Regulation of vesicular monoamine and glutamate transporters by vesicle-associated trimeric G proteins: new jobs for long-known signal transduction molecules.

Neurotransmitters of neurons and neuroendocrine cells are concentrated first in the cytosol and then in either small synaptic vesicles ofpresynaptic terminals or in secretory vesicles by the activity of specific transporters of the plasma and the vesicular membrane, respectively. In the central nervous system the postsynaptic response depends--amongst other parameters-on the amount of neurotransmitter stored in a given vesicle. Neurotransmitter packets (quanta) vary over a wide range which may be also due to a regulation of vesicular neurotransmitter filling. Vesicular filling is regulated by the availability of transmitter molecules in the cytoplasm, the amount of transporter molecules and an electrochemical proton-mediated gradient over the vesicular membrane. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Galphao2 and Galphaq. Galphao2 and Galphaq regulate vesicular monoamine transporter (VMAT) activities in brain and platelets, respectively. Galphao2 also regulates vesicular glutamate transporter (VGLUT) activity by changing its chloride dependence. It appears that the vesicular content activates the G protein, suggesting a signal transduction from the luminal site which might be mediated by a vesicular G protein-coupled receptor or as an alternative possibility by the transporter itself. Thus, G proteins control transmitter storage and thereby probablylink the regulation of the vesicular content to intracellular signal cascades.

Regulation of vesicular neurotransmitter transporters.

Neurotransmitters are key molecules of neurotransmission. They are concentrated first in the cytosol and then in small synaptic vesicles of presynaptic terminals by the activity of specific neurotransmitter transporters of the plasma and the vesicular membrane, respectively. It has been shown that postsynaptic responses to single neurotransmitter packets vary over a wide range, which may be due to a regulation of vesicular neurotransmitter filling. Vesicular filling depends on the availability of transmitter molecules in the cytoplasm and the active transport into secretory vesicles relying on a proton gradient. In addition, it is modulated by vesicle-associated heterotrimeric G proteins, Galphao2 and Galphaq, which regulate VMAT activities in brain and platelets, respectively, and may also be involved in the regulation of VGLUTs. It appears that the vesicular content activates the G protein, suggesting a signal transduction form the luminal site which might be mediated by a vesicular G-protein coupled receptor or, as an alternative, possibly by the transporter itself. These novel functions of G proteins in the control of transmitter storage may link regulation of the vesicular content to intracellular signal cascades.

Plasticity of rat central inhibitory synapses through GABA metabolism.

1. The production of the central inhibitory transmitter GABA (gamma-aminobutyric acid) varies in response to different patterns of activity. It therefore seems possible that GABA metabolism can determine inhibitory synaptic strength and that presynaptic GABA content is a regulated parameter for synaptic plasticity. 2. We altered presynaptic GABA metabolism in cultured rat hippocampal slices using pharmacological tools. Degradation of GABA by GABA-transaminase (GABA-T) was blocked by gamma-vinyl-GABA (GVG) and synthesis of GABA through glutamate decarboxylase (GAD) was suppressed with 3-mercaptopropionic acid (MPA). We measured miniature GABAergic postsynaptic currents (mIPSCs) in CA3 pyramidal cells using the whole-cell patch clamp technique. 3. Elevated intra-synaptic GABA levels after block of GABA-T resulted in increased mIPSC amplitude and frequency. In addition, tonic GABAergic background noise was enhanced by GVG. Electron micrographs from inhibitory synapses identified by immunogold staining for GABA confirmed the enhanced GABA content but revealed no further morphological alterations. 4. The suppression of GABA synthesis by MPA had opposite functional consequences: mIPSC amplitude and frequency decreased and current noise was reduced compared with control. However, we were unable to demonstrate the decreased GABA content in biochemical analyses of whole slices or in electron micrographs. 5. We conclude that the transmitter content of GABAergic vesicles is variable and that postsynaptic receptors are usually not saturated, leaving room for up-regulation of inhibitory synaptic strength. Our data reveal a new mechanism of plasticity at central inhibitory synapses and provide a rationale for the activity-dependent regulation of GABA synthesis in mammals.

The expression of vesicular glutamate transporters defines two classes of excitatory synapse.

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

The neuronal monoamine transporter VMAT2 is regulated by the trimeric GTPase Go(2).

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go(2). We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, dense-core vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by Galphao(2), whereas comparable concentrations of Galphao(1) are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go(2). By electron microscopic analysis from prefrontal cortex we show that VMAT2 and Galphao(2) associate preferentially to locally recycling small synaptic vesicles in serotonergic terminals. In addition, Go(2)-dependent modulation of VMAT2 also works when using a crude synaptic vesicle preparation from this brain area. We conclude that regulation of monoamine uptake by the heterotrimeric G proteins is a general feature of monoaminergic neurons that controls the content of both large, dense-core and small synaptic vesicles.

The synaptophysin-synaptobrevin complex is developmentally upregulated in cultivated neurons but is absent in neuroendocrine cells.

Regulated secretion requires the formation of a fusion complex consisting of synaptobrevin, syntaxin and SNAP 25. One of these key proteins, synaptobrevin, also complexes with the vesicle protein synaptophysin. The fusion complex and the synaptophysin-synaptobrevin complex are mutually exclusive. Using a combination of immunoprecipitation and crosslinking experiments we report here that the synaptophysin-synaptobrevin interaction in mouse whole brain and defined brain areas is upregulated during neuronal development as previously reported for rat brain. Furthermore the synaptophysin-synaptobrevin complex is also upregulated within 10-12 days of cultivation in mouse hippocampal neurons in primary culture. Besides being constituents of small synaptic vesicles in neurons synaptophysin and synaptobrevin also occur on small synaptic vesicle analogues of neuroendocrine cells. However, the synaptophysin-synaptobrevin complex was not found in neuroendocrine cell lines and more importantly it was also absent in the adrenal gland, the adenohypophysis and the neurohypophysis although the individual proteins could be clearly detected. In the rat pheochromocytoma cell line PC 12 complex formation between synaptophysin and synaptobrevin could be initiated by adult rat brain cytosol. In conclusion, the synaptophysin-synaptobrevin complex is upregulated in neurons in primary culture but is absent in the neuroendocrine cell lines and tissues tested. The complex may provide a reserve pool of synaptobrevin during periods of high synaptic activity. Such a reserve pool probably is less important for more slowly secreting neuroendocrine cells and neurons. The synaptophysin on small synaptic vesicle analogues in these cells appears to resemble the synaptophysin of embryonic synaptic vesicles since complex formation can be induced by adult brain cytosol.

Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of Gbeta- and Ggamma2/3-subunits suggests that region-specific combinations of G-protein subunits mediate signal transduction in the central nervous system. The different subcellular distribution of Gbeta-subunits in cultivated neurons reflects that observed in tissue where Gbeta5 and Gbeta2 associate preferentially with the perikarya and the neuropil, respectively, and suggests an additional association of Gbeta2 with secretory vesicles.

The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation.

Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity.