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Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892, USA
1Correspondence: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, 9000 Rockville Pike, Bldg. 36, Rm. 2A-11, Bethesda, MD 20892, USA. E-mail: eiden{at}codon.nih.gov
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INTRODUCTION |
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 TOP INTRODUCTION CONCLUDING REMARKSREFERENCES |
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. The vesicular inhibitory amino acid transporter
(VIAAT/VGAT) is a member of a second family with limited homology
to plant amino acid permeases (2
, 3)
. A vesicular
transporter for the excitatory amino acid glutamate (VGlut) has been
cloned and functionally characterized (4
, 5)
, and is a
member of a separate, third gene family. These three transporter
families account for the vesicular transport of catecholamines,
serotonin, histamine, acetylcholine, GABA, glycine, and glutamate. The
vesicular neurotransmitter transporters (VNTs) are the final arbiters
of neurotransmitter entry into the secretory vesicle: they specify the
quality and quantity of its content. VNT targeting to specific types of
vesicles determines the subcellular locations from which transmitters
are mobilized and thus the synaptic dynamics of neurotransmission. Loss
of chemical neurotransmission in nematodes deficient in vesicular
transmitters and loss of transmitter release in cells cultured from
VNT-deficient mice have provided reverse genetic proof of the vesicular
hypothesis of neurotransmission (6
, 7)
. Neurodegenerative
processes as they occur during human life are being imaged with
neurotransmitter transporter ligands (8)
. The chemical
neuroanatomy of the vesicular transporters has revealed novel neuronal
phenotypes and demonstrated the existence of amine-handling capability
in inflammatory as well as neuroendocrine cells (9)
.
Future prospects include elucidating gene regulatory mechanisms for
cell-specific expression of the vesicular transporters; identifying
novel proteins required for vesicular transporter targeting; and
characterizing discrete classes of neurotransmitter release sites based
on vesicular transporter targeting to several types of synaptic
vesicle, required for the dynamic range of neurotransmission used by
different neuronal subtypes.
Vesicular neurotransmitter transporters are the gatekeepers of the
secretory vesicle, responsible for regulated secretion of informational
molecules from neurons (i.e., neurotransmitters) and neuroendocrine
cells (i.e., hormones, paracrine, or autocrine factors). The history of
the vesicular transporter field began with the discovery of
neurotransmitter secretory vesicles themselves by Hillarp in 1958
(10)
, and several reviews have been written on the early
development of the field, describing the isolation of catecholamine
granules and cholinergic vesicles, development of pharmacological
agents for the study of vesicular transport, and the biochemical
characterization of the transport process itself (1
, 11
12
13
14
15
16
17
18)
. The cloning of the vesicular amine transporters has
revealed much about their involvement in neurotransmission and its
regulation. Perhaps most important, vesicular transporters are no
longer viewed as static filters for admitting a fixed amount of
neurotransmitter into a fixed number of standard-sized vesicles, but as
dynamic regulators of where, how, what kind, and how much
neurotransmitter is released during synaptic transmission
(19)
. The full extent of how the regulation of vesicular
transporter activity fine-tunes neuronal and endocrine informational
output is just now being delineated.
Information obtained about the neuroanatomical localization and
expression of vesicular transporters is reviewed here by Weihe and
Eiden (9)
. The question of why two monoamine transporters
should exist at all in mammals, for example, is answered by the
observation that the VMAT1 isoform is restricted to endocrine cells and
VMAT2 is the only form expressed in neurons, although VMAT2 is
expressed in some neuroendocrine cells, depending on the species
examined. A strict neuroendocrine division between VMAT1 and VMAT2
expression exists in the enterochromaffin cells of the gut in all
species. Only VMAT1 is expressed in serotonin-accumulating
enterochromaffin cells, whereas only VMAT2 is found in
enterochromaffin-like histaminocytes of the stomach. The major
difference between the two transporters is that VMAT2 efficiently
transports histamine and VMAT1 does not. VMAT1 may have evolved during
the evolutionary emergence of histamine as a secreted effector
molecule, restricting the storage of histamine in enterochromaffin
cells of the lower gut.
The ability to visualize the vesicular transporters in individual neurons has revealed that several groups of neurons possess some aminergic traits, but lack a complete aminergic phenotype. ‘Orphan neurons’ include those that possess biosynthetic enzymes for biogenic amines but no vesicular transporters, and the reverse. Will these turn out in the first case to represent neurons with new modes of vesicular secretion and, in the second, neurons that store biogenic amines synthesized by novel biosynthetic pathways?
It has long been known that reserpine-sensitive transporter activity
exists in amine-handling cells of the immune/inflammatory system,
including histamine-secreting basophils and mast cells and
serotonin-secreting platelets. All three cell types express VMAT2, as
predicted from the substrate preference for histamine of VMAT2. The
presence of VMAT2 in platelets validates the study of amine storage and
amine storage defects in these accessible cells as models for
amine-handling in central nervous system (CNS) neurons
(20)
. An intriguing aspect of VMAT2 expression in
histamine-storing mast cells is that little or no VMAT2 is expressed in
mast cells of the lamina propria of the stomach, whereas VMAT2 is
easily visualized in mast cells of other tissues. Perhaps VMAT2 is
down-regulated in mast cells entering specific tissue milieux, to allow
acute histamine release but prevent chronic histamine secretion.
Transport assays in cells into which wild-type and mutant transporter
cDNAs are admitted and expressed have accelerated the study of
transporter function at the molecular level. Insights have been gained
into the evolution of the vesicular transporters as a diverse set of
proteins that all exploit the presence of vacuolar ATPase, and the
relative proton impermeability of the storage vesicle, to drive
transport of positively charged, zwitterionic, and negatively charged
species into a positively charged environment. The driving force for
transport is generated by the proton gradient in all cases, but
dominated by proton loss per se (chemical gradient) for
positively charges species, by both proton loss and charge dissipation
(electrochemical) for zwitterions, and by dissipation of the
electrical and loss of the chemical gradient for negatively charged
species. Here, Stan Parsons analyzes the bioenergetics and
structure–activity relationships for the vesicular amine transporters,
focusing mainly on VAChT and the common mechanisms of transport shared
by VAChT and VMATs (21)
. Experimental verification of the
exchange of two protons for one amine for both VAChT and VMAT transport
has emphasized the experimental validity of using information from the
study of both VAChT and the VMATs to develop a common mechanistic
explanation of VAT transport of amines. Understanding the molecular
pathway that protons must take to exit the vesicle through the
vesicular transporter should reveal the essential commonalities among
all proton-coupled transporters, along with the unique properties of
VATs, VIAATs, and VGlut that are potentially exploitable
pharmacologically.
The current status of how the transport machinery is mobilized to
subpopulations of secretory vesicles is reviewed here by Erickson and
colleagues (22)
. Key issues in this area include the
trafficking of vesicular transporters to their final destinations on
different classes of secretory vesicles. The original observation made
in PC12 cells that VAChT may be transported to small synaptic vesicles
via large dense-core vesicles (LDCVs) (23)
has received
additional support from studies that indicate VAChT phosphorylation may
reversibly direct it either to LDCVs or SSVs (24)
. What
remains to be seen is whether the LDCV is always a trafficking pathway
for VAChT and if, in some neurons, it is VAChT’s final destination.
Resolution of this issue not only in PC12 cells, but also in actual
neurons, will have major implications for understanding the cell
biology of chemically coded neurotransmission.
Rand and colleagues review in this issue the neurogenetics of
transporter function in Caenorhabditis elegans
(25)
. Historically, worm genetics has played a prominent
role in the study of vesicular neurotransmitter transporters. The
strong homology of unc-17 to mammalian VMAT, along with other evidence,
allowed Alfonso et al. to posit that the protein encoded by unc-17 was
a vesicular acetycholine transporter even without direct evidence of
transport activity (6)
. The unc-17 cDNA in turn provided a
probe for mammalian VAChTs that could be directly assayed for
acetylcholine transporter activity (26)
. The discovery of
unc-17 as part of a primary transcript also generating choline
acetyltransferase by alternative splicing in the worm
(27)
, in turn led to the discovery of the cholinergic gene
locus in mammals (28)
.
C. elegans is the first complex metazoan organism with a
fully defined genome (25)
. In C. elegans,
‘saturation’ genomics predicts the total number of transporters and
transporter-related gene-encoded proteins in the genome and allows
functional evaluation of each. Thus, the presence of a single VMAT in
the C. elegans genome indicates that VMAT1 and VMAT2 likely
emerged as vesicular monoamine transporter variants after duplication
of a single ancestral gene. Chalfie and Jorgensen have pointed out that
the worm nervous system is ideally suited to both forward and reverse
genetic analysis of neuronal function, because even severe neuronal
dysfunction does not cause developmental lethality in this organism
(29)
. Consequently, testing of the nonvesicular hypothesis
of neurotransmission can be carried out in whole organisms completely
lacking a given vesicular neurotransmitter transporter. Worm mutants in
which either VAChT or VIAAT/VGAT are nonfunctional or absent exhibit a
complete loss of cholinergic or GABAergic function, respectively, even
though cytoplasmic levels of ACh or GABA are in fact elevated rather
than reduced (6
, 30)
. The same is true for dopaminergic
function in VMAT-deficient (cat-1) worms (31)
. Thus, the
nonvesicular secretion of acetylcholine through the mediatophore (a
subunit of the vacuolar ATPase that powers vesicular uptake of all
known neurotransmitters), once proposed as a potential alternative to
secretion by exocytosis of acetylcholine from synaptic vesicles,
appears to be without physiological relevance (32)
.
However, nonvesicular secretion of other neurotransmitters, as proposed
for GABA via reversal of the plasma membrane GABA transporter, may well
occur (33)
. Undoubtedly, knockout or conditional knockout
models for the vesicular GABA transporter will be useful in further
testing of nonexocyotic models for neurotransmission. The issue of
‘orphan’ neurons, in this case expressing VMAT but not dopamine or
serotonin, is again raised in C. elegans as in mammals.
These neurons may be histaminergic, octopaminergic, or they may store
and secrete a novel neurotransmitter, perhaps synthesized by one of the
four amino acid decarboxylases of C. elegans with an as yet
unassigned biosynthetic function.
In contrast to the cat-1 mutation in C. elegans, the VMAT2
null mutation is lethal in mice soon after birth, and thus adult nulls
cannot be studied (7
, 34
, 35)
. The absence of biogenic
amine storage in the CNS of neonatal VMAT2-deficient mice, however,
confirms that VMAT2 is the only functional biogenic amine vesicular
transporter in the mammalian CNS (36)
and that vesicular
transporter activity is absolutely required for storage of significant
amounts of biogenic amines, even when their synthesis is completely
unimpaired. VMAT2 heterozygous mice do exhibit
haploinsufficiency at the VMAT2 gene. Remarkably, mice with a
single VMAT2 gene express about half the normal amount of VMAT2
protein, and nervous tissue contains about half the normal amount of
serotonin, dopamine, and norepinephrine (7
, 34
, 35)
. Uhl
and colleagues review here the VMAT2 heterozygous knockout
haploinsufficiency syndrome in mice, and its implications for VMAT2 as
a potential target in drug-seeking behavior and neuropsychiatric
disease in humans (37)
. Monoamine oxidase inhibition
increases serotonin and norepinephrine, but not dopamine levels, while
amphetamine causes significant dopamine release in neurons cultured
from VMAT2 null mice and rescues VMAT2-deficient mice for several days
after birth (7)
. These observations have direct relevance
to the neuropharmacology of these agents and the potential etiologies
of the human diseases that they effectively treat (7)
.
Amine transport by the VATs can be blocked by particularly potent
heterocyclic organic inhibitors: vesamicol for VAChT and tetrabenazine
and reserpine for VMATs. These ligands also bind the VATs with high
affinity when administered in vivo, and thus provide a way
to image the nerve terminal and study synaptic patency in human brain
in neurodegenerative and other diseases. Simon Efange reviews the
development of vesamicol- and tetrabenazine-based radioligands for the
visualization of VAChT and VMAT2, respectively, in primate and human
brain (38)
. The use of these agents in the in
vivo imaging of human brain during neurodegenerative and
psychiatric illness, and after traumatic brain injury, is potentially
enormous. Frey and colleagues, for example, have demonstrated in mild
Parkinson’s disease (PD) that tetrabenazine binding is greatly
decreased in posterior but not yet in anterior putamen as in later
stages of PD, suggesting an anterograde progression of dopaminergic
neuronal destruction in this disease (8)
. Efange reviews
the critical aspects of ligand development for in vivo
studies that allow mechanistic conclusions to be drawn from these
somewhat empirical imaging studies. Fully interpretable imaging
studies, in turn, allow a host of interesting and important questions
about human neurodegenerative disease to be raised. These include
asking what neurodegeneration really consists in (e.g., loss of
neurons, loss of neurotransmitter synthetic capacity, loss of storage
capacity, dysregulation of targeting or synapse formation with neuronal
death secondary to synapse loss, etc.). Imaging allows these questions
to be addressed in early stages of the illness, and moves
neuropathological assessment from attempts to deduce disease mechanism
from autopsy findings alone, to clinical diagnosis and monitoring of
disease in a dynamic way.
|
CONCLUDING REMARKS |
|---|
|
TOPINTRODUCTIONCONCLUDING REMARKSREFERENCES |
|---|
First, the vesicular neurotransmitter transporters are now recognized
as providing a critical avenue of experimental access to the study of
the cell biology of vesicular function that focuses on the
environmental content of secretory vesicles. The analysis of SNARE
proteins and their function in guiding synaptic and secretory vesicles
to the nerve terminal and positioning them for exocytotic release is
recognized as a mature area of research rapidly nearing completion
(39)
. Studying the molecular mechanisms for vesicular
neurotransmitter transporter trafficking now promises to provide the
tools required for identification of the additional set of proteins
that sorts the VNTs to their final destinations, so that they can
determine the informational content of individual types of synaptic
vesicles, much as the SNARE proteins and their interactors determine
where the vesicles themselves end up in each type of chemically coded
neuron or neuroendocrine cell.
Second, the regulation of VNT abundance, location, and activity by transcriptional and posttranslational mechanisms and by interaction with other vesicle-associated proteins is now appreciated to play a major role in determining vesicular quantal size, the ‘currency’ of synaptic neurotransmission.
Third, the generality of proton antiport as the engine that
drives vesicular uptake of neurotransmitters is now fully appreciated
through mechanistic studies of the type pioneered by Parsons and
co-workers. Although proton exchange powers amine uptake, however, it
may be merely permissive for the uptake of glutamate and aspartate by
providing charge neutralization for each substrate molecule
transported. Thus, although the universal driver for neurotransmitter
uptake into vesicles is the proton gradient generated by the vesicle’s
vacuolar ATPase, proton antiport through the transporter itself may be
unique to the amine transporters compared to other subfamilies of
vesicular transporters. Several other vesicular transporters remain to
be characterized besides the excitatory amino acid carriers. These
include the carrier for vesicular ATP and the carrier for
intravesicular calcium. SV2, which is strikingly homologous to the
VATs, is a potential vesicular calcium transporter
(40
41
42)
. If so, it would be predicted to be itself a
proton antiporter since it carries a net positively charged cation into
the vesicle. For each of these transporters, drugs that modulate VNT
activity, and thus increase or decrease quantal size and the efficacy
of neurotransmission, can be envisioned.
Fourth, the study of VNT expression during development and in the
mature nervous system have provided important insight into the
regulation of genomic neuronal regulatory units and how they are
mobilized to provide functional phenotypes for the chemical coding of
neurotransmission underlying the complex ionotropic and metabotropic
intercellular communication in which nervous system function wholly
consists. The most obvious example is the cholinergic gene locus, the
first known neuronal operon. Here, cholinergic chemical coding is
ensured by coregulation of the transcripts for VAChT and ChAT from the
same gene locus (43)
. The ‘regulons’ that are
transcriptionally activated to produce serotonergic, GABAergic,
glycinergic, dopaminergic, and other chemically coded neurons must also
involve differential regulation of VNT genes in various neuronal
populations. The recent discovery that the transcriptional activator
unc-30 regulates the genes encoding glutamic acid decarboxylase and the
vesicular GABA transporter in C. elegans is an important
example, and the regulation of dopamine-ß-hydroxylase and other
components of the noradrenergic phenotype by Phox2a is another
(44
, 45)
. However, transcriptional regulation of the VNTs
that are shared by multiple chemically coded phenotypes such as VMAT2
will be particularly interesting to examine. Their regulation in
serotonergic vs. noradrenergic neurons, for example, will reveal
important facets of the transcriptional regulator cascades operating
during the establishment of chemical coding in the nervous system. It
is noteworthy that at present, the VGlut is the only known protein
marker associated with the excitatory amino acid neuronal phenotype.
Understanding the regulation of the VGlut gene will provide an
important avenue into the identification of transcriptional activators
that determine the chemical phenotype of glutamatergic neurons, one of
the most ubiquitous chemically coded phenotypes in the nervous system.
Finally, the ability to image vesicular transporters, if extended to those for inhibitory and excitatory neurons, will make the synaptic organization of the brain, and its derangement in nervous system dysfunction transparent to clinicians at stages of disease amenable to actual prevention of neurodegeneration, rather than repair or limiting of overt damage.
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REFERENCES |
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TOPINTRODUCTIONCONCLUDING REMARKSREFERENCES |
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