INVITED REVIEW ABSTRACT: The release of neurotransmitter from neurons represents one

of the pivotal events in synaptic transmission. Neurotransmitters are re-

leased from synaptic vesicles in presynaptic neurons in response to neural
activity, diffuse across the synaptic cleft, and bind specific receptors in order
to bring about changes in postsynaptic neurons. Some of the molecular
processes that govern neurotransmitter release are now becoming better
understood. The steps involved can be broken down into two partially over-
lapping presynaptic cycles, the neurotransmitter cycle and the synaptic
vesicle cycle. The neurotransmitter cycle involves transmitter biosynthesis,
storage, reuptake, and degradation. The synaptic vesicle cycle involves tar-
geting to the nerve terminal, docking, fusion, endocytosis, and recycling.
Biochemical and structural studies have yielded important insight into our
understanding of each of these two cycles. Further, both pharmacological
and genetic interference with either of these cycles results in profound al-
terations in synaptic transmission and behavior, demonstrating the crucial
role of neurotransmitter release.
© 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 581–601, 2001

MOLECULAR MECHANISMS OF
NEUROTRANSMITTER RELEASE
EDWARD A. FON, MD, FRCP(C),1 and ROBERT H. EDWARDS, MD2

1
Centre for Neuronal Survival, Montreal Neurological Institute, McGill University,
3801 University Street, Montreal, Quebec H3A 2B4, Canada
2
Departments of Neurology and Physiology, UCSF School of Medicine,
San Francisco, California, USA

Accepted 1 December 2000

Chemical signaling between neurons and their tar-
get cells mediates information processing in the ner-
vous system. Neurotransmitter released from a pre-
synaptic neuron in response to neural activity
Abbreviations: ACh, acetylcholine; AChE, acetylcholinesterase; ALS,
amyotrophic lateral sclerosis; AP, clathrin adaptor protein; ATP, adeno-
sine triphosphate; BNPI, brain-specific sodium-dependent inorganic
phosphate cotransporter; CaMK, Ca2+/calmodulin-dependent protein ki-
nase; CAPS, Ca2+-dependent activator of secretion; cDNA, complemen-
tary DNA; ChAT, choline acetyltransferase; CHT1, choline transporter 1;
COMT, catechol-o-methyl transferase; CSP, cysteine string protein; DA,
dopamine; DAT, plasma membrane DA transporter; DAG, diacylglycerol;
DBH, dopamine ␤-hydroxylase; EAAT, excitatory amino acid transporter;
ER, endoplasmic reticulum; GABA, ␥-aminobutyric acid; GAD, glutamic
acid decarboxylase; GAT, plasma membrane GABA transporter; GDI,
guanine nucleotide dissociation inhibitor; GTP, guanosine triphosphate;
5-HT, serotonin; LDCV, large, dense-cored vesicle; LTP, long-term poten-
tiation; MAO, monoamine oxidase; MPP+, 1-methyl-4-phenylpyridinium;
MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NE, norepinephrine;
NET, plasma membrane NE transporter; NSF, N-ethylmaleimide-sensitive
factor; PKA, cAMP-dependent protein kinase A; PKC, protein kinase C; PI,
phosphatidylinositol; PITP, phosphatidylinositol transfer protein; SERT,
plasma membrane 5-HT transporter; SLMVs, synaptic-like microvesicles;
SN1, System N transporter; SNARE, SNAP receptor; ␣-SNAP, soluble NSF
attachment protein; SV, synaptic vesicle; TGN, trans-Golgi network; TH,
tyrosine hydroxylase; VAChT, vesicular ACh transporter; VGAT, vesicular
GABA transporter; VMAT, vesicular monoamine transporter
Key words: synaptic transmission; exocytosis; synaptic vesicles; trans-
porters; quantal release
Correspondence to: E.A. Fon; e-mail: ted@mni.lan.mcgill.ca
© 2001 John Wiley & Sons, Inc.
diffuses across the synaptic cleft and transduces the
physiological signal by binding to postsynaptic recep-
tors. The receptors thus determine the nature of the
physiological signal. Classical neurotransmitters
such as ␥-aminobutyric acid (GABA), glutamate, and
acetylcholine (ACh) activate ion channels and hence
mediate fast synaptic transmission. In contrast, neu-
romodulators, such as monoamines and peptides (as
well as GABA, glutamate, and ACh), activate G-
protein–coupled receptors, which activate second
messengers and act on a much longer time scale.
Chemical neurotransmission is particularly well
suited for the complex computation performed by
the nervous system. Compared to other modes of
communication, such as electrical transmission, syn-
aptic transmission allows greater flexibility and regu-
lation. Indeed, the signal produced by chemical neu-
rotransmission decays very little and can vary in
intensity, in speed, in the effect on membrane po-
tential, and in the modulation of second messenger
pathways. This flexibility contributes to synaptic plas-
ticity and, at many synapses, prior activity influences
synaptic function, providing a mechanism for learn-
ing and memory. However, appropriate synaptic
function requires the precise coupling of transmitter

Mechanisms of Neurotransmitter Release MUSCLE & NERVE May 2001 581

release to neural activity. The biochemical processes
that mediate neurotransmitter release are thus cru-
cial for information processing.
Insight into the mode of neurotransmitter re-
lease initially derived from the work of Bernard Katz
on the frog neuromuscular junction. Katz showed
that release occurs in packets or quanta containing
several thousand molecules of transmitter.114 It has
since become clear that synaptic release in the cen-
tral nervous system is also quantal in nature.108 Fur-
ther, one quantum corresponds to the release of a
single synaptic vesicle (SV) filled with neurotransmit-
ter.99 SVs accumulate neurotransmitter to high con-
centrations133 and cluster in close proximity to the
sites of release at active zones in the presynaptic
nerve terminal.35,223 Neural activity then causes the
exocytotic fusion of SVs with the plasma membrane,
resulting in transmitter release.
Neurotransmitter release involves two partially
overlapping cycles, the neurotransmitter cycle and
the SV cycle (Fig. 1).269 The neurotransmitter cycle
involves transmitter biosynthesis, storage, reuptake,
and degradation. SVs contain extremely high con-
centrations of neurotransmitter relative to the cyto-
plasm. Because classical transmitters are made in the
cytoplasm, SV filling requires active transport from
the cytoplasm. Further, the high rates of exocytosis
observed at many synapses require a constant infu-
sion of cytoplasmic transmitter to maintain SV fill-
ing. Although biosynthesis produces the various
transmitters de novo, plasma membrane reuptake
systems recycle the released transmitter and the re-
cycled transmitter generally contributes much more
to SV filling than newly synthesized transmitter. In
addition, transmitter degradation by metabolic en-
zymes and their removal from the extracellular space
by reuptake systems both terminate signaling.
The SV cycle involves targeting vesicles to the
nerve terminal where docking, fusion, endocytosis,
and recycling occur. High rates of exocytosis require
the efficient regeneration of SVs at the nerve termi-
nal. Long distances from the cell body make it im-
possible to replenish released SVs by retrieval of
their constituents to the Golgi complex. Rather, SVs
recycle locally, in the nerve terminal. The local re-
cycling nonetheless involves sorting of SV proteins

FIGURE 1. A model illustrating the relationship between the neurotransmitter (nt) cycle and the synaptic vesicle (sv) cycle at the nerve
terminal. The nt cycle involves transmitter biosynthesis, storage, reuptake, and degradation (red arrows). The sv cycle involves targeting
vesicles to the nerve terminal where docking, fusion, endocytosis, and recycling occur (green arrows). High rates of exocytosis require
the efficient packaging of neurotransmitter into SVs (vesicular neurotransmitter transporters, shown as blue triangles) as well as rapid
recycling of SVs at the nerve terminal, either directly from the plasma membrane or through an endosomal intermediate. The local
recycling involves sorting of SV proteins from plasma membrane proteins, clathrin-mediated endocytosis (blue), as well as docking of SVs
at the plasma membrane. Regulated fusion occurs in response to locally elevated Ca2+ entering the nerve terminal through voltage-gated
Ca2+ channels (orange), which cluster near the site of vesicle fusion.

582 Mechanisms of Neurotransmitter Release MUSCLE & NERVE May 2001

from plasma membrane and other proteins, as well
as the docking of SVs at the plasma membrane and
regulated fusion in response to an intracellular sig-
nal, usually elevated Ca2+. Importantly, pharmaco-
logical or genetic interference with either the neu-
rotransmitter or SV cycle profoundly alters synaptic
transmission and behavior, demonstrating the im-
portance of each for signaling by neurons. Molecu-
lar and biochemical studies have now begun to illu-
minate the cellular mechanisms involved in these
two cycles.
THE NEUROTRANSMITTER CYCLE
Modulation of the neurotransmitter cycle can im-
pact neurotransmitter release and signaling in two
important ways. First, it can affect signaling qualita-
tively by determining the site and the mode of neu-
rotransmitter release. Second, it can exert a pro-
found influence on the magnitude of signaling by
regulating the quantity of neurotransmitter available
for release. For each neurotransmitter, analogous,
albeit distinct, sets of proteins carry out the distinct
steps in the cycle (Table 1). This diversity allows flex-
ibility in how release is regulated for each of the
neurotransmitters. The diversity contrasts to the rela-
tively conserved processes implicated in the SV cycle,
which are similar not only in different types of neu-
rons but also for distinct organelle fusion and bud-
ding events within the same cell.
Site and Mode of Neurotransmitter Release. No
feature more clearly defines the phenotype of a neu-
ron than the identity of its neurotransmitter. Trans-
mitter identity depends on the expression of specific
biosynthetic, storage, reuptake, and degradation ma-
chinery. The localization of this machinery within
neurons in turn determines the site and mode of
neurotransmitter release. Indeed, the machinery dif-
fers substantially for distinct classes of neurotrans-
mitters, reflecting differences in their mode of ac-
tion. Classical neurotransmitters such as ACh,
GABA, and glutamate recycle locally in the nerve
terminal, where they accumulate in SVs. SVs un-
dergo exocytosis within a specialized domain of the
plasma membrane overlying the synapse known as
the active zone, consistent with the role of classical
transmitters in rapid, precise computational signal-
ing. In contrast, neural peptides are synthesized as
larger precursor proteins in the lumen of the endo-
plasmic reticulum (ER). They transit through the
Golgi complex and sort directly from the trans-Golgi
network (TGN) into large, dense-cored vesicles
(LDCVs, or secretory granules in endocrine cells)
where they undergo proteolytic processing into ma-
ture, biologically active peptides. Like SVs, LDCVs
undergo regulated exocytosis in response to elevated
cytoplasmic Ca2+. However, unlike SVs, LDCVs fuse
with the plasma membrane at virtually any site in the
cell body or dendrite as well as the nerve terminal
and hence are not restricted to an active zone. The
exocytosis of LDCVs also requires stronger stimula-
tion than the release of SVs and takes place over a
longer time scale, consistent with the role of neural
peptides as modulators of synaptic activity rather
than as fast neurotransmitters. Monoamines appear
to represent a special case because, like other clas-
sical transmitters, their synthesis occurs in the cyto-
plasm, but they undergo storage and release from
LDCVs as well as SVs. Indeed, monoamine release
from these two different compartments presumably
subserves distinct roles in signaling.64
Amount of Neurotransmitter Release. Changes in
the rate of transmitter biosynthesis, storage, reup-
take, and degradation each have the potential to in-
fluence extracellular neurotransmitter concentra-

Table 1. Proteins involved in the neurotransmitter cycle.

Biosynthesis Vesicular uptake Storage vesicle Reuptake Degradation

Monoamines
Norepinephrine D␤H VMAT2 LDCVs NET MAO and COMT
Dopamine TH VMAT2 SVs and LDCVs DAT MAO and COMT
Serotonin TPH VMAT2 SVs SERT MAO
Acetylcholine ChAT VAChT SVs CHT1 AChE
Amino acids
GABA GAD65, 67 VGAT SVs GAT GABA transaminase
Glutamate Glutaminase VGLUT1 SVs EAATs Glutamine synthetase
Neural peptides ER and Golgi None LDCVs None Proteases

AChE, acetylcholinesterase; ChAT, choline acetyltransferase; CHT1, choline transporter 1; COMT, catechol-o-methyltransferase; DAT, dopamine
transporter; D ␤H, dopamine ␤-hydroxylase; EAATs, excitatory amino acid transporters; ER, endoplasmic reticulum; GAD, glutamic acid decarboxylase;
GAT, GABA transporter; LDCVs, large, dense-cored vesicles; MAO, monoamine oxidase; NET, norepinephrine transporter; SERT, serotonin transporter;
SVs, synaptic vesicles; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; VAChT, vesicular acetylcholine transporter; VGAT, vesicular GABA
transporter; VGLUT1, vesicular glutamate transporter; VMAT2, vesicular monoamine transporter 2.

Mechanisms of Neurotransmitter Release MUSCLE & NERVE May 2001 583

tions, and hence signaling. Until recently, however,
the role of these activities in regulating transmitter
release has remained unclear. Indeed, it had been
widely held that the amount of neurotransmitter re-
leased from individual vesicles vastly exceeds the
amount required to saturate postsynaptic receptors,
precluding a regulatory role for this machinery. On
the other hand, recent experiments suggest that re-
ceptors at central synapses are often not saturated by
single release events.140 Similarly, at the developing
neuromuscular junction, increasing SV filling with
ACh increases the amplitude of quantal events re-
corded from the muscle.220 Further, the amount of
neurotransmitter stored in individual SVs has gener-
ally been considered invariant. However, recent
work demonstrating the variable neurotransmitter
content of individual vesicles, even within the same
terminal, challenges this view.131 Individual steps in
the neurotransmitter cycle may therefore have an
important influence, not only on the site and mode
of transmitter release, but also on the amount of
neurotransmitter released. The observed effects on
postsynaptic signaling support the significance of
this form of regulation.
Neurotransmitter Biosynthesis. Monoamines.
Regulation of the biosynthetic pathway influences
monoamine release. In mice, disruption of the gene
encoding tyrosine hydroxylase, the rate-limiting en-
zyme in catecholamine biosynthesis, results in
midgestational lethality, illustrating the critical role
of transmitter release for survival.267 In addition, ty-
rosine hydroxylase undergoes intricate control at the
transcriptional and posttranslational levels, includ-
ing modulation by cofactors, feedback inhibition of
the enzyme by catecholamine products, and, impor-
tantly, phosphorylation.120 Indeed, phosphorylation
by multiple protein kinases appears to couple tyro-
sine hydroxylase activity with synaptic activity as mea-
sured by autoreceptor stimulation.268 Activation of
D2-like dopamine receptors in particular inhibits ad-
enylcyclase, reduces the activity of cAMP-dependent
protein kinase A (PKA), and hence reduces the
phosphorylation of tyrosine hydroxylase, which nor-
mally serves to increase enzyme activity. In addition,
the enzyme, dopamine ␤-hydroxylase (DBH), which
converts dopamine (DA) to norepinephrine (NE),
resides inside secretory vesicles. NE biosynthesis
therefore requires prior DA transport into vesicles,
and the targeting of this and other biosynthetic en-
zymes to secretory vesicles presumably contributes to
their role in neurotransmitter release.
GABA. The conversion of glutamic acid to
GABA requires the enzyme, glutamic acid decarbox-
584 Mechanisms of Neurotransmitter Release
ylase (GAD), which occurs in two distinct isoforms in
the mammalian brain, GAD65 and GAD67. Al-
though GAD65 is by far the most abundant isoform,
GAD67 appears responsible for most GABA biosyn-
thesis.212 GABA levels remain normal in GAD65
knockout mice but fall to 10% of wild-type litter-
mates in GAD67 knockout animals. Nonetheless,
GAD65 associates with brain membranes through an
N-terminal domain and appears more tightly regu-
lated than GAD67, which occurs throughout the cy-
toplasm of inhibitory neurons. It has therefore been
suggested that GAD67 contributes to GABA synthesis
for general metabolic activity, such as flux through
the tricarboxylic acid cycle, whereas GAD65 contrib-
utes to synaptic transmission. Indeed, GAD65 knock-
out mice do not display major behavioral defects but
rather have seizures and increased anxiety,112,113
presumably related to a reduction in GABA re-
lease.238 Interference with GAD in humans also im-
pairs synaptic transmission with important conse-
quences on health. Indeed, about 90% of patients
with stiff-person syndrome have autoantibodies di-
rected against GAD, resulting in impaired GABAer-
gic transmission and continuous axial muscle con-
tractions.33,213,214
Acetylcholine. Choline acetyltransferase (ChAT),
the enzyme responsible for ACh synthesis, also asso-
ciates with membranes in the nerve terminal.259 De-
tergent extraction shows that the membrane associa-
tion depends on amphipathic rather than ionic or
covalent interactions.167 Further, ChAT may specifi-
cally localize to SVs,37 suggesting a mechanism to
couple ACh biosynthesis with storage, although the
physiological significance of the membrane associa-
tion remains unclear.
Neurotransmitter Storage. Large, Dense-Cored
Vesicles. Neural peptides and classical neurotrans-
mitters differ in their site of storage. As noted pre-
viously, the storage of neural peptides occurs in
LDCVs (or secretory vesicles in endocrine cells).
Precursors to the neural peptides translocate into
the lumen of the endoplasmic reticulum during
translation and hence already reside within the lu-
men of the secretory pathway. After transit through
the Golgi complex, the proteins sort selectively into
LDCVs through unknown mechanisms prior to regu-
lated exocytosis. Cleavage of the precursors by endo-
proteases such as furin and prohormone convertases
may promote sorting to LDCVs or the regulated (as
opposed to constitutive) secretory pathway.153 In-
deed, it has been proposed that sorting to LDCVs
involves interactions with lumenal granule proteins
MUSCLE & NERVE May 2001
or membrane lipids42,119 rather than with a cytoplas-
mic sorting machinery that mediates most other
membrane-trafficking events within the cell.
Synaptic Vesicles. In contrast to neural peptides,
classical neurotransmitters are synthesized in the cy-
toplasm and therefore need to be packaged into the
lumen of SVs prior to release. Since neurotransmit-
ter concentrations within the lumen of neurosecre-
tory vesicles can approach the molar range, storage
requires active transport across the membrane of the
vesicle. Indeed, previous biochemical and pharma-
cological studies have identified four distinct trans-
port activities expressed by native brain synaptic
vesicles — for monoamines, ACh, GABA, and gluta-
mate.133 All of these activities rely on the proton
electrochemical gradient generated by a vacuolar
H+–adenosine triphosphatase (ATPase) that pumps
protons into the vesicles. In particular, transport in-
volves the exchange of lumenal protons for cytoplas-
mic neurotransmitter. Drugs that interfere with ve-
sicular transport also have the potential to modulate
neurotransmitter storage and release, and thus influ-
ence behavior. Indeed, reserpine and tetrabenazine,
which directly inhibit vesicular monoamine trans-
port, have profound effects on behavior by depleting
the pool of stored monoamines, resulting in a clini-
cal state resembling depression and Parkinson’s dis-
ease.76,83 Recent cloning of the proteins responsible
for vesicular monoamine, ACh, GABA and gluta-
mate transport activities now allows a molecular
analysis of their role in transmitter release.
VMATs and VAChT. Selection in the potent par-
kinsonian neurotoxin, 1-methyl-4-phenylpyridinium
(MPP+; the active metabolite of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine [MPTP]), led to identifi-
cation of the first vesicular neurotransmitter trans-
porter, vesicular monoamine transporter 1
(VMAT1). Both VMAT1 and VMAT2 protect against
MPP+ by sequestering the toxin, like normal mono-
amine substrates, inside secretory vesicles and away
from its primary site of action in the mitochon-
dria.135 Interestingly, the sequence indicates similar-
ity to a family of bacterial antibiotic resistance pro-
teins that extrude antibiotics and also act as proton
exchangers that show sensitivity to reserpine.134 The
VMATs thus appear to have evolved from ancient
detoxification mechanisms and may have two dis-
tinct roles, one in transmitter release and the other
in detoxification. The VMATs may indeed protect
against an endogenous toxin, such as the normal
neurotransmitter dopamine, which is quite toxic to
many cells in culture. A defect in VMAT function
may thus predispose one to Parkinson’s disease. The
sequences of the cDNAs encoding vesicular mono-
Mechanisms of Neurotransmitter Release
amine transport also predict polytopic membrane
proteins with 12 transmembrane domains and de-
fine a new family of proteins that includes the ve-
sicular ACh transporter (VAChT)66,184 as well as the
the nonneural VMAT1 and neuronal VMAT2.65,134
Interestingly, reserpine inhibits both VMATs,
whereas tetrabenazine inhibits primarily VMAT2,169
possibly accounting for the more substantial side-
effects of reserpine.
Genetic manipulation in mice indicates that very
little redundancy exists at the step of vesicular trans-
port. VMAT2 knockout mice move very little spon-
taneously, do not feed, and die within a few days
after birth.75,230,251 Neurons cultured from the mid-
brain of these mice are unable to package mono-
amines into synaptic vesicles and therefore cannot
release the transmitter in response to neural activ-
ity.75 Further, cultured midbrain neurons from
VMAT2+/− mice that express half the wild-type levels
of VMAT2 protein, store and release half as much
dopamine as wild-type animals,75 indicating that
VMAT2 limits monoamine storage and release.
Overexpression of VMAT2 and VAChT in cultured
neurons also increases quantal size by several-
fold,175,220 suggesting that vesicular transport limits
release even under physiological conditions.181
Overexpression increases the frequency of release
events as well, suggesting an increase in the number
of vesicles containing transmitter. The expression of
vesicular transport activity may thus determine not
only the amount of neurotransmitter stored per
vesicle but also the number of filled vesicles available
for release.
Expression of vesicular transport proteins on
different types of storage vesicles has the potential
to influence the site and mode of release as well as
the amount of transmitter released. Classic studies
have suggested that rat pheochromocytoma PC12
cells release monoamines from LDCVs and ACh
from synaptic-like microvesicles (SLMVs),14 suggest-
ing the differential localization of VMAT2 and
VAChT. Indeed, VAChT occurs mostly on SVs in the
brain,81,254 whereas VMAT2 occurs on both SVs and
LDCVs.158–160 In PC12 cells, VMATs reside almost
exclusively on LDCVs, whereas VAChT localizes pref-
erentially to SLMVs.132,254 Since LDCVs do not re-
lease at a specialized active zone, and release more
slowly and in response to different stimuli than SVs,
the localization of the transport proteins presumably
contributes to important differences in the mode of
signaling by monoamines and ACh. The strong se-
quence similarity between VMAT2 and VAChT has
further enabled the identification of critical regions
responsible for the differential targeting.246 Indeed,
MUSCLE & NERVE May 2001 585
acidic residues upstream of dileucine motifs present
in the cytoplasmic C-termini of both proteins appear
important for the differential localization to LDCVs
and SVs.233 Interestingly, VAChT also undergoes
phosphorylation on a residue upstream of its dileu-
cine motif, and this modification redirects VAChT to
LDCVs,117 indicating the potential to regulate the
site of transmitter storage and hence its role in sig-
naling.
VGAT and Related Transporters. Although the
transport of all classical transmitters into SVs de-
pends on a proton electrochemical gradient, the
transport of monoamines and ACh depends primar-
ily on the chemical component ⌬pH, whereas the
transport of glutamate and other amino acids de-
pends to a greater extent on the electrical compo-
nent ⌬␺ It is thus not surprising to find that the
sequence of a cDNA encoding vesicular GABA trans-
port (VGAT) showed no sequence similarity to the
VMATs or to VAChT. 147 Originally identified
through molecular genetic analysis in C. elegans,
VGAT encodes a novel protein with 11 predicted
transmembrane domains. In addition to GABAergic
neurons, it resides in glycinergic neurons and ap-
pears to recognize glycine as well as GABA.48 VGAT
also defines a novel family of membrane proteins
that includes the classically described amino acid
transport Systems N and A (see later).
Vesicular Glutamate Transporter. The uptake of
glutamate into SVs is mediated by a previously char-
acterized brain-specific sodium-dependent inorganic
phosphate cotransporter (BNPI).16 BNPI was ini-
tially thought to influence glutamate biosynthesis by
regulating glutaminase, the enzyme that converts
glutamine to glutamate. Indeed, the glutaminase iso-
form occurring in neurons depends exquisitely on
intracellular inorganic phosphate levels. BNPI could
therefore regulate glutamate levels indirectly by me-
diating phosphate uptake into neurons, which in
turn could activate glutaminase. Interestingly, how-
ever, BNPI resides largely on SVs, where it presum-
ably cannot function in inorganic phosphate up-
take.15 Further, a selective defect in glutamate
release by the C. elegans BNPI homolog eat-4 has also
suggested a more direct role for BNPI in glutamate
release.123 Indeed, measurements show that BNPI
(renamed vesicular glutamate transporter 1, or
VGLUT1) mediates glutamate uptake into vesicles
with kinetics and bioenergetics very similar to endog-
enous brain vesicular glutamate transport activity. In
addition, VGLUT1 exhibits a conductance for chlo-
ride that is blocked by glutamate, a feature also re-
ported for glutamate transport into brain SVs.16 It
remains possible nonetheless that VGLUT1 func-
586 Mechanisms of Neurotransmitter Release
tions both as an inorganic phosphate transporter
when it is translocated to the plasma membrane after
exocytosis and as a vesicular glutamate transporter
when internalized on SVs.
Neurotransmitter Reuptake. The reuptake of clas-
sical neurotransmitters across the plasma membrane
serves to remove extracellular transmitter from the
synaptic cleft and thereby terminate signaling.
Plasma membrane transport proteins, which medi-
ate reuptake of distinct neurotransmitters, can be
classified into two large families.145 Both rely on the
Na+ gradient across the cell membrane to drive up-
take, but plasma membrane monoamine and GABA
transporters (DAT, NET, SERT, and GAT) also re-
quire Cl− for transport (Na+/Cl−-dependent trans-
porters).157 The localization of this family of trans-
porters on the presynaptic neuron allows reuptake
to replenish stores by recycling transmitter released
by exocytosis.110 In contrast, plasma membrane
transporters for the excitatory amino acid transmit-
ters glutamate and aspartate (excitatory amino acid
transporters, or EAATs) require K+, instead of Cl−,
for transport (Na+/ K+-dependent transporters) and,
in most cases, do not occur on presynaptic neurons,
precluding a direct role in neurotransmitter recy-
cling.203
Monoamine Transport. Interference with plasma
membrane DA transport with psychostimulants such
as cocaine and amphetamines has profound effects
on behavior.4,204 Indeed, cocaine inhibits the reup-
take of catecholamines and hence prolongs its ac-
tion on the postsynaptic neuron,12 whereas amphet-
amines promote flux reversal by DAT, which
increases extracellular dopamine.74 Genetic disrup-
tion of the DAT gene in mice mimics the behavioral
phenotype of cocaine use, with dramatic locomotor
hyperactivity.82 Interestingly, these animals show a
dramatic reduction in total brain dopamine levels
despite the high extracellular concentrations.110
This suggests that reuptake through DAT may nor-
mally contibute more DA to the intracellular pool
than even de novo synthesis, highlighting its impor-
tant role in signaling. These transporters also have
the potential to modulate synaptic efficacy depend-
ing on the level of transporter expression at the cell
surface of neurons. In the case of SERT, DAT, and
NET, protein kinase C (PKC)-mediated phosphory-
lation promotes internalization from the cell sur-
face, thereby reducing neurotransmitter reuptake
into cells.7,27,57,178 For SERT, both PKC-mediated
phosphorylation and sequestration were inhibited by
serotonin, but not inhibitors such as cocaine and
antidepressants, suggesting a homeostatic mecha-
MUSCLE & NERVE May 2001
nism for regulation of extracellular monoamine con-
centrations.177
Excitatory Amino Acid Transport. The plasma
membrane transport of the excitatory amino acids
glutamate and aspartate differs in important ways
from the transport of monoamines and GABA. The
EAATs also depend on Na+ but not Cl− to drive re-
uptake.145,203 Further, they catalyze the inward trans-
location of a proton in exchange for cytoplasmic K+
coupled to the uptake of glutamate.264 In addition to
providing an extremely strong driving force for glu-
tamate uptake, this ionic dependence presumably
reflects the function of a class of proteins entirely
distinct from the monoamine and GABA transport-
ers. Interestingly, the EAATs also exhibit a gluta-
mate-activated chloride conductance uncoupled
from transport activity that would serve to hyperpo-
larize the neuron.69,219,249 The postsynaptic location
of neuronal EAATs (EAAT3 and EAAT4 isoforms)
would enable them to act as glutamate receptors as
well as transport proteins, and the uncoupled chlo-
ride conductance appears to have a physiological
role in phototransduction. 68,124,218 In contrast,
EAAT1 and 2 reside on astrocytes, where they ac-
count for the bulk of glutamate reuptake in the
brain.191,234 Importantly, this localization is also con-
sistent with recent work suggesting a more direct
role for astrocytes in glutamatergic synaptic transmis-
sion.248 In addition, removal of glutamate from the
extracellular space by EAAT1 and 2 appears to help
shape the postsynaptic response and protect against
excitotoxicity. 20,21,61 Indeed, disruption of the
EAAT2 gene in mice produces seizures and hippo-
campal degeneration.234 In amyotrophic lateral scle-
rosis (ALS), aberrant RNA processing of EAAT2 re-
duces transporter levels in affected brain regions,
presumably resulting in excitotoxic injury and motor
neuron degeneration.28,127 Of all the EAATs, how-
ever, only EAAT5 appears to reside on the presyn-
aptic nerve terminal,9,176 but this isoform occurs
only on photoreceptors. Thus, the EAATs do not
appear to have a direct role in the recycling of glu-
tamate in the same way that DAT contributes to do-
pamine recycling. Rather, synaptically released glu-
tamate is taken up by glia, converted to glutamine,
and then delivered back to the neuron for conver-
sion to glutamate.53,88,139,165,237 Inhibition of glial
glutamine synthetase and neuronal glutaminase re-
duces glutamate release, supporting a role for this
cycle in synaptic transmission.192 However, the pro-
teins responsible for glutamine export from glia and
uptake by neurons have not been identified until
recently. Interestingly, a novel protein related to the
vesicular GABA transporter occurs on the plasma
Mechanisms of Neurotransmitter Release
membrane of astrocytes.49 Like VGAT, it acts as a
proton exchanger but also mediates the Na + -
dependent uptake of glutamine and apparently
corresponds to classically described amino acid
transport System N.116,232 Under physiological cir-
cumstances, however, the System N transporter
(SN1) more likely mediates the efflux of glutamine
from astrocytes and hence contributes to the gluta-
mine–glutamate cycle. Also, another protein closely
related to SN1, encoding System A, occurs on neu-
rons and may mediate the uptake of glutamine by
these cells that is also required for the glutamine–
glutamate cycle.180,247
Neurotransmitter Degradation. Acetylcholine. Deg-
radation can also influence extracellular neurotrans-
mitter levels and synaptic transmission. Acetylcholin-
esterase (AChE), the enzyme that hydrolyzes ACh, is
the target of drugs used in the diagnosis and treat-
ment of myasthenia gravis. In myasthenia, autoanti-
bodies directed against acetylcholine receptors at
the neuromuscular junction impair synaptic trans-
mission. Drugs that inhibit AChE prolong the half-
life of ACh in the neuromuscular junction and
thereby partially rescue the synaptic defect in myas-
thenia.67,150 AChE occurs in multiple forms, classi-
fied as either globular (G) or asymmetric (A), each
of which can be membrane-bound or soluble.54 At
certain synapses, AChE binds to the plasma mem-
brane through a glycophospholipid anchor. At oth-
ers, such as the neuromuscular junction, the asym-
metric (A12) form containing a collagen tail
covalently attached to complexes containing 12
globular subunits, binds to the synaptic basal lamina
rather than the plasma membrane.193 This localiza-
tion involves a very tight association with extracellu-
lar matrix proteins such as perlacan and dystoglycan
and mirrors the clustering of ACh receptors at the
neuromuscular junction. Localization of AChE to
this site couples degradation with neurotransmitter
release, and may also contribute to regulation. The
recycling of ACh thus differs from other classical
transmitters, which undergo reuptake at the nerve
terminal. In contrast to direct reuptake of the
transmitter, the Na+-dependent uptake of choline
produced by AChE is mediated by CHT1, a novel
protein unrelated to other neurotransmitter trans-
porters.162
Monoamines. In contrast to ACh, monoamine
degradation occurs at cytoplasmic as well as extracel-
lular sites. Monoamine oxidase (MAO), an enzyme
localized to the outer membrane of mitochondria,
mediates the cytoplasmic metabolism of mono-
amines and also produces hydrogen peroxide.209 In-
MUSCLE & NERVE May 2001 587
hibition of the principal neuronal isoform MAO-A
enhances monoamine signaling, particularly for se-
rotonin (5-HT). Genetic disruption of MAO-A in
mice leads to high 5-HT levels and causes aggressive
behavior and defects in cortical development.38
Monoamine levels do, however, eventually decline to
near wild-type in MAO-A knockout mice, presumably
due to expression later in life of the glial isoform,
MAO-B. Under normal circumstances, MAO-B pre-
dominantly degrades catecholamines rather than
5-HT. MAO-B also activates the parkinsonian toxin
MPTP by converting it to MPP+, and either pharma-
cological inhibition or genetic interuption of
MAO-B completely prevents MPTP toxicity.85,122
However, MAO-B is present in glia and therefore can
degrade only the dopamine released from neurons.
If cytoplasmic dopamine contributes to the patho-
genesis of Parkinson’s disease, inhibiting MAO-B
would have little protective effect, consistent with the
results of recent large-scale clinical studies.210 Since
it is expressed by dopamine neurons, MAO-A may
have a much larger role, but MAO-A may actually
protect against dopamine toxicity by preventing the
spontaneous, nonenzymatic oxidation of dopamine
that yields more highly reactive products.63 In addi-
tion, catecholamines are degraded by catechol-o-
methyltransferase (COMT) which, like AChE, occurs
in the synaptic cleft.155 Thus, catecholamine metabo-
lism may occur outside as well as inside neurons. The
redundancy in metabolic pathways suggests that the
cell may require multiple mechanisms to protect
against monoamine and in particular dopamine tox-
icity.
THE SYNAPTIC VESICLE CYCLE
Synaptic transmission depends on the efficient coor-
dination of multiple events, including SV fusion, re-
trieval, and docking prior to a second round of
fusion. SV fusion involves highly conserved mecha-
nisms responsible for essentially all the fusion events
that contribute to membrane trafficking in eukary-
otic organisms.19,190 However, SV exocytosis differs
from many other forms of membrane fusion in terms
of its regulation. SV fusion depends on an elevation
in cytoplasmic Ca2+ mediated by voltage-sensitive
Ca2+ channels.156 Indeed, the regulation of exocyto-
sis by neural activity accounts for the morphological
accumulation of SVs and has enabled their purifica-
tion.107 SV fusion also exhibits a high degree of spa-
tial specificity, with exocytosis essentially restricted to
the active zone. Further, high rates of exocytosis re-
quire the retrieval of SVs locally, in the nerve termi-
nal. At both the neuromuscular junction and central
synapses, the time from exocytosis to retrieval of the
588 Mechanisms of Neurotransmitter Release
fused membrane and a second round of exocytosis
can take as little as 20 seconds.25,194,195 Membrane
retrieval and SV biogenesis must therefore occur
very efficiently. Retrieval and translocation to the ac-
tive zone also appear to undergo regulation196 that
contributes to synaptic plasticity and perhaps learn-
ing and memory.141 Long-term potentiation (LTP)
in the mossy fiber terminals of region CA3 in the
hippocampus involves presynaptic changes, and the
genetic manipulation of transmitter release inhibits
mossy fiber LTP.39
Our understanding of the SV cycle has evolved
rapidly over the past two decades. The purification
of SVs has allowed identification of many protein
constituents223 and these proteins form complexes
that have suggested possible functions (Table 2). For
example, association of an SV protein with a plasma
membrane protein suggests that these proteins me-
diate SV docking and fusion.35,216 The discovery that
botulinum and tetanus toxins inhibit neurotransmit-
ter release by specifically cleaving proteins in this
complex has supported a physiological role for these
interactions.189,199 In contrast to the clostridial tox-
ins that inhibit release, the black widow spider
venom toxin, ␣-latrotoxin, promotes massive neuro-
transmitter release by Ca2+-dependent and -indepen-
dent mechanisms.97,164 Two presynaptic ␣-latrotoxin
receptors, neurexin and latrophilin/CIRL, appear to
mediate these effects and may also control neuro-
transmitter release under physiological circum-
stances.118,226 To understand the function of these
novel proteins, their genes have been disrupted in
organisms ranging from C. elegans and D. melanogas-
ter to mice and the phenotypes characterized by elec-
trophysiological methods.260 In addition, similar ge-
netic and biochemical approaches have also
reconstituted various other steps in the SV cycle.73
Targeting and Docking. The Readily Releasable Pool.
Tightly regulated exocytosis requires the localization
of SVs to the nerve terminal and, in particular, to the
active zone. The active zone aligns precisely with the
synaptic cleft and the postsynaptic density, a mem-
brane specialization enriched in neurotransmitter
receptors.99,100 At the active zone, a few SVs are in
direct contact with the plasma membrane, a state
known as docked.170 Docked vesicles are irreversibly
bound to the cell surface but not necessarily compe-
tent for fusion.98,187 Invasion of the nerve terminal
by action potentials activates voltage-sensitive Ca2+
channels and the resulting increase in cytoplasmic
Ca2+ causes fusion of a small fraction of the docked
vesicles. In contrast, application of hypertonic su-
crose results in fusion all docked vesicles, indepen-
MUSCLE & NERVE May 2001
 Table 2. Proteins involved in the synaptic vesicle cycle.

Protein Function

Targeting and docking Synapsins Phosphoproteins linking SVs to the actin cytoskeleton
Piccolo and bassoon Presynaptic cytomatrix proteins; role in active zone assembly
Sec6/8 complex Mammalian exocyst homolog; defines the site of the active zone
Fusion Synaptobrevin v-SNARE
SNAP-25 t-SNARE
Syntaxin t-SNARE
NSF ATPase; dissociates SNARE complex
␣-SNAP NSF binding protein
Modulation and Ca2+ dependence Rab3a Small GTPase; regulates SV fusion via multiple effectors
Uso1/ p115 Rab effector; modifies lipids; “tethers” SVs
nSec-1 Inhibits SNARE assembly by binding syntaxin; Rab effector
Munc13 C1 and C2 domain protein; required for neurotransmitter release
N, P/Q-type Ca2+ channels Bind SNAP-25 and Syntaxin; occur near the site of SV fusion
Synaptotagmin C2 domain SV protein; Ca2+ sensor
SV2 Transporter; inhibits SV fusion
CAPS Facilitates LDCV exocytosis
Endocytosis and recycling Clathrin Coats nascent endocytic vesicles and induces their invagination
AP-2 Adaptor protein recruits clathrin to the plasma membrane
Dynamin GTPase; “pinches” the neck of endocytic vesicles
Amphiphysin Binds AP-2 and clathrin; recruits dynamin to endocytic vesicles
Synaptojanin Binds amphiphysin and endophilin; inositol 5-phosphatase
Endophilin SH3 domain protein; lysophosphatidic acid arachidonate transferase
AP-3 Adaptor protein for SV recycling from endosomal intermediates

dent of Ca2+, and this population of SVs is known as
the readily releasable pool.188
The Reserve Pool. In addition to the docked
vesicles, a large number of SVs cluster over the syn-
aptic cleft. Although not physically docked, these
vesicles aggregate over the active zone as a result of
interactions with the actin cytoskeleton through pro-
teins such as synapsins and the enormous cytomatrix
proteins piccolo and bassoon.101,239 Genetic dis-
ruption of synapsins in mice causes seizures and a
decrease in short-term synaptic plasticity, most
likely due to a reduction in the reserve pool of
SVs.126,186 Interestingly, stimulation at physiological
frequencies does not release SVs from this reserve
pool.173 However, it remains unclear whether re-
cycled SVs have a higher likelihood of release
than vesicles in the reserve pool.195 The clustering of
SVs over the active zone may also undergo regula-
tion that contributes to synaptic plasticity. Indeed,
synapsin binding to SVs is negatively regulated by
PKA and Ca2+/calmodulin-dependent protein ki-
nases (CaMKs).41,103,221
The Exocyst. Additional mechanisms also appear
to restrict SV docking and fusion to the nerve termi-
nal active zone. In yeast, polarized secretion involves
a large protein complex, known as the exocyst,
which appears to function as a landmark for recruit-
ing secretory vesicles to the site of exocytosis.236 In
neurons, the homologous sec6/8 complex localizes
to the synaptic plasma membrane.104 However, the
exocyst localizes at this site prior to the development
of a presynaptic element, and regresses with the
maturation of the nerve terminal.95 The exocyst may
therefore define the subsequent site of the active
zone, but does not appear to be directly involved in
the SV cycle.
Fusion. SNAREs. Regulated fusion with the
plasma membrane is the central event in the SV
cycle. A little more than 10 years ago, a number of
synaptic proteins were identified that together
formed a stable complex, known as the SNARE (for
SNAP receptor). The complex consists of a vesicle-
associated SNARE (v-SNARE), synaptobrevin (or
VAMP),240 and two target or plasma membrane
SNAREs (t-SNAREs), SNAP-25166 and syntaxin.17 In-
deed, v- and t-SNAREs form extremely stable protein
complexes resistant to detergents that require boil-
ing to dissociate.94 It was also shown that the ATPase,
N-ethylmaleimide-sensitive factor (NSF), and its as-
sociated protein, soluble NSF attachment protein (␣-
SNAP), both previously implicated in membrane fu-
sion, specifically bind the SNARE complex. 217
Further, NSF-dependent ATP hydrolysis dissociated
the complex, possibly serving as a trigger initiating
membrane fusion.215

Mechanisms of Neurotransmitter Release MUSCLE & NERVE May 2001 589

 SNAREs and Fusion. The SNARE hypothesis ex-
tends to the fusion of many other membranes in the
cell, such as those involved in endoplasmic reticu-
lum (ER) to Golgi transport and transport to the
lysosome. Distinct v- and t-SNAREs occur in these
different compartments, suggesting that they confer
the specificity required for appropriate membrane
fusion. Also, SNARE proteins mediate membrane
trafficking and secretion in yeast as well as mamma-
lian cells,18 indicating the generality of the SNARE
hypothesis. However, the precise role of the SNARE
complex remains unclear and it has been proposed
to function in SV docking, fusion, or both.215 De-
spite the identification of different v- and t-SNAREs
on different compartments, they appear to form
complexes promiscuously, raising questions about a
role in the specificity of docking.263 Indeed, disrup-
FIGURE 2. The molecular targets of tetanus and botulinum toxins
tion of the SNARE complex with clostridial toxins
are proteins in the SNARE complexes for SV exocytosis. Tetanus
does not prevent docking106 and morphological toxin cleaves synaptobrevin, whereas, of the seven classes of
docking is not prevented in Drosophila mutants lack- botulinum toxins, four also cleave synaptobrevin, two cleave
ing syntaxin.30 Further, recent observations have led SNAP-25, and the remaining one cleaves syntaxin.
to a revision in the role of the ATPase NSF as a
fusion protein.223 Indeed, NSF-mediated disruption
The conserved residues contributed by each of the
of the SNARE complex after docking neither trig-
helices create an ionic layer within the bundle, pre-
gers nor inhibits fusion, but rather induces a state of
sumably contributing to the high affinity of the com-
membrane hemifusion that does not involve the re-
plex. As type II membrane proteins, the N-termini of
lease of vesicle contents.244 Other work using the
all the SNARE proteins protrude into the cytoplasm
Drosophila temperature-sensitive NSF mutant comatose
and the parallel arrangement suggests that the heli-
suggests that NSF disassembles the SNARE complex
ces “zipper up,” driving the SV and plasma mem-
after fusion in order to retrieve v- but not t-SNAREs
to the synaptic vesicle.129
Clostridial Toxins and SNARE Structure. The ac-
tions of tetanus and botulinum toxins further dem-
onstrate the importance of SNARE complexes for SV
exocytosis.199 These clostridial toxins are composed
of disulfide-linked heavy and light chains. The heavy
chains mediate translocation of the toxins into the
cytoplasm, where the reducing environment sepa-
rates the two chains. The free light chains then func-
tion as endoproteases that inhibit exocytosis by se-
lectively cleaving a SNARE protein at a single site
(Fig. 2). Tetanus toxin cleaves synaptobrevin,
whereas, of the seven classes of botulinum toxins,
four also cleave synaptobrevin, two cleave SNAP-25,
and the remaining one cleaves syntaxin. In addition,
FIGURE 3. A model of the synaptic fusion complex as it joins the
structural studies have recently begun to indicate
membranes of the synaptic vesicle and plasma membrane,
how these cleavage events destabilize the SNARE based on the crystal structure of the SNARE complex. Although
complex and thereby inhibit neurotransmitter re- originally proposed to interact in an anti-parallel manner, which
lease.105,229,261 The crystal structure of the SNARE would suggest a role in docking, v- and t-SNAREs actually ap-
complex also supports a specific role in fusion. Al- pear to form a parallel helix bundle. The N-termini of all the
SNARE proteins protrude into the cytoplasm and the parallel ar-
though originally proposed to interact in an antipa-
rangement suggests that the helices “zipper up,” driving the SV
rallel manner, which would suggest a role in dock- and plasma membranes together. Indeed, the strain placed on
ing, this work shows that v- and t-SNAREs actually the parallel-bound SNAREs may drive membranes already in
form a four-helix bundle in parallel (Fig. 3).89,128,229 close proximity with one another to fuse.

590 Mechanisms of Neurotransmitter Release MUSCLE & NERVE May 2001

branes together. Indeed, the strain placed on the
parallel bound SNAREs may drive membranes al-
ready in close proximity with one another to fuse.
Further supporting a direct role for the SNAREs in
fusion, the addition of a SNARE peptide to the com-
plex cleaved by a clostridial toxin directly triggers
fusion,51 and the v- and t-SNAREs reconstituted into
separate artificial membranes suffice to drive fusion
in vitro.253 However, fusion in this system occurs at a
rate much slower than observed in native mem-
branes, suggesting modulation by other factors.
Modulation and Ca2+ Dependence. Rab Proteins.
Rab proteins regulate membrane trafficking at a
number of different sites within the cell. They be-
long to the ras superfamily of small guanosine tri-
phosphatases (GTPases) and, like the SNAREs, mul-
tiple, distinct isoforms occur on different membrane
compartments.84 In neurons, the rab3a isoform pre-
dominates on SVs.80 Similar to other rabs, rab3a
cycles between a soluble and membrane-bound state.
In the GTP-bound form, rab3a associates with SVs.
After hydrolysis of the GTP to GDP, rab3a dissociates
from the vesicles, a process dependent on the pro-
tein, guanine nucleotide dissociation inhibitor
(GDI).242 It is likely that GTP hydrolysis serves as a
timer that monitors the completion of one reaction
in the SV cycle before initiation of the next, but the
role remains unclear. Rab3a knockout mice show an
initial increase in neurotransmitter release, suggest-
ing that rab3a initially acts to inhibit exocytosis.79
Intriguingly, the knockouts also appear defective in a
presynaptic form of LTP39 and exhibit a profound
depression in neurotransmitter release upon repeti-
tive stimulation, suggesting a depletion of the re-
serve pool of SVs.77 Indeed, alterations in rab func-
tion redistribute SVs away from the nerve terminal in
C. elegans.161
Rab Effectors. Consistent with this role in modu-
lation of SV targeting, a yeast rab homolog binds to
the sec6/8 exocyst complex, possibly contributing to
reversible tethering of vesicles to the plasma mem-
brane.87 Interestingly, yeast rabs also interact with
the soluble factor, Uso1/p115, to promote reversible
vesicle attachment in preparation for SNARE com-
plex formation.36 These fibrous proteins appear to
correspond to the “string” structures observed by
electron microscopy, which may also tether vesicles
to the plasma membrane.163 Uso1/p115 has also
been characterized as a CTP–phosphocholine-
cytidyltransferase-enhancing factor, suggesting that
at least one rab effector modifies lipids.72 In yeast,
rabs may also contribute to the dissociation of inhibi-
tory nsec-1 from the t-SNARE, syntaxin.138 In neu-
Mechanisms of Neurotransmitter Release
rons, a number of rab effectors have been identified,
including rabphilin and RIM, but their precise role
in transmitter release remains unknown.80,84
Ca2+ Channels and SVs. Synaptic vesicle exocyto-
sis differs from most other forms of membrane fu-
sion in its speed and responsiveness to Ca2+. In the
Lambert–Eaton myasthenic syndrome, an autoim-
mune disorder of neuromuscular transmission in
which antibodies are directed against P/Q-type volt-
age-dependent Ca2+ channels, quantal release of
ACh is reduced.198,262 Interference with Ca2+ chan-
nel function appears to impair the tight coupling of
neural activity with Ca2+ entry into the presynaptic
neuron. Indeed, neurotransmitter release occurs
within 1 ms of Ca2+ entry into the nerve terminal,
suggesting that a significant fraction of the docked
SVs exist in a metastable, possibly hemifused, state.34
Proximity to the Ca2+ entry sites also contributes to
the speed of release. SV fusion actually has a rela-
tively low sensitivity to Ca2+ in the 50–100-µmol/L
range, but vesicle docking occurs in close proximity
to the Ca2+ channels. SV exocytosis thus does not
depend on diffusely elevated cytoplasmic Ca2+ but
responds primarily to local increases in Ca2+ near the
active zone.
Ca2+ Channels and SNAREs. Both syntaxin and
SNAP-25 bind to the N- and P/Q-type voltage-
dependent Ca2+ channels known to be responsible
for transmitter release, indicating a link between
Ca2+ entry and SNARE complex formation.40 Fur-
ther, syntaxin and SNAP-25 appear to inactivate Ca2+
channels when expressed individually, but not when
they are assembled into a SNARE complex.26,266 This
suggests that free t-SNAREs inhibit Ca2+ entry, but
vesicle docking relieves this inhibition, restricting SV
fusion to the sites of Ca2+ entry. Recent work also
suggests that Ca2+ entry through P/Q-type channels
influences syntaxin levels by regulating transcription
via mechanisms involving internal Ca2+ stores and
phosphorylation.227 An unusual SV protein known
as the cysteine string protein (CSP) also associates
with Ca2+ channels and may provide an additional
link between SV docking and channel activa-
tion.146,179,243 Interestingly, CSP also contains a DnaJ
domain that suggests a role in protein folding, but
the function of the protein remains unclear.
Synaptotagmin. How do SVs sense a local in-
crease in Ca2+ and use this to drive exocytosis? The
rapid kinetics of fusion suggest that a conforma-
tional or electrostatic change in a Ca2+ sensor, rather
than a large-scale rearrangement of proteins, trig-
gers the final stages of fusion. Both genetic and bio-
chemical evidence further implicate the SV protein
synaptotagmin I as such a Ca2+ sensor.80 Synap-
MUSCLE & NERVE May 2001 591
totagmin I belongs to a family of integral membrane
proteins expressed predominantly in neurons,
where they localize to SVs and other neurosecretory
vesicles such as LDCVs that also undergo regulated
exocytosis.125 The data suggest that synaptotagmin
does not constitute part of the basic fusion machin-
ery, an idea further supported by the absence of
synaptotagmin isoforms in yeast. Importantly, synap-
totagmin I knockout mice lack the fast component
of Ca2+-triggered neurotransmitter release, but show
no abnormality in the slower asynchronous phase of
Ca2+-independent release.78 Synaptotagmin mutants
also show increased spontaneous release of SVs, sug-
gesting that synaptotagmin normally acts to inhibit
transmitter release, but can be overriden by in-
creased Ca2+.29,62 Consistent with this, overexpres-
sion of synaptotagmin in neuromuscular cultures de-
creases spontaneous release, but increases Ca2+-
dependent paired-pulse facilitation.152
C2 Domains. The cytoplasmic region of synap-
totagmins contains two C2 domains (originally iden-
tified in PKC) that bind to phospholipids and Ca2+
with varying affinity.183,224 The affinity of synaptotag-
min I for Ca2+ clearly corresponds to the known,
relatively low sensitivity of SV fusion to Ca2+. The
crystal structure of one of the C2 domains in synap-
totagmin I (C2A) suggests that it binds three Ca2+
ions with the expected affinities.228,241 Further, Ca2+
binding seems to induce electrostatic changes that
may act as a switch to drive exocytosis.206,207 Synap-
totagmin has thus been proposed to act as the Ca2+
sensor for SV exocytosis.59 The existence of multiple
synaptotagmin isoforms with widely varying sensitiv-
ity to Ca2+ may also contribute to the differences in
sensitivity to Ca2+ observed between different popu-
lations of neurosecretory vesicles. Although they
generally require more stimulation to induce fusion
than SVs, LDCVs have an extremely high sensitivity
to Ca2+. They presumably require more stimulation,
because, unlike SVs, LDCVs do not reside in close
proximity to the sites of Ca2+ entry. Rather, LDCVs
sense diffuse elevations in cytoplasmic Ca2+, and sev-
eral synaptotagmin isoforms show the high affinity
for Ca2+ that corresponds to physiological observa-
tions of LDCV exocytosis. However, it remains un-
clear how synaptotagmin contributes to the exocyto-
sis of SVs.
Function of Synaptotagmin. The two C2 domains
may act in very different ways to promote regulated
exocytosis. The C2A domain interacts with syntaxin
as well as phospholipids, and this may promote SV
docking as well as fusion. The C2B domain is also
predicted to bind Ca2+, but this reaction stimulates
synaptotagmin dimerization.43,225 The C2B domain
592 Mechanisms of Neurotransmitter Release
also interacts with SV2,200 a synaptic vesicle protein
with homology to transporters but as yet unknown
function.13,71 Interestingly, SV2 knockout mice show
a Ca2+-sensitive facilitation of neurotransmitter re-
lease, suggesting that SV2 may itself negatively regu-
late fusion by binding Ca2+ or pumping excess pre-
synaptic Ca2+ into SVs.56,109 In one model, Ca2+ may
simply shift the affinity of interactions made by syn-
aptotagmin. At low Ca2+ concentrations, synaptotag-
min binds SV2, which might inhibit fusion. With
Ca2+ entry, synaptotagmin dimerizes via its C2B do-
main and interaction of the C2A domain with syn-
taxin may organize and promote the assembly of
SNARE proteins. The C2A domain also appears to
penetrate the bilayer in a Ca2+-dependent fashion,
which may promote fusion directly.44 Interestingly,
synaptotagmin IV, an isoform with a substitution in a
highly conserved residue of the C2A domain that
abolishes its ability to bind membranes in response
to Ca2+, is upregulated during chronic depolariza-
tion or seizure activity. By assembling with synapto-
tagmin I, synaptotagmin IV may prevent the com-
plex from penetrating the lipid bilayer, reduce
evoked neurotransmitter release, and perhaps con-
tribute to a novel mechanism of synaptic plasticity.
However, many proteins in the nerve terminal in
addition to synaptotagmin could serve as the Ca2+
sensor for exocytosis. The calcium-binding protein,
calmodulin, has been shown to promote Ca 2+ -
regulated exocytosis.222 More recently, two studies in
yeast have suggested that both calmodulin and pro-
tein phosphatase 1 are required for a late step in
constitutive membrane trafficking.171,172 In addi-
tion, a number of proteins in the nerve terminal
contain C2 domains, and thus have the same poten-
tial as synaptotagmin to serve as Ca2+ sensors.
Other C2-Domain–Containing Proteins. Several
proteins involved in synaptic transmission contain
C2 domains that may also bind Ca2+.183 For example,
the evolutionarily conserved neuron-specific verte-
brate ortholog of the C. elegans Unc-13 (Munc-13),
binds syntaxin and contains both C1 and C2 do-
mains.24,32,144,197 Interestingly, the C1 rather than
the C2 domain of Munc-13 appears critical for a late
step in SV fusion and the C1 domain binds the sec-
ond messenger, diacylglycerol (DAG), as well as
phorbol esters.115 Both DAG and phorbol esters in-
duce the translocation of Munc-13 to the plasma
membrane, and Munc-13 mediates the sensitivity of
neurotransmitter release to phorbol esters.23 Impor-
tantly, recent reports have shown that C. elegans, Dro-
sophila, and mouse Unc-13 mutants all have dramatic
reductions in both action potential-evoked and
spontaneous, Ca 2+ -independent release. 8,10,182
MUSCLE & NERVE May 2001
Munc-13 may also link G-protein–coupled neuro-
transmitter receptors with neurotransmitter release.
ACh and serotonin receptors couple to G proteins
that activate the production of DAG. C. elegans mu-
tants in the corresponding receptors show differ-
ences from wild-type in neurotransmitter release via
the DAG/Unc-13 pathway.121,151 Together with a role
in coupling neurotransmitter release to receptor sig-
naling, the requirement for both evoked and spon-
taneous release suggests that Munc-13 may have a
central role in SV fusion. The synaptic proteins
DOC2, rabphilin, and RIM also contain C2 domains,
but their role in transmitter release remains less
clear. Further, Ca2+-dependent regulation may occur
via proteins without C2 domains such as the Ca2+-
dependent activator of secretion (CAPS), a phospha-
tidylinositol 4,5-bisphosphate-binding protein that is
selectively required for catecholamine secretion
from LDCVs but not glutamate release from SVs (dis-
cussed in what follows).64
Large, Dense-Cored Vesicles. The release of neu-
ral peptides from LDCVs shares certain similarities
with release of classical transmitters from SVs, but
also exhibits a number of important differences.143
Like SVs, LDCVs contain the v-SNARE, synaptobre-
vin, and use the same t-SNAREs, syntaxin and SNAP-
25.258 LDCVs also contain synaptotagmin I, although
different isoforms may be responsible for the high
sensitivity of LDCV fusion to Ca2+.125 However,
LDCV exocytosis appears to involve several mecha-
nisms distinct from those involved in SV release. In
particular, an elegant biochemical analysis of LDCV
fusion has indicated two distinct phases.92 The first
phase involves a priming step that requires ATP and
probably follows SV docking at the plasma mem-
brane. The factors responsible for this step include
the phosphatidylinositol (PI) transfer protein
(PITP), PI-4-kinase, and PI(4-P)-5-kinase.91,93,255
Priming thus appears to involve sequential lipid
modification that forms PI(4,5)-bisphosphate. Inter-
estingly, the second phase of LDCV exocytosis in-
volves a triggering step that requires Ca2+. Purifica-
tion of the factor responsible for Ca2+-dependent
triggering has yielded the Ca2+-dependent activator
of secretion protein (CAPS). CAPS is the vertebrate
ortholog of the C. elegans Unc-31 protein, which has
been implicated in the release of neural peptides,
serotonin, and perhaps ACh, supporting a role for
CAPS in LDCV exocytosis.6,11,250 Further, CAPS is a
soluble protein that associates with LDCVs and the
plasma membrane by binding to PI(4,5)-bisphos-
phate,137 the product of the priming reaction. CAPS
does not bind to SVs or appear important for their
exocytosis.22,235 However, sequential lipid modifica-
Mechanisms of Neurotransmitter Release
tion may nonetheless contribute to SV exocytosis,
possibly through the involvement of a protein dis-
tinct from Unc-31.256 In addition, it is appears that
SV recycling prominently involves modulation by
both Ca2+ and lipids.
Endocytosis and Recycling. Clathrin and AP-2.
How does the nerve terminal maintain a relatively
constant surface area despite high rates of exocyto-
sis? After exocytosis, empty SVs are rapidly endocy-
tosed, a process dependent on the formation of a
clathrin coat on nascent endocytotic vesicles. Cla-
thrin presumably initiates invagination after recruit-
ment to the plasma membrane by the clathrin adap-
tor protein AP-2.55,142 According to one model, Ca2+
signals endocytosis as well as exocytosis, and Ca2+
does appear to be required for internalization.96 Al-
though it remains unclear how Ca2+ promotes SV
endocytosis, it is of interest that synaptotagmin, the
putative Ca2+ sensor for exocytosis, has also been
implicated in endocytosis. Synaptotagmin mutants
indeed show reduced numbers of SVs, implicating
the protein in endo- as well as exocytosis, but the SV
depletion could also result from increased, unregu-
lated fusion.111 Importantly, synaptotagmin binds
AP-2, and thereby provides a mechanism to link Ca2+
signaling with clathrin-mediated endocytosis.45,90,265
The simple appearance of SV proteins, such as syn-
aptotagmin or synaptobrevin, on the plasma mem-
brane may thus activate the endocytic machinery.
Dynamin. The late phase of endocytosis re-
quires dynamin, which binds to the nascent vesicles
at the margin of the clathrin coat.201 At nonpermis-
sive temperature, the Drosophila temperature-
sensitive dynamin mutant shibire rapidly becomes
paralyzed and electron microscopy has shown a
marked depletion of SVs.50,174,245 Dynamin func-
tions as a GTPase and blocking dynamin-mediated
GTP hydrolysis in a preparation of nerve terminals
induces the formation of a spiral collar around the
neck of vesicles invaginating from the plasma mem-
brane.102,231 This suggests a role for dynamin as the
“pinchase” involved in SV retrieval.252 Although re-
cent work has questioned a direct mechanical role
for dynamin as the pinchase, it nonetheless appears
to have a role in orchestrating endocytosis and the
recycling of SVs.205 In particular, dynamin may func-
tion by associating with a series of accessory endo-
cytic proteins such as amphiphysin, synaptojanin,
and endophilin.31,58,60,149 Disruption of the interac-
tion with amphiphysin interupts the SV cycle at a
position shortly after membrane invagination.211
Synaptojanin exhibits phosphotydilinositol 5-phos-
phatase activity that may reverse the lipid modifica-
MUSCLE & NERVE May 2001 593
tions involved in priming.148 Endophilin appears to
function as a lysophosphatidic acid arachidonate
transfer protein, which may contribute to the nega-
tive membrane curvature required at the margin of
the nascent endocytotic vesicle.202
“Kiss-and-Run” versus Full Fusion. The local recy-
cling of SVs at the nerve terminal is another special-
ization of neurons, which obviates the need for a
continuous supply of new SVs to be transported from
the neuronal soma, thereby also enhancing effi-
ciency. Indeed, SVs may be recycled in a number of
different ways. Instead of full exocytosis, SVs may
form a transient fusion pore, allowing transmitters to
diffuse out, and then recycle instantly by closing the
pore (“kiss-and-run” exocytosis).3,52 After transmit-
ter release, closure of the pore would regenerate the
original SV, albeit without or with less transmitter.
This “kiss-and-run” model of transmitter release has
not been demonstrated directly for SVs, but the
study of LDCV exocytosis does support its existence.
In particular, measurements of cell capacitance,
which provide a direct measure of plasma mem-
brane surface area, coupled with electrochemical
measurement of monoamine release from LDCVs by
amperometry, indicate that a slow leak of transmitter
often precedes the bulk release from an LDCV.1,2
This slow leak presumably begins with the “flicker-
ing” of a fusion pore and the leakage of soluble
transmitter. However, the bulk of monoamine in an
LDCV cannot be released until the pore dilates
enough to allow solubilization of the dense core,
followed by its diffusion into the extracellular me-
dium.257 Also, solubilization and release of the core
appear to depend on presynaptic autoreceptor activ-
ity, suggesting that the fusion pore constitutes a ma-
jor locus for the regulation of transmitter release.5
Since SVs are considerably smaller than LDCVs, it is
more difficult to detect the exocytosis of an indi-
vidual SV, but SV fusion may involve flicker as well.
“Kiss-and-run” also greatly simplifies the recycling of
SVs, which are regenerated directly from the plasma
membrane.
Recycling and Biogenesis. In neurons, the regen-
eration of SVs directly from the plasma membrane
also appears to predominate after full fusion.154
However, some SVs undergo a more complex indi-
rect recycling pathway that involves endosomal inter-
mediates. Biochemical analysis has implicated the
adaptor protein AP-3 specifically in sorting certain
proteins to SVs through this indirect pathway.70,208
This suggests either that multiple trafficking path-
ways converge on a single population of SVs, or that
multiple, distinct, populations of SVs exist. Interest-
ingly, although many proteins occur on all SVs in the
594 Mechanisms of Neurotransmitter Release
nerve terminal, certain proteins occur only on sub-
populations further away from the synaptic cleft that
may correspond to the reserve pool. The functional
significance of these observations remains unclear,
but they indicate the potential for novel modes of
signaling and regulation.
CONCLUSIONS
Neurotransmitter release constitutes a central yet
poorly understood aspect of synaptic transmission.
Two distinct cycles involve the neurotransmitter it-
self and the membrane components, including lip-
ids as well as proteins. Within each of these cycles,
additional cycles monitor passage through the re-
quired biochemical reactions. Many of the proteins
involved in transmitter release have now been iden-
tified but it is still not understood how many of them
integrate into these cycles. Together with biochemi-
cal analysis, future work will address the physiologi-
cal consequences of these cycles and contribute to
our understanding of synaptic plasticity and neuro-
psychiatric disease.
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