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Neuroscience Center and Department of Pharmacology, Louisiania State University Health Sciences Center, New Orleans, Louisiana 70112, USA
1Correspondence: LSUHSC/Neuroscience, 2020 Gravier St., Suite D, New Orleans, LA 70112, USA. E-mail: jerick{at}lsumc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESIDUES IMPLICATED IN TRANSPORT...DOMAINS IMPORTANT FOR SUBSTRATE...TARGETING IN FIBROBLASTSTARGETING IN NEUROENDOCRINE PC12...TARGETING IN NEURONAL CAD...DOMAINS IMPORTANT FOR TARGETING...VESICULAR TRANSPORT ACTIVITY,...FUTURE PERSPECTIVESREFERENCES |
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Key Words: CAD • PC12 • biogenic amines • acetylcholine • small synaptic vesicle • large dense core vesicle • synaptic-like microvesicle • trafficking • quantal release
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESIDUES IMPLICATED IN TRANSPORT...DOMAINS IMPORTANT FOR SUBSTRATE...TARGETING IN FIBROBLASTSTARGETING IN NEUROENDOCRINE PC12...TARGETING IN NEURONAL CAD...DOMAINS IMPORTANT FOR TARGETING...VESICULAR TRANSPORT ACTIVITY,...FUTURE PERSPECTIVESREFERENCES |
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, 2
). These biogenic amines, with the exception of
histamine, are also transported by the VMAT1 isoform, found only in
neurohormone-containing large dense core vesicles (LDCV) in various
endocrine and neuroendocrine cells. The vesicular acetylcholine (ACh)
transporter (VAChT) is a third member of this transporter gene family
and represents a single isoform expressed exclusively on small synaptic
vesicles (SSV) in all cholinergic neurons (reviewed in ref
3
). These vesicular amine transporters share a similar
structure and use similar bioenergetic mechanisms for substrate
accumulation into secretory organelles. Various drugs specifically
interact with these proteins and decrease transmitter levels in
synaptic vesicles. Reserpine is a competitive antagonist of both VMAT
isoforms, but once bound becomes occluded in the protein and
irreversibly blocks transport, vesicular storage, and quantal release
(4
, 5)
. Tetrabenazine and vesamicol are noncompetitive
antagonists of VMAT2 and VAChT, respectively, have short durations of
action, and block vesicular transport and secretion
(6
7
8)
. Psychostimulants such as amphetamine also directly
interact with VMAT2 (9
, 10)
and act, at least in part, by
promoting release of vesicular stores of biogenic amines into the
cytoplasm (11)
.
VMAT2 is unique in that it is expressed on several classes of regulated
secretory organelles. In most instances, biogenic amines are stored in
LDCVs (120–160 nM) together with neural peptide hormones. In
noradrenergic neurons of the CNS, for instance, VMAT2 is preferentially
found on LDCVs and but it is also seen on SSVs (45–50 nM) by
immunoelectron microscopy (12)
. In dopaminergic neurons of
the substantia nigra, VMAT2 is found primarily on SSVs in axons and in
tubulovesicular structures, i.e., the smooth endoplasmic reticulum, in
dendrites (13
, 14)
. In these dendritic organelles, storage
and release of dopamine is reserpine sensitive, similar to that
described for the dopaminergic nerve terminals in striatum
(15)
. In peripheral neurons, monoamines are stored in
secretory vesicles that contain markers for both SSV and LDCV membranes
(16)
, are intermediate in size (60–80 nM), and referred
to as small dense core vesicles (SDCV) (17)
. The different
subcellular localizations of VMAT2 in various cells may be due, in
part, to the types of regulated secretory organelle present in a given
cell type.
Membrane proteins destined for LDCVs or SSVs differ in their
trafficking from the cell body. Since LDCVs contain neuropeptide
hormones, they must be produced at the trans-Golgi network
(18)
. SSV proteins on the other hand are thought to reach
the nerve terminal via constitutive (nonregulated) vesicles (19
, 20)
. In peripheral neurons, VMAT2 is thought to reach the SDCV
via the LDCVs (21)
. A second degree of membrane
trafficking occurs in the nerve terminal. There, SDCVs and SSVs can
recycle via endocytosis and budding from ‘early’ endosomes or from an
intermediate endosome-like compartment that is continuous with the
plasmalemma (22
23
24
25
26
27)
.
In this study, we review work on the structure and function of the
vesicular amine transporters with emphasis on the domains of the
proteins that might participate in the amine/H+
translocation pore and be involved in differential substrate
specificity. In addition, we review the evidence that the trafficking
of these proteins to different secretory organelles in vitro
depends on the class of organelle present in the cell and relies, in
part, on information contained in their cytoplasmic tails. Since
different levels of transporter expression on vesicles may affect the
rate at which the vesicles are filled with transmitter and perhaps the
maximal level of accumulation possible, the size of the releasable pool
in vivo may not be constant (28
29
30)
. Thus,
quantal amine secretion may depend on several factors including the
intrinsic activity of the transporters, their level of expression and
subcellular localization, in addition to the secretory activity of the
cell.
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RESIDUES IMPLICATED IN TRANSPORT FUNCTION |
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, 31
, 32)
and SSVs isolated from the marine ray Torpedo
(8)
. The movement of amine substrates across membranes of
secretory organelles is powered by a transmembrane electrochemical
H+ gradient generated by the vacuolar
H+-ATPase. The complete transport cycle depends
on the counter-transport of two protons (33
, 34)
. The
first proton to efflux from the vesicle interior is thought to generate
a high-affinity amine uptake recognition site. In the case of VMATs,
this is also the reserpine binding site. Efflux of a second proton then
induces a second conformational change that moves the amine across the
membrane and into the vesicle. In this process, the uptake site is
converted into a substrate discharge site that exhibits low affinity
for substrates.
Molecular analysis of vesicular amine transporter function has enabled
the testing of this model in heterologous systems. Initially,
Schuldiner and co-workers identified a histidine residue in the
cytoplasmic loop between TMDs 10 and 11 of VMAT1, which upon
protonation is thought to induce the conformation shift that exposes
the high-affinity amine binding site (35)
. Aspartic acid
residues that are predicted to lie in the middle of putative
membrane-spanning domains (TMD) 1,6,10 and 11 of both VMAT1 and VMAT2
were then examined for their importance in transport function.
Site-directed mutagenesis of these residues indicates that some of them
play discrete roles in the transport cycle. Edwards and colleagues have
shown that the aspartate in TMD 11 forms an ion pair with a conserved
lysine residue in TMD 2 (36)
. This interaction increases
slightly the affinity of the protein for substrate but is not required
for transport as the double alanine substitution retains functional
activity. The salt bridge may therefore indirectly promote
high-affinity substrate recognition and be important for overall
structure of VMAT2 (37)
. Replacement of aspartic acid
residues in TMDs 1 with asparagine or alanine results in proteins that
cannot catalyze the transport of 5HT (2
, 37)
. Reserpine
binding is unaffected, indicating that the coupling to the first
H+ and subsequent conformational change occurs.
However, the ability of 5HT to displace reserpine is significantly
reduced, suggesting a specific defect in substrate recognition
(37)
. Replacement of the aspartate in TMD 1 with glutamate
reduces the maximal level of transport but does not affect the affinity
for 5HT or reserpine (37)
. Likewise, a negative charge in
TMD 10 is essential for transport function. However, replacement of
this aspartate in VMAT1 with glutamate modifies the pH profile and
tetrabenazine sensitivity (38)
. TMD 10 may therefore be
important in steps after ligand recognition and coupling to the first
H+ such as H+/antiport. The
aspartic acid in TMD 6 is not important for transport function
(36)
and may not actually reside within a
membrane-spanning domain. Together, these studies suggest that the
aspartic acid residues in TMD 1 and 10 bind protonated substrates and
protons to facilitate their exchange across the vesicle membrane.
VAChT contains an additional conserved aspartic acid in the fourth TMD.
Hersh and co-workers have recently reported that aspartate residues in
TMD 1 and TMD 4 of VAChT do not participate in transport of ACh into
synaptic-like microvesicles when transiently expressed in PC12 cells
(39)
. We have confirmed these results with alanine
replacement of these residues in human VAChT using stable PC12
transformants (unpublished data). These results contrast with those
obtained with VMAT2, where a negative charge in TMD 1 is essential for
biogenic amine transport. They are also at odds with the work of Song
et al. (29)
, which suggests that the aspartic acid residue
found in TMD 4 of VAChT is essential for transport function.
Microinjection of rat VAChT in Xenopus embryos results in a
significant increase in miniature end-plate currents at neuromuscular
junctions that was not observed when TMD 4 aspartate of rat VAChT was
changed to asparagine. Hersh and co-workers propose that this
discrepancy may indicate a novel role of this aspartate in mediating
ACh release (39)
. Although neither aspartate residues in
TMD 1 and 4 of VAChT alone are required for ACh transport, when both
are replaced with alanine the affinity of VAChT for ACh and vesamicol
decreases dramatically (Zhu et al., unpublished results). This
indicates that these aspartate residues are in fact necessary for ACh
transport, and having two may simply be redundant in VAChT. That is,
the additional aspartate residue found in VAChT compared to the VMAT
isoforms may have evolved in an attempt to increase the low-affinity
VAChT displays for ACh (Km
1 mM compared to µM
range for VMATs) or the efficiency of transport compared to VMAT1 and
2. The role of TMD 1 in the ACh transport function of VAChT is further
supported by chimeric analysis in which a decreased affinity for ACh is
observed when TMD1 of VMAT2 is present (40)
. As VAChT and
the VMAT isoforms share similar structure and function, it would not be
surprising that TMDs 1 and 10 serve similar roles in
amine/H+ exchange.
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10- to 20-fold compared to VMAT2. VMAT2 displays similar affinity
for these substrates as it does for 5HT, except that histamine is
10-fold lower (9
, 10
, 41)
.
Edwards and colleagues have mutated numerous residues in VMAT2 in an
effort to decrease high-affinity recognition of 5HT, histamine, and
tetrabenazine to that displayed by VMAT1. They have concluded that
multiple residues contribute to the interaction of VMAT2 with ligands
(42
, 43)
. These studies have suggested that tyrosine-434
in TMD 11 of rat VMAT2 interacts with the hydroxyl group of 5HT.
Previously, it was thought that the hydroxyl groups on the catechol or
indole ring interact with a group of serines in the third TMD
(37)
. Whereas many mutations decrease the apparent
affinity of VMAT2 for 5HT, histamine, and tetrabenazine, introduction
of these residues from VMAT2 into the equivalent positions of VMAT1
does not confer high-affinity recognition of ligands (43)
.
It is therefore difficult to determine whether these residues interact
directly with the substrate or whether they exert an indirect effect on
protein structure.
A chimeric approach to determine the basis for the substrate
specificity of VMAT1 and VMAT2 may also yield information regarding the
putative TMDs involved in the translocation of amine substrates across
the vesicle membrane. Simple chimeric transport proteins were first
constructed between rat VMAT1 and VMAT2, and it was determined that two
domains (TMDs 5–8 and 9–12) are apparently both required for
high-affinity interaction of ligands with VMAT2 (44)
. We
followed this approach and have constructed a series of double chimeric
transport proteins between human VMAT1 and VMAT2 in an effort to
localize VMAT2 TMDs that confer high-affinity substrate recognition. We
had shown previously that a chimera (2/1/2) in which the region
containing half of TMD6 through TMD 10 of VMAT2 is replaced with VMAT1
sequences is important for histamine recognition (45)
.
This chimera retains high-affinity recognition of amphetamine,
indicating that discrete domains of VMAT2 are important for the
interaction of histamine and other unsubstituted aromatic amines.
Even though histamine is a relatively poor substrate for VMAT2
compared to amphetamine, it too is competitive with 5HT for transport.
When expressed in permeabilized fibroblasts, VMAT1 and VMAT2 display
affinities for histamine that are 1 mM and 95 µM, respectively, at
pH 8. Thus, VMAT2 shows approximately a 10-fold greater affinity for
histamine than VMAT1. Using these uptake conditions, we have further
localized the domain important for histamine recognition to the region
containing half of TMD6 to the beginning of TMD8. Again, this chimera
retains high-affinity recognition of phenylethylamine and tetrabenazine
similar to VMAT2. The corresponding 1/2/1 chimera does not display
high-affinity histamine recognition, which means that other regions of
VMAT2 are important as well. Preliminary observations suggest that a
2/1/2 chimera in which the 11th TMD of VMAT1 is present in VMAT2
display reduced affinity toward histamine similar to VMAT1 (Yao et al.,
unpublished results). In agreement with earlier studies, it is likely
that multiple TMDs interact to form substrate binding sites and that
differential sensitivity to unsubstituted aromatic amines, such as
histamine and amphetamine relies on unique differences within different
TMDs. Chimeric molecules where individual TMDs are swapped may
ultimately yield a ‘gain of function’ phenotype for VMAT1, similar to
VMAT2, and provide conclusive information as to the domains comprising
the amine translocation pore.
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, 47)
.
The ability of fibroblasts to support transport of biogenic amines and
the energetic requirement of this process indicates that these
transporters are targeted to intracellular compartments that contain an
electrogenic vacuolar type H+ pump. Whereas
studies of VAChT transport properties are usually determined in
transfected PC12 cells (28)
, like the VMAT isoforms it is
also targeted to the endosomal compartment in fibroblasts
(48)
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TARGETING IN NEUROENDOCRINE PC12 CELLS |
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.
SLMVs in PC12 cells are thought to be the neuroendocrine equivalent of
cholinergic SSVs. Since VMAT2 is found on both LDCVs and SSVs in CNS
neurons, several groups have questioned whether VMAT2 might be targeted
to both LDCVs and the SLMVs in stably transfected PC12 cells. By
immunoelectron microscopy, VMAT2 is found exclusively on LDCVs in PC12
cells differentiated with nerve growth factor (50
, 51)
.
Western blot analysis of subcellular fractions from sucrose equilibrium
density gradients of homogenates of VMAT2-expressing cells indicates
that VMAT2 is found in heavy fractions containing VMAT1 and
chromogranin B, markers of LDCVs and absent in lighter fractions
containing synaptophysin (p38), a marker of SSVs (51)
.
VMAT2 is targeted to LDCVs, similarly to the endogenous expression of
the endocrine-specific VMAT1 isoform, and VAChT is preferentially
targeted to SLMVs in PC12 cells, similarly to the endogenous expression
of the rat VAChT protein (50
51
52)
. Thus, VMATs appear to
be excluded from SLMVs of PC12 cells.
When VAChT is overexpressed in PC12 cells, it can be detected on LDCVs
(48
, 51)
. However, the endogenous VAChT protein is rarely
detected on LDCVs in control PC12 cells (50
, 53)
and is
predominantly on SSVs in cholinergic axon terminals in situ
(54)
. It has been hypothesized that VAChT travels from the
cell body to the nerve ending via an LDCV (2
, 3)
. In
autonomic postganglionic sympathetic cholinergic fibers, however,
vasoactive intestinal polypeptide is present in rather large granular
structures whereas VAChT is present mostly in small vesicles
(55)
. Based on studies with transfected PC12 cells, it has
been purported that phosphorylation-dependent trafficking of VAChT to
LDCVs may endow LDCVs with the ability to accumulate and release ACh
(56)
. In central cholinergic nerves and those of
Torpedo, SSVs far outnumber LDCVs, in contrast to the
situation in PC 12 cells, where LDCVs predominate. Furthermore, ACh and
vasoactive intestinal polypeptide can selectively be released after
stimulation of peripheral cholinergic nerves innervating the myenteric
plexus (57)
. This differential release depends on the two
secretory organelles having different content and differential
responsiveness to stimulation. Furthermore, this requires efficient
sorting of VAChT only to SSVs. It is possible that phosphorylated VAChT
exits the trans-Golgi via an LDCV in PC12 and is then
dephosphorylated in the nerve terminal allowing it to reach the SMLV
compartment.
Since VMAT2 is expressed on LDCVs and not on SSVs in PC12 cells,
cholinergic and monoaminergic small vesicles may be fundamentally
different and have unique biosynthetic origins. In peripheral
noradrenergic nerves, once the LDCVs reach the nerve ending and release
their content by exocytosis, the membrane proteins are able to recycle
and SDCVs are formed (21
, 58)
. Secretagogue-triggered
transfer of membrane proteins from LDCVs to SLMVs has been shown in
PC12 cells (59)
. These studies examined the trafficking of
P-selectin, a protein that is targeted to both the LDCV and the SLMV
when transfected in PC12 cells. To determine whether VMAT2 might also
be transferred to SLMVs after activation of the LDCV recycling pathway,
stable VMAT2-expressing PC12 cells were treated with 10 mM carbachol
for 10 min at 37°C in the presence of DMEM, washed, and incubated in
medium without carbachol for 30 min prior to harvesting. SLMVs were
then purified by glycerol velocity centrifugation (60)
and
assayed by Western blotting and 3H-TBZ binding.
Neither an increase in VMAT2 immunoreactivity nor an increase in the
binding of 3H-TBZ was observed in fractions that
were positive for synaptophysin after carbachol stimulation
(unpublished observation). These results indicate that VMAT2 does not
recycle in the SSV pathway in transfected PC12 cells and suggest that
it is specifically retrieved to LDCVs (via the trans-Golgi
network) after its appearance at the plasma membrane.
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TARGETING IN NEURONAL CAD CELLS |
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. The smaller, clear vesicles in CAD are of
approximately the same size as SDCVS that are found in peripheral
noradrenergic neurons. SDCVs do not contain soluble neuropeptides but
do contain the membrane bound form of dopamine ß-hydroxylase. They
also show an electron dense core when certain chemical fixations (for
example, permanganate) are used that prevent the loss of
catecholamines. Since CAD cells do not express dopa decarboxylase, they
do not contain dopamine or norepinephrine; hence, these small secretory
vesicles are clear. Furthermore, CAD cells have lost expression of
VMAT2 that is normally expressed in noradrenergic neurons of the locus
coeruleus. Transient expression of hVMAT2 in undifferentiated CAD cells
reveals that it is targeted to two populations of organelles when the
cells are fractionated by sucrose density gradients (unpublished
observation). When postnuclear supernatants of VMAT2-expressing CAD
cells are incubated with 3H-5HT and then
fractionated by sucrose density gradients, two peaks are observed that
correspond to fractions that contain VGF, a neural peptide and marker
of LDCVs (62)
, and synaptophysin, a marker of SSVs. When
VMAT2 is assayed by Western blots, a predominant labeling is seen in
light fractions positive for synaptophysin and synaptotagmin. This
situation is clearly different than that observed with VMAT2-expressing
PC12 cells (51)
. Thus, the lack of monoaminergic SSVs (or
SDCVs) in PC12 cells could be explained by the absence from PC12 cells
of additional proteins or factors present in CAD cells that allow VMAT2
to reach the SSV compartment or allow the biogenesis of a specific
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. In contrast, a VAChT chimera that
contains the cytoplasmic tail of hVMAT2 is relatively excluded from the
SLMVs in PC12 cells. Thus, the cytoplasmic tails of the vesicular
transporters contain information that allows or restricts the
expression of VAChT and VMAT2 on recycling SLMVs in PC12 cells. It is
not known whether the cytoplasmic tails of these proteins are necessary
or even sufficient for SSV targeting in neuronal cell lines or in
vivo. Since many sorting decisions are made during the transit
from the Golgi stacks, in endosomal compartments and during recycling,
additional cytoplasmic regions likely play important roles as well.
Deletions of the cytoplasmic COOH terminus of VMAT2 have been examined
to determine whether a domain within it is required for small secretory
vesicle targeting in the neuronal CAD cell line (unpublished
observations). For these studies, VMAT2 contains a myc epitope tag
inserted into the large lumenal loop between TMD 1 and 2 that does not
interfere with transport activity or subcellular localization in CAD
cells. Transient expression of various carboxyl-terminal VMAT2
truncations in undifferentiated CAD cells and visualization under
epifluorescence microscopy revealed no differences in the punctate
pattern of distribution except when the 13 amino acids in the
cytoplasmic tail of VMAT2 closest to the 12th TMD are deleted. Within
these amino acids is a highly conserved KEEKMAIL sequence that is found
in both VMAT isoforms in bovine, rat, and human proteins. When this
region is deleted, accumulation of VMAT2 at the plasma membrane is
observed and the punctate pattern of fluorescence is lost. Recently, it
has been shown that the isoleucine-leucine pair within this KEEKMAIL
sequence of VMAT2 functions as a signal for endocytosis
(63)
. In these studies, VMAT2 constructs were transfected
into fibroblasts as well as neuroendocrine PC12 cells. Our results in
neuronal CAD cells support these observations. Dileucine-based
internalization signals in many membrane proteins are recognized by
clathrin adaptor proteins AP-1, 2, and 3 (64)
. Selective
sorting of vesicular proteins may require AP3 (27
, 65
, 66)
. Since the sequences immediately preceding the leucine-based
endocytosis signals of VAChT and VMAT2 are highly conserved across
various species, these differences may contribute to a selective
interaction with APs and therefore to differences in trafficking of
these proteins within the secretory pathway.
Recently, it has been shown that VAChT is phosphoryated by protein
kinase C (67)
and that the residue responsible (serine;
480) lies in the cytoplasmic tail (56
, 68)
.
This serine is located 5 amino acids upstream of a dileucine motif in
VAChT. VMAT2 has two glutamic acid residues (E478/E479) at the
corresponding position, which is located 5 amino acids upstream of the
isoleucine-leucine pair. These dileucine-like pairs may be important
for efficiency of endocytosis (63)
. Using a transient
expression system, Hersh and co-workers demonstrate that mutation of
serine (480) to alanine shifts the localization of
immunoreactivity in sucrose density gradients to fractions intermediate
of those containing synaptophysin and secretogranin II, indicating that
phosphorylation of VAChT is required for SMLV targeting in PC12 cells
(68)
. In the stably transfected PC12 lines of Edwards and
colleagues, however, neither the serine (180) to alanine
mutation nor the serine (180) to glutamate mutation affect
the relative level of targeting of VAChT to SMLVs (56)
.
Instead, these mutations in VAChT, as well as the double glutamate
(E478A/E479A) to alanine mutation in the cytoplasmic tail of VMAT2
appear, to some degree, to affect the relative amounts of these
proteins on LDCVs (56)
.
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The cytoplasmic concentration of transmitter available for
sequestration is an important determinant for the level of vesicular
storage and amount available for secretion. The levels of dopamine
(0.5–2 µM) and ACh (0.2–1 mM) in the cytoplasm are around the
half-maximal substrate concentrations for their respective transporters
(8
, 69)
. Thus, changes in biosynthesis or degradation of
these transmitters could affect the rate that vesicles could fill and
the levels attained at equilibrium. Sulzer and co-workers have shown in
tissue culture that the number of dopamine molecules per quantum
released from LDCVs (in PC12 cells) and SSVs (in midbrain dopamine
neurons) increases severalfold after exposure to the dopamine precursor
L-dihydroxyphenylalanine (70)
. Similar effects are seen in
cholinergic systems in vitro and in vivo, where
administration of choline is rapidly accumulated by cholinergic
neurons, enhancing both the biosynthesis and release of ACh
(71
72)
. Furthermore, genetic mutants of choline
acetyltransferase in nematodes and flies that have reduced enzyme
activity exhibit behavioral phenotypes consistent with reduced
cholinergic neurotransmission (73
, 74)
.
Alterations in the molecular structures that determine intrinsic
activity, such as the substrate affinity or the efficiency of transport
(Vmax/Km), also
affect vesicular accumulation of transmitter and subsequent regulated
neurotransmitter release. Rand and colleagues have assembled a
collection of unc-17 point mutants that express normal
amounts of VAChT on SSVs but show resistance to esterase inhibition and
exhibit behavior changes consistent with a deficit in ACh
neurotransmission (75)
. Expression of the corresponding
mutant VAChT proteins in a heterologous system has revealed that
several display reduced affinity for ACh, indicating that the mutations
were in TMDs directly involved in ACh recognition (unpublished data).
Others are in regions that affect Vmax
only or the binding of vesamicol, and may be important in steps of the
ACh transport cycle beyond substrate recognition such as
H+ exchange. These results indicate that specific
structural changes in VAChT can be correlated with specific alterations
in the biochemical parameters of transport, neurosecretion, and
behavioral severity of the cholinergic deficit.
The level of expression of the vesicular transporters in whole
organisms and in transfected cells is yet another way to influence the
amount of transmitter stored in each synaptic vesicle and therefore the
amount released from neurons. Heterozygotic VMAT2 knockout mice display
a 50% decrease in protein expression, which results in corresponding
decreases in vesicular transport activity and quantal release
(30)
. In heterozygotic VAChT knockout flies, a 50%
decrease causes an abnormality in cholinergic transmission when
stressed by a high-frequency stimulus (76)
. Overexpression
of VAChT in PC12 cells and in Xenopus embryos increases ACh
accumulation in SSVs and quantal size by two- to threefold (28
, 29)
. Different levels of transporter expression on synaptic
vesicles may account for the heterogeneity of transmitter storage and
release observed in several systems (77
78
79
80)
.
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82
83)
of the
vesicular transporters is a relatively unexplored area, but recent data
suggest that it may be a general feature of monoaminergic neurons that
controls the content of both LDCVs and SSVs (82)
. Evidence
that ACh storage and release can be modified by protein kinases
suggests that VAChT might be regulated at the level of vesicular
transport or targeting to secretory organelles (83
84
85
86)
.
VAChT is phosphorylated in rat brain synaptosomes and in PC12 cells by
protein kinase C (56
, 67
, 68)
. VMAT2 is also
phosphorylated by casein kinase II (87)
. Whether
phosphorylation modulates intrinsic activity and/or regulates
trafficking to secretory organelles, the rate at which vesicles
accumulate transmitter, and the amount of transmitter released on
neurosecretion may be altered. Identifying potential sites for
regulation of vesicular amine transport function may therefore be
important during development of cholinergic and monoaminergic synapses
and an important mechanism for synaptic plasticity. |
ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESIDUES IMPLICATED IN TRANSPORT...DOMAINS IMPORTANT FOR SUBSTRATE...TARGETING IN FIBROBLASTSTARGETING IN NEUROENDOCRINE PC12...TARGETING IN NEURONAL CAD...DOMAINS IMPORTANT FOR TARGETING...VESICULAR TRANSPORT ACTIVITY,...FUTURE PERSPECTIVESREFERENCES |
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