The loading of neurotransmitters into synaptic vesicles
Introduction
The release of neurotransmitters by nerve terminals requires their previous accumulation into secretory vesicles. This accumulation follows quite distinct routes depending on the nature of the transmitter. Neuropeptides are synthesized as large polypeptide precursors. As constitutive secretory proteins, these precursors enter the secretory pathway at early steps, by co-translational import into the lumen of the endoplasmic reticulum. Subsequently, they pass to and through the Golgi complex and, at the level of the trans-Golgi, they are recruited into nascent secretory granules by co-agregation with other intragranular proteins such as chromogranins. The acidification of maturing progranules then activates specific proteases, which release neuropeptides from their precursors (see the review by Gomblik and Gerdes in this issue).
In contrast, the accumulation of non-peptide transmitters, which are essentially amino acids or amino acid derivatives, occurs at late steps of the secretory granule or synaptic vesicle biogenesis, by active transport from the cytosol. This transport is ensured by two proteins of the vesicular membrane: i) a vacuolar H+-ATPase [1], [2], present on all acidic organelles, which acidifies and charges positively the vesicle lumen; and ii) a neurotransmitter transporter, which translocates cytosolic neurotransmitters into the vesicle lumen in exchange for protons, thus allowing the uptake of transmitters against their concentration gradient. In the case of anionic transmitters such as glutamate and ATP, the inside-positive electric potential created by the H+-ATPase may be sufficient to drive their uptake without exchanging for protons (see table I).
The existence of such distinct mechanisms has important physiological consequences for the dynamics of neurotransmitter release, since the sites of release can be very distant from those of protein synthesis in neurons (up to ≈ 1 m in the case of some human motoneurons). Whereas the release of neuropeptides is limited by the supply of newly formed dense-core granules via the axonal flow, the release of non-peptide transmitters can be ensured by repeated cycles of exo/endocytosis and transmitter refilling of the pre-existing pool of synaptic vesicles present at nerve terminals. Recent experiments on brain clathrin-coated vesicles indicate that the ability to refill vesicles with transmitters occurs at early steps of the recycling pathway [3]. As a consequence, nerve terminals can sustain the release of non-peptide transmitters at high frequency with a limited number of vesicles (typically, a few hundreds per terminal [4], [5]).
This review will focus on the transporter proteins responsible for the active uptake of transmitters into secretory vesicles. Since this topic has been covered by several exhaustive reviews in recent years [6], [7], [8], [9], the present study will emphasize latest developments, such as the identification of the first vesicular amino acid transporter [10], [11] and the emerging issue of a possible control of neurotransmission at the level of vesicular loading.
Section snippets
An overview
Table I synthesizes our current knowledge on vesicular neurotransmitter uptake. Five distinct uptake activities have been described to date, based on in vitro studies of purified secretory vesicles. The monoamine- and ATP-containing chromaffin granules from bovine adrenal medulla, the cholinergic synaptic vesicles from Torpedo electric organ and rat brain synaptic vesicles, which represent a heterogeneous mixture of vesicles with distinct neurotransmitter specificities, were the main sources of
Identification of the nematode and mammalian transporters
Recently, two independent studies [10], [11] reported the identification of a vesicular inhibitory amino acid transporter, based on the previous characterization of C. elegans mutants. In this animal, the selective impairment of GABAergic neurotransmission, using cell ablation techniques or mutagenesis, induces several characteristic phenotypes, including the ‘shrinker’ behavior: when prodded, treated or mutant animals shrink along their body axis whereas untreated, wild-type animals move
Presynaptic mechanisms
Several mechanisms are susceptible to alter the amount of transmitter packaged into secretory vesicles and hence, its secretion. A first possibility is by changing the cytosolic concentration of transmitter. Because transporters use the vesicular proton motive force to build up a neurotransmitter gradient, variations in the cytosol will be reflected in the vesicle lumen at steady-state. This has recently been illustrated in the case of monoamines. Indeed, exposure of PC12 cells to L-DOPA, the
Acknowledgements
The immunofluorescence photographs shown in figure 1 were kindly provided by A. Dumoulin and A. Triller. I am grateful to J.-P. Henry for his constant support. I also thank M.F. Isambert, C. Sagné, C. Bedet, S. El Mestikawy, B. Giros, S. Lévi, P. Rostaing, A. Dumoulin and A. Triller for fruitful collaborations.
References (99)
Structure and properties of the vacuolar (H+)-ATPases
J. Biol. Chem.
(1999)- et al.
Glutamate uptake occurs at an early stage of synaptic vesicle recycling
Curr. Biol.
(1997) - et al.
The vesicular monoamine transporter: from chromaffin granule to brain
Neurochem. Int.
(1998) - et al.
Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases
FEBS Lett.
(1997) - et al.
A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter
Cel
(1992) - et al.
The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors
J. Biol. Chem.
(1994) - et al.
Transport of histamine by vesicular monoamine transporter-2
Neuropharmacology.
(1995) - et al.
Cloning and expression of the vesamicol binding protein from the marine ray Torpedo. Homology with the putative vesicular acetylcholine transporter UNC-17 from Caenorhabditis elegans
FEBS Lett.
(1994) - et al.
Active transport of acetylcholine by the human vesicular acetylcholine transporter
J. Biol. Chem.
(1996) - et al.
Expression of a putative vesicular acetylcholine transporter facilitates quantal transmitter packaging
Neuron.
(1997)
Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts
J. Biol. Chem.
Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles
J. Biol. Chem.
The amino acid/auxin: proton symport permease family
Biochim. Biophys. Acta
Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission
Cell
Cloning and functional identification of a neuronal glutamine transporter
J. Biol. Chem.
Uptake of L-glutamate into synaptic vesicles: competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups
Biochem. Pharmacol.
Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH
J. Biol. Chem.
Glutamate uptake by brain synaptic vesicles. Energy dependence of transport and functional reconstitution in proteoliposomes
J. Biol. Chem.
Characterization of ATP transport into chromaffin granule ghosts. Synergy of ATP and serotonin accumulation in chromaffin granule ghosts
J. Biol. Chem.
Identification, purification, and characterization of the rat liver golgi membrane ATP transporter
J. Biol. Chem.
Neurotransmitter transporters: three distinct gene families
Curr. Opin. Neurobiol.
Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence
J. Biol. Chem.
Specificity and mechanism of nucleotide uptake by adrenal chromaffin granules
Neuroscience
Vesicular L-glutamate transporter in microvesicles from bovine pineal glands. Driving force, mechanism of chloride anion activation, and substrate specificity
J. Biol. Chem.
GABA and glycine in synaptic vesicles: storage and transport characteristics
Neuron
Presynaptic calcium channels and field-evoked transmitter exocytosis from cultured cerebellar granule cells
Neuroscience
Inhibition of gamma-aminobutyrate and glycine uptake into synaptic vesicles
Eur. J. Pharmacol.
Uptake of glycine, GABA and glutamate by synaptic vesicles isolated from different regions of rat CNS
Neurosci. Lett.
The inhibitory glycine receptor: architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex
Curr. Opin. Neurobiol.
Cloning and expression of a spinal cord- and brain-specific glycine transporter with novel structural features
J. Biol. Chem.
Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action
Neuron
Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine
Neuron
Long term stimulation changes the vesicular monoamine transporter content of chromaffin granules
J. Biol. Chem.
Expression and regulation of the bovine vesicular monoamine transporter gene
FEBS Lett.
Saturation of postsynaptic glutamate receptors after quantal release of transmitter
Neuron
Saturation of postsynaptic receptors at central synapses?
Curr. Opin. Neurobiol.
Modulation of decay kinetics and frequency of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons
Neuroscience
Variation in GABA mini amplitude is the consequence of variation in transmitter concentration
Neuron
Differences in synaptic GABA(A) receptor number underlie variation in GABA mini amplitude
Neuron
Synaptic transmission at single visualized hippocampal boutons
Neuropharmacology
Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices
Neuron
Radioligands of the vesicular monoamine transporter and their use as markers of monoamine storage vesicles
Biochem. Pharmacol.
Vacuolar and plasma membrane proton-adenosinetriphosphatases
Physiol. Rev.
Dendritic spines of rat cerebellar Purkinje cells: serial electron microscopy with reference to their biophysical characteristics
J. Neurosci.
Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics
J. Neurosci.
Vesicular neurotransmitter transporters: from bacteria to humans
Physiol. Rev.
The role of vesicular transport proteins in synaptic transmission and neural degeneration
Annu. Rev. Neurosci.
Vesicular neurotransmitter transporters. Potential sites for the regulation of synaptic function
Mol. Neurobiol.
Identification and characterization of the vesicular GABA transporter
Nature
Cited by (127)
Caenorhabditis elegans: An elegant model organism for evaluating the neuroprotective and neurotherapeutic potential of nutraceuticals
2021, Nutraceuticals: Efficacy, Safety and Toxicityγ-Aminobutyric acid transporters as relevant biological target: Their function, structure, inhibitors and role in the therapy of different diseases
2020, International Journal of Biological MacromoleculesCorrecting miR92a-vGAT-Mediated GABAergic Dysfunctions Rescues Human Tau-Induced Anxiety in Mice
2017, Molecular TherapyCitation Excerpt :To understand the mechanisms underlying the htau-induced GABA reduction, we measured the factors regulating GABA synthesis, release, uptake, and transport, and selective reduction of vGAT was found. The normal function of vGAT is to transport GABA from cytoplasm into the vesicles,37,38 thus loss of vGAT disrupts GABA loading and eventually decreases GABA release. This point was further validated by the data showing that the intracellular GABA clusters were not changed much, although the released GABA level was significantly decreased.