several factors that appear to be important in the regulation of GABA synthesis have been identified by relating studies of purified glutamate decarboxylase to conditions in vivo. 2. The interaction of glutamate decarboxylase with its cofactor, pyridoxal 5'-phosphate, is a regulated process and appears to be one of the major means of controlling enzyme activity. The enzyme is present in brain predominantly as apoenzyme (inactive enzyme without bound cofactor). Studies with purified enzyme indicate that the relative amounts of apo- and holoenzyme are determined by the balance in a cycle that continuously interconverts the two. 3. The cycle that interconverts apo- and holoenzyme is part of the normal catalytic mechanism of the enzyme and is strongly affected by several probable regulatory compounds including pyridoxal 5'-phosphate, ATP, inorganic phosphate, and the amino acids glutamate, GABA, and aspartate. ATP and the amino acids promote apoenzyme formation and pyridoxal 5'-phosphate and inorganic phosphate promote holoenzyme formation. 4. Numerous studies indicate that brain contains multiple molecular forms of glutamate decarboxylase. Multiple forms that differ markedly in kinetic properties including their interactions with the cofactor have been isolated and characterized. The kinetic differences among the forms suggest that they play a significant role in the regulation of GABA synthesis.     

Structure of glutamate decarboxylase from E. coli: spectral studies

Structure of recombinant glutamate decarboxylase (GAD alpha) was studied by optical methods and electron microscopy. The active (pH 4.6) and inert (pH 6.3) holoGAD and apoGAD were investigated. Absorption and CD spectra were recorded in the range of 190 - 500 nm. Visible spectra were resolved into the bands corresponding to individual electron transitions using lognormal curves. The structures of predominant tautomers of internal aldimines were determined as ketoenamine at pH 4.6 and enolimine at pH 6.3. CD spectra show that holoGAD and apoGAD exhibit a negative band at 204 - 245 nm and a positive band near 190 - 204 nm. The contents of the secondary structure elements were estimated on the basis of the values of the mean residue ellipticity. Evidently, the main difference between the GAD forms studied is in the content of alpha-helix and random coil. HoloGAD has 50% of alpha-helix at pH 4.6 and 67% at pH 6.3, whereas apoGAD - 17 and 27%, respectively. Thus presented data establish the essential role of pyridoxal phosphate (PLP) in the organization of the GAD secondary structure due to tightening its polypeptide chain. It seems possible, that conformational changes induced by PLP binding stabilize the protein structure and promote the assembly of subunits into macromolecule, which was confirmed by electron microscopy.     

Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo

In this study we tested the hypothesis that the 65-kDa isoform of glutamate decarboxylase (GAD(65)) mediates activity-dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA-transaminase inhibition by gabaculine using spatially localized (1)H NMR spectroscopy before and after bicuculline-induced seizures. Experiments were conducted with animals pre-treated with vigabatrin 24 h earlier in order to reduce GAD(67) protein and also with non-treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin-treated rats compared to non-treated controls. In vigabatrin-treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin-treated, 0.043 +/- 0.011 micromol/(g min); non-treated, 0.060 +/- 0.014 micromol/(g min)] and GAD(67) was 60% lower. No difference between groups was observed for GAD(65). Seizures increased GABA synthesis in both control [174%; control, 0.060 +/- 0.014 micromol/(g min) vs. seizures, 0.105 +/- 0.043 micromol/(g min)] and vigabatrin-treated rats [214%; control, 0.043 +/- 0.011 micromol/(g min); seizures, 0.092 +/- 0.018 micromol/(g min)]. GAD(67) could account for at least half of basal GABA synthesis but only 20% of the two-fold increase observed in vigabatrin-treated rats during seizures. The seizure-induced activation of GAD(65) in control cortex occurs concomitantly with a 2.3-fold increase in inorganic phosphate, known to be a potent activator of apoGAD(65)in vitro. Our results are consistent with a major role for GAD(65) in activity-dependent GABA synthesis.     

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.     

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.     

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.     

Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.     

Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.     

Mammalian retinal horizontal cells are unconventional GABAergic neurons

Lateral interactions at the first retinal synapse have been initially proposed to involve GABA by transporter-mediated release from horizontal cells, onto GABA(A) receptors expressed on cone photoreceptor terminals and/or bipolar cell dendrites. However, in the mammalian retina, horizontal cells do not seem to contain GABA systematically or to express membrane GABA transporters. We here report that mouse retinal horizontal cells express GAD65 and/or GAD67 mRNA, and were weakly but consistently immunostained for GAD65/67. While GABA was readily detected after intracardiac perfusion, it was lost during classical preparation for histology or electrophysiology. It could not be restored by incubation in a GABA-containing medium, confirming the absence of membrane GABA transporters in these cells. However, GABA was synthesized de novo from glutamate or glutamine, upon addition of pyridoxal 5'-phosphate, a cofactor of GAD65/67. Mouse horizontal cells are thus atypical GABAergic neurons, with no functional GABA uptake, but a glutamate and/or glutamine transport system allowing GABA synthesis, probably depending physiologically from glutamate released by photoreceptors. Our results suggest that the role of GABA in lateral inhibition may have been underestimated, at least in mammals, and that tissue pre-incubation with glutamine and pyridoxal 5'-phosphate should yield a more precise estimate of outer retinal processing.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity

GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which exists in two isoforms, that is, GAD65 and GAD67. In line with GAD65 being located in the GABAergic synapse, several studies have demonstrated that this isoform is important during sustained synaptic transmission. In contrast, the functional significance of GAD65 in the maintenance of GABA destined for extrasynaptic tonic inhibition is less well studied. Using GAD65-/- and wild type GAD65+/+ mice, this was examined employing the cortical wedge preparation, a model suitable for investigating extrasynaptic GABA(A) receptor activity. An impaired tonic inhibition in GAD65-/- mice was revealed demonstrating a significant role of GAD65 in the synthesis of GABA acting extrasynaptically. The correlation between an altered tonic inhibition and metabolic events as well as the functional and metabolic role of GABA synthesized by GAD65 was further investigated in vivo. Tonic inhibition and the demand for biosynthesis of GABA were augmented by injection of kainate into GAD65-/- and GAD65+/+ mice. Moreover, [1-(13) C]glucose and [1,2-(13) C]acetate were administered to study neuronal and astrocytic metabolism concomitantly. Subsequently, cortical and hippocampal extracts were analyzed by NMR spectroscopy and mass spectrometry, respectively. Although seizure activity was induced by kainate, neuronal hypometabolism was observed in GAD65+/+ mice. In contrast, kainate evoked hypermetabolism in GAD65-/- mice exhibiting deficiencies in tonic inhibition. These findings underline the importance of GAD65 for synthesis of GABA destined for extrasynaptic tonic inhibition, regulating epileptiform activity.     

Glutamate Decarboxylases in Nonneural Cells of Rat Testis and Oviduct: Differential Expression of GAD65 and GAD67

gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.     

Quantum chemistry studies of the catalysis mechanism differences between the two isoforms of glutamic acid decarboxylase

The production of gamma-aminobutyric acid (GABA) is catalyzed by two isoforms of glutamic acid decarboxylase (GAD), using pyridoxal 5′-phosphate (PLP) as the cofactor. Between the two enzymes, GAD67 accounts for normal GABA requirement, while GAD65 stays inactive until emergent demand for GABA. Recent crystal structure findings revealed that the distinct conformation of a common catalytic loop of the enzymes may account for their different functions (Fenalti et al Nat Struct Mol Biol, 14:280-286, 2007). Enlightened by their inferences, we studied the underlying reaction mechanism of the two GAD isoforms using density functional theory (DFT). A rather complete reaction pathway is identified, including nine transition state (TS) structures and 14 intermediate (IM) structures. The rate limiting step occurs early during the reaction and involves a proton transfer. In the late stage, there are two pathways that involve C_4’ and C_α protonation by Tyr or Lys. Our calculations show that the reaction barriers corroborate the conjecture made by Fenalti et al.