A model depicting a structural and functional coupling between GABA synthesis and vesicular GABA transport into SV. GAD 65 is anchored to SVs fi rst through forming a protein complex with the chaperone protein, HSC70, followed by association of HSC70 ⅐ GAD 65 complex to CSP, VGAT, and CaMKII on SVs. The sequence of events leading from neuronal stimulation to activation of GAD 65 and packaging of GABA into SVs is discussed in the text. 

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... These two isoforms, through formation of a homodimer or heterodimer, are found in the membrane- associated as well as the soluble extracts and are responsible for the formation of the major inhibitory neurotransmitter GABA (2). Previously, we have shown that SGAD, presumably GAD 67 , is activated by protein dephosphorylation through the Ca 2 ϩ - dependent phosphatase, calcineurin, and is inhibited by phos- phorylation through protein kinase A (23). Conversely, MGAD, presumably GAD 65 , is activated by protein phosphorylation, which is catalyzed by an unidentified membrane-associated kinase (7). Site-directed mutagenesis studies have also shown that several serine residues at the N terminus of MGAD are involved in protein phosphorylation (14). Furthermore, our results indicate that the ATP activation of MGAD activity depends on the integrity of the electrochemical gradient of the SVs (7). This ATP-mediated activation is abolished under conditions that disrupt the vesicular proton gradient, such as in 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). Newly synthesized neurotransmitters were found to be prefer- entially released upon stimulation (25, 26). Furthermore, Angel et al. (26) reported that in a reconstituted system GABA taken up into SVs is derived from newly synthesized GABA. The same authors further postulated that the same molecule, namely GAD, is responsible for both GABA synthesis and transport into SVs. Although this is an intriguing hypothesis, separate clonings of VGAT and GAD 65 ͞ GAD 67 suggest that these are distinct proteins encoded by different genes (1, 16). VGAT is an integral membrane protein with 10 transmembrane domains (16), whereas GAD 65 and GAD 67 are soluble proteins lacking any transmembrane regions (1). Hence, it is clear that two separate protein entities are responsible for synthesis and packaging of GABA into SVs. Recently, we have shown that GAD 65 may become anchored to SVs through protein complex formation first with HSC70, followed by interaction with CSP, an intrinsic SV protein (15). In this article, we have shown the presence of HSC70 and CSP in purified GABAergic SV (Fig. 5 d ). In addition, we provide evidence to show that the protein complex includes two additional SV proteins, namely, VGAT and CaMKII (Fig. 5 a–c ). Furthermore, we have used purified GABAergic-specific SVs to demonstrate that [ 3 H]GABA, newly synthesized from [ 3 H]Glu by SV-associated GAD, is taken up preferentially into SV over the preexisting cytosolic GABA (Fig. 4 a ). The material accumulated inside SVs has also been posi- tively identified as GABA (Fig. 4 b ). Based on these observations, we proposed that there is a structural and functional coupling between the synthesis and packaging of GABA into SV. This hypothesis is further supported by the following lines of evidence. First, the newly synthesized [ 3 H]GABA from [ 3 H]Glu by SV-associated GAD is taken up efficiently by VGAT. The VGAT activity is markedly inhibited when the GAD inhibitor, hydrazine or aminooxyacetate, is included in the reaction system, suggesting a functional coupling between MGAD and VGAT. Second, although GAD is a soluble protein, it can anchor to SV through its interaction with HSC70 and several members of SV proteins including CSP, CaMKII, and VGAT (Fig. 5 and ref. 15). This protein machinery provides a structural basis for an effi- cient coupling between GABA synthesis by GAD and GABA packaging into SV by VGAT. Third, a good correlation between the expression of VGAT and GAD 65 in various brain regions as well as at various developmental stages (data not shown) is compatible with the above hypothesis. Fourth, in addition to the GABA system, we have found that a similar mechanism may also be involved in the dopaminergic systems.** Recently, we re- ported that the membrane-associated tyrosine hydroxylase, the rate-limiting enzyme involved in dopaminergic synthesis, is activated by ATP through protein phosphorylation and inacti- vated by protein dephosphorylation. Furthermore, the activation of tyrosine hydroxylase mediated by ATP is abolished under conditions disrupting the proton gradient on the SV. Because the uptake of dopaminergic into SVs also depends on the proton gradient of the SV, these observations also support a coupling mechanism between dopaminergic synthesis and packaging into SV. In conclusion, we propose that GAD 65 is anchored to SVs by forming a protein complex first with HSC70, followed by the association with proteins on SVs, e.g., CSP, VGAT, and CaMKII. This protein complex functions as a machine to ensure that GABA biosynthesis and packaging into the SV is efficiently coupled. A model that depicts a functional and structural coupling of GABA synthesis, regulation, and packaging into SVs is proposed (Fig. 6). The physiological events leading from neuronal stimulation to activation of SV-associated GAD and subsequent packaging of GABA into the SV can be described as follows. GABA is released by exocytosis after the arrival of an action potential (stage 1). The SV is recycled by means of clathrin-coated pits (stage 2). The clathrin coat is then dissoci- ated from the vesicles through interaction with HSC70 (27). Vesicles are then returned to the resting state of SVs, where the proton gradient is restored by V-ATPase (stage 3). GAD 65 is activated through protein phosphorylation by a proton gradient- dependent protein kinase (stage 4). One of the candidates of protein kinase is CaMKII. GABA newly synthesized by GAD 65 is then transported into SVs by VGAT (stage 5). These refilled GABA-containing SVs are ready to be released upon arrival of a new action potential (stage 6). Previously, we reported that SGAD is activated by calcineurin-mediated dephosphorylation and inhibited by protein kinase A-mediated protein phosphor- ylation (22, 23). Here, we propose that when GABA neurons are stimulated, the influx of Ca 2 ϩ into the terminal results in dephosphorylation and activation of SGAD ͞ GAD 67 (stage 7). GABA synthesized by SGAD ͞ GAD 67 in the cytosol may also be transported into SVs, although it represents a minor pathway (stage 8). Cytosolic GABA may also be metabolized to generate ATP through the GABA shunt pathway, which may be used to maintain electrochemical proton gradient for GABA transport (stage 9). The model proposed here could explain the efficiency of vesicular transport of GABA newly synthesized by SV- associated GAD 65 , but not GABA in the cytosolic pool. One can imagine that the active site of VGAT and the catalytic site of GAD 65 are tightly coupled in a key and lock manner. This tight structural coupling would allow an efficient transfer of GABA from its site of synthesis to the site of transport. It also prevents the access of GABA in the cytosol to reach the active site of VGAT, resulting in the minimum rate of transport of cytosolic GABA. However, in the absence of GAD 65 , the active site of VGAT will become available to cytosolic GABA, and vesicular transport of GABA can be restored to a certain extent. This proposed mechanism can explain the observation that GAD 65 knockout mice can survive and grow relatively normal, except with signs of deficiencies in GABA transmission such as increase in seizure susceptibility (28), absence of long-term depression (29), increase in anxiety-like behavior (30, 31), and lack of cortical plasticity (32). However, GAD 67 knockout is lethal. GAD 67 -deficient mice did not survive after birth because of cleft palate (28, 33). These observations are compatible with the notion that GAD 65 is primarily responsible for synthesis of GABA to be used as a neurotransmitter, whereas GAD 67 is responsible for GABA to be used for other functions such as serving as a signaling molecule in development, a source of energy, and a source of GABA released via nonvesicular mech- anism. Further studies on the 3D structure of GAD and its protein complex are needed to elucidate the molecular mecha- nism of coupling between GABA synthesis and its packaging into ...