Che-Chang Hsu

Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles

l-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of γ-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [3H]GABA from [3H]l-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca2+/calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.

Role of taurine in regulation of intracellular calcium level and neuroprotective function in cultured neurons

Glutamate‐induced excitotoxicity has been implicated as an important mechanism underlying a variety of brain injuries and neurodegenerative diseases. Previously we have shown that taurine has protective effects against glutamate‐induced neuronal injury in cultured neurons. Here we propose that the primary underlying mechanism of the neuroprotective function of taurine is due to its action in preventing or reducing glutamate‐induced elevation of intracellular free calcium, [Ca2+]i. This hypothesis is supported by the following findings. First, taurine transport inhibitors, e.g., guanidinoethyl sulfonate and β‐alanine, have no effect on taurines neuroprotective function, suggesting that taurine protects against glutamate‐induced neuronal damage through its action on the extracellular membranes. Second, glutamate‐induced elevation of [Ca2+]i is reduced to the basal level upon addition of taurine. Third, pretreatment of cultured neurons with taurine prevents or greatly suppresses the elevation of [Ca2+]i induced by glutamate. Furthermore, taurine was found to inhibit the influx but not the efflux of 45Ca2+ in cultured neurons. Taurine has little effect on the binding of [3H]glutamate to the agonist binding site and of [3H]MDL 105,519 to the glycine binding site of the N‐methyl‐D‐aspartic acid receptors, suggesting that taurine inhibits 45Ca2+ influx through other mechanisms, including its inhibitory effect on the reverse mode of the Na+/Ca2+ exchangers (Wu et al. [ 2000 ] In: Taurine 4: taurine and excitable tissues. New York: Kluwer Academic/Plenum Publishers. p 35–44) rather than serving as an antagonist to the N‐methyl‐D‐aspartic acid receptors. J. Neurosci. Res. 66:612–619, 2001.

Role of Synaptic Vesicle Proton Gradient and Protein Phosphorylation on ATP-mediated Activation of Membrane-associated Brain Glutamate Decarboxylase

Previously, we have shown that the soluble form of brain glutamic acid decarboxylase (GAD) is inhibited by ATP through protein phosphorylation and is activated by calcineurin-mediated protein dephosphorylation (Bao, J., Cheung, W. Y., and Wu, J. Y. (1995) J. Biol. Chem. 270, 6464–6467). Here we report that the membrane-associated form of GAD (MGAD) is greatly activated by ATP, whereas adenosine 5′-[β,γ-imido]triphosphate (AMP-PNP), a non-hydrolyzable ATP analog, has no effect on MGAD activity. ATP activation of MGAD is abolished by conditions that disrupt the proton gradient of synaptic vesicles, e.g. the presence of vesicular proton pump inhibitor, bafilomycin A1, the protonophore carbonyl cyanidem-chorophenylhydrazone or the ionophore gramicidin, indicating that the synaptic vesicle proton gradient is essential in ATP activation of MGAD. Furthermore, direct incorporation of32P from [γ-32P]ATP into MGAD has been demonstrated. In addition, MGAD (presumably GAD65, since it is recognized by specific monoclonal antibody, GAD6, as well as specific anti-GAD65) has been reported to be associated with synaptic vesicles. Based on these results, a model linking γ-aminobutyric acid (GABA) synthesis by MGAD to GABA packaging into synaptic vesicles by proton gradient-mediated GABA transport is presented. Activation of MGAD by phosphorylation appears to be mediated by a vesicular protein kinase that is controlled by the vesicular proton gradient.

Role of Protein Phosphorylation in Regulation of Brain L-Glutamate Decarboxylase Activity.

In the brain, the gamma-aminobutyric acid (GABA) level is primarily controlled by the activity of its synthesizing enzyme, L-glutamate decarboxylase (GAD). At present, mechanisms responsible for regulation of GAD activity remain largely unknown. Here we report that GAD activity is inhibited by conditions favoring protein phosphorylation, and this inhibition can be reversed by phosphatase treatment. Furthermore, this inhibition appears to result from the suppression of a Ca(2+)-dependent phosphatase. Phosphorylation of GAD is demonstrated by direct incorporation of (32)P into the GAD protein. These results suggest that GAD activity in the brain is inhibited by phosphorylation and activated by dephosphorylation. A model for regulation of GABA synthesis related to neuronal excitation is discussed. Copyright 1994 S. Karger AG, Basel

Mode of Action of Taurine and Regulation Dynamics of Its Synthesis in the CNS

The regulation of taurine biosynthesis can be summarized as following: (i) When neurons are stimulated, the arrival of action potential will open the voltage-dependent Ca2+-channel, resulting in an increase of intracellular free Ca2+, [Ca2+]i, (ii) Elevation of [Ca2+]i will trigger release of taurine as well as activation of PKC, which in turn activates CSAD through protein phosphorylation; (iii) The activated CSAD then synthesizes more taurine to replenish that lost due to stimulation-mediated release; (iv) When intracellular taurine reach a certain level, it then inhibits the activation of PKC directly or indirectly (possibly through regulating Ca2+ availability), thus shutting down activation of CSAD through inhibition of CSAD phosphorylation by PKC; and (v) CSAD soon returns to its inactive state through the action of a protein phosphatase, most likely PrP-2C. The mode of action of taurine inlowering the level of [Ca2+]i, is at least partially due to its inhibition on the reverse mode of Na+-Ca2+ exchanger activity

An integral membrane protein form of brain l-glutamate decar☐ylase: purification, characterization and its relationship to insulin-dependent diabetes mellitus

A new and novel form of L-glutamate decarboxylase (GAD; EC 4.1.1.15) was purified from whole porcine brain to apparent homogeneity by a combination of column chromatographies on DE-52, ultragel AcA 34, hydroxylapatite and Sephadex G-200, and native gel electrophoresis. The purified GAD was established as an integral membrane protein based on hydrophobic interaction chromatography and membrane extraction studies. This membrane GAD (MGAD) has a native molecular weight of 120 +/- 5 kDa and is a homodimer of 60 +/- 2 kDa. Immunoprecipitation and immunoblotting tests using the sera from insulin-dependent diabetes mellitus (IDDM) patients revealed the presence of antibodies against this newly identified MGAD in IDDM. The role of MGAD in the pathogenesis of IDDM and related autoimmune disorders is also discussed.

Multiplicity of brain cysteine sulfinic acid decarboxylase — Purification, characterization and subunit structure

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in: (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn(2+), methione, and other sulfur-containing amino acids, and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5-20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE, and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 +/- 2 kDa subunits. CSAD appears to require Mn(2+) for its maximum activity. Other divalent cations fail to replace Mn(2+) in activation of CSAD activity. However, the precise role of Mn(2+) in the action of CSAD remains to be determined. Copyright 1996 S. Karger AG, Basel

Membrane Associated L-Glutamate Decarboxylase and Insulin-Dependent Diabetes Mellitus (IDDM)

Recently, three novel forms of membrane associated L-glutamate decarboxylase (M-GAD) referred to as M-GADI, M-GADII and M-GADIII were isolated and purified from porcine brain. M-GADI and M-GADII appear to be integral membrane proteins whereas M-GADIII is a peripheral protein attached to membranes in a Ca2+ dependent manner. In addition, M-GADI and M-GADII were found to be the major autoantigens in insulin-dependent diabetes mellitus (IDDM), also known as type 1 diabetes, even more potent than GAD65/GAD67, as demonstrated in immunoprecipitation and immunoblotting tests. M-GADI has a native molecular weight of 96 kDa and consists of two identical subunits of 48 kDa whereas both M-GADII and M-GADIII are homodimers of 60 kDa. M-GADI and M-GADII have fulfilled all three criteria routinely used to determine whether a protein is an integral membrane protein including partitioning in the detergent phase in Triton X-114 partitioning assay, shift in electrophoretic mobility in the presence of ionic detergent and elution by low ionic strength buffer in hydrophobic interaction chromatography. Based on these observations, it is hypothesized that M-GADI and M-GADII may be involved in the pathogenesis of IDDM.