Elsevier

Biochimie

Volume 82, Issue 4, April 2000, Pages 327-337
Biochimie

The loading of neurotransmitters into synaptic vesicles

https://doi.org/10.1016/S0300-9084(00)00221-2Get rights and content

Abstract

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H+-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na+-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.

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.

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