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

Hsu, Che‐Chang; Thomas, Charles W.; Chen, Weiqing; Davis, Kathleen; Foos, Todd; Chen, Jeffrey L.; Wu, Elliott; Floor, Erik; Schloss, John V.; Wu, Jang‐Yen

doi:10.1074/jbc.274.34.24366

Cited by 64 publications

(60 citation statements)

References 40 publications

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“…Data are presented as mean Ϯ SD (n ϭ 3). the presence of V-type ATPase inhibitor, protonophore, or ionophore uncouplers (7). It has been well established that GABA uptake into SVs by VGAT depends on the integrity of electrochemical proton gradients (e.g., ⌬pH and ⌬ ) of SVs (24).…”

Section: Discussionmentioning

confidence: 99%

“…The ratio of GAD 65 to GAD 67 is higher in synaptic vesicle (SV) fractions than in the cytosol (5). Some studies suggest that GAD 65 binds to the membranes (6,7) and that GAD 67 subsequently interacts with MGAD 65 (2,6). However, the nature of anchorage of GAD to membranes and its physiological significance is still not well understood.…”

mentioning

confidence: 98%

“…The interaction of GAD with membranes was reported to be through ionic (9)(10)(11), hydrophobic (12,13), protein phosphorylation (14), or proteinprotein interaction (15). Previously, we reported that MGAD is activated by phosphorylation that requires an electrochemical gradient across the SV membrane (7). A model for the anchoring mechanism of GAD to SV and its role as a link between GABA synthesis and storage in nerve terminals was also proposed (15).…”

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confidence: 99%

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

Osterhaus

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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L-Glutamic acid decarboxylase (GAD) exists as both membraneassociated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of ␥-aminobutyric acid (GABA) by membraneassociated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [ 3 H]GABA from [ 3 H]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 Ca 2؉ ͞ 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.T he rate-limiting enzyme L-glutamic acid decarboxylase (GAD, EC 4.1.1.15) is involved in the synthesis of ␥-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian brain. There are two well-characterized GAD isoforms in the human brain, namely GAD 65 and GAD 67 (referring to GAD with a molecular mass of 65 kDa and 67 kDa, respectively) (1). Both GAD 65 and GAD 67 are present as homodimers or heterodimers in soluble GAD (SGAD) and membrane-associated GAD (MGAD) pools (2-4). The ratio of GAD 65 to GAD 67 is higher in synaptic vesicle (SV) fractions than in the cytosol (5). Some studies suggest that GAD 65 binds to the membranes (6, 7) and that GAD 67 subsequently interacts with MGAD 65 (2, 6). However, the nature of anchorage of GAD to membranes and its physiological significance is still not well understood. GAD is not considered to be an integral membrane protein because it lacks a stretch of hydrophobic amino acids long enough to span the membrane. Subpopulations of GAD 65 and GAD 67 remain firmly anchored to membranes despite various ionic extraction methods (2,4,8). The interaction of GAD with membranes was reported to be through ionic (9-11), hydrophobic (12, 13), protein phosphorylation (14), or proteinprotein interaction (15). Previously, we reported that MGAD is activated by phosphorylation that requires an electrochemical gradient across the SV membrane (7). A model for the anchoring mechanism of GAD to SV and its role as a link between GABA synthesis and storage in nerve terminals was also proposed (15). The evidence presented here will demonstrate that GABA synthesized by SV-associated GAD is preferentially transported into the SV by vesicular GABA transporters (VGATs). We have also demonstrated that VGAT, a 10-transmembrane helix protein (16), forms a protein complex with GAD on the SV and could be involved in the anchorage of MGAD to the SV. The formation of this GAD protein complex ensures an efficient coupling between GABA synthesis and packaging into the SV. Materials and MethodsPreparation of SVs. SVs were purified from whole rat brain (Sprague-Dawle...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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

Osterhaus

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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220

204

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“…2). GAD 67 is mostly soluble and is distributed evenly throughout the cell, whereas GAD 65 is concentrated at the nerve terminals (3) and constitutes the majority of the membrane-associated GAD (MGAD) (4,5). Despite its importance, our knowledge regarding the regulation of GAD activity is quite limited.…”

mentioning

confidence: 99%

“…Recently, we have shown that soluble GAD (SGAD) is activated by dephosphorylation, mediated by a Ca 2ϩ -dependent phosphatase, calcineurin, and is inhibited by phosphorylation, mediated by a cAMP-dependent protein kinase A (6,7). Conversely, MGAD is activated by protein phosphorylation, which depends on the integrity of the electrochemical gradient of synaptic vesicles (5). Hence, GAD activity appears to be regulated differently depending on whether it exists as a soluble or membrane-anchored protein.…”

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confidence: 99%

Association of l-Glutamic Acid Decarboxylase to the 70-kDa Heat Shock Protein as a Potential Anchoring Mechanism to Synaptic Vesicles

Hsu¹,

Davis²,

Ji³

et al. 2000

Journal of Biological Chemistry

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Recently we have reported that the membrane-associated form of the ␥-aminobutyric acid-synthesizing enzyme, L-glutamate decarboxylase (MGAD), is regulated by the vesicular proton gradient (Hsu, C. C., Thomas, C., Chen, W., Davis, K. M., Foos, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., and Wu, J. Y. (1999) J. Biol. Chem. 274, 24366 -24371). In this report, several lines of evidence are presented to indicate that L-glutamate decarboxylase (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 70 (HSC70), followed by interaction with cysteine string protein (CSP), an integral protein of the synaptic vesicle. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form complex with GAD 65 as shown by gel mobility shift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipitation studies, again, HSC70 was co-immunoprecipitated with GAD by a GAD 65 -specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD 65 and GAD 67 are associated with synaptic vesicles along with HSC70 and CSP. Based on these findings, a model is proposed to link anchorage of MGAD to synaptic vesicles in relation to its role in ␥-aminobutyric acid neurotransmission.L-Glutamate decarboxylase (GAD 1 ; EC 4.1.1.15) is the ratelimiting enzyme involved in the synthesis of ␥-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian brain (1). There are two well characterized GAD isoforms in the brain, namely GAD 65 and GAD 67 , referring to GAD with molecular masses of 65 and 67 kDa, respectively (for review, see Ref.2). GAD 67 is mostly soluble and is distributed evenly throughout the cell, whereas GAD 65 is concentrated at the nerve terminals (3) and constitutes the majority of the membrane-associated GAD (MGAD) (4, 5). Despite its importance, our knowledge regarding the regulation of GAD activity is quite limited. Recently, we have shown that soluble GAD (SGAD) is activated by dephosphorylation, mediated by a Ca 2ϩ -dependent phosphatase, calcineurin, and is inhibited by phosphorylation, mediated by a cAMP-dependent protein kinase A (6, 7). Conversely, MGAD is activated by protein phosphorylation, which depends on the integrity of the electrochemical gradient of synaptic vesicles (5). Hence, GAD activity appears to be regulated differently depending on whether it exists as a soluble or membrane-anchored protein. Judging from the amino acid sequences, it is unlikely that GAD 65 and/or GAD 67 can be integral membrane components, since neither contains a stretch of hydrophobic amino acids long enough to span the membrane (greater than 20 residues), a typical feature for integral membrane proteins. Furthermore, both isoforms lack the appropriate consensus se...

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Amygdalar activation alters the hippocampal GABA system: ?Partial? modelling for postmortem changes in schizophrenia

2001

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Abnormalities in amygdala and hippocampus have been shown to coexist in schizophrenia (SZ). In the hippocampus, compelling evidence suggests that a disruption of GABA neurotransmission is present mainly in sectors CA4, CA3, and CA2. The amygdala sends important inputs to the hippocampus and is also believed to have a defective GABA system in schizophrenia. To explore the possibility that changes in the hippocampal GABAergic system could be related to an increased inflow of activity originating in the amygdala, a "partial" animal model has been developed. In awake, freely moving, rats a GABA(A) receptor antagonist was infused locally into the basolateral nuclear complex of the amygdala (BLn). Within 2 hours, a decreased density of both the 65- and 67-kDa isoforms of glutamate decarboxylase (GAD(65) and GAD(67)) -immunoreactive (IR) terminals was detected on neuron somata in sectors CA3 and CA2, but not in CA1, CA3, or dentate gyrus. An increase of GAD(67)-IR somata was also found in the dentate gyrus and CA4. In anterograde tracer studies, amygdalo-hippocampal projection fibers were exclusively found in CA3 and CA2, but not CA1. Taken together, these results indicate that activation of amygdalo-hippocampal afferents is associated with the induction of significant changes in the GABA system of the hippocampus, with a subregional distribution that is remarkably similar to that found in SZ. Under pathologic conditions, an excessive discharge of excitatory activity emanating from the amygdala could be capable of altering inhibitory modulation along the trisynaptic pathway. This mechanism may potentially contribute to disturbances of GABAergic function in the major psychoses. Such "partial" rodent modelling provides an important strategy for deciphering the effect of altered cortico-limbic circuits in SZ.

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

Cited by 64 publications

References 40 publications

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

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

Association of l-Glutamic Acid Decarboxylase to the 70-kDa Heat Shock Protein as a Potential Anchoring Mechanism to Synaptic Vesicles

Amygdalar activation alters the hippocampal GABA system: ?Partial? modelling for postmortem changes in schizophrenia

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