The two closely related vesicular monoamine
transporters (VMATs) 1 and 2 differ substantially in ligand
recognition. The neuronal VMAT2 exhibits a higher affinity for
monoamine substrates and in particular for histamine as well as a
greater sensitivity to the inhibitor tetrabenazine than the nonneuronal
VMAT1. The analysis of chimeric transport proteins has previously shown
that two major domains, one spanning transmembrane domains (TMDs) 5-8
(TMD5-8) and the other, TMDs 9-12 (TMD9-12), are required for the
high affinity interactions characteristic of VMAT2. Using site-directed mutagenesis to replace residues in TMD5-8 of VMAT2 with the equivalent residues from VMAT1, we now show that the sensitivity of VMAT2 to
tetrabenazine requires Ala-315, and this interaction occurs independently of the interaction with residues in TMD9-12. The ability
to recognize histamine as a substrate depends on Pro-237, and the
contribution of TMD9-12 to histamine recognition appears to involve a
common mechanism. In contrast, the replacement of many residues in
TMD5-8 of VMAT2 with equivalent residues from VMAT1 improves the
recognition of both serotonin and tryptamine, and these mutations show
a dominant effect on the recognition of both tryptamine and serotonin
over mutations in TMD9-12. The results indicate that different ligands
interact through distinct mechanisms with the VMATs and that the
recognition of each ligand involves multiple, independent interactions
with the transport protein.
 |
INTRODUCTION |
Neurotransmitters are packaged into secretory vesicles so that
their release can be regulated by exocytosis. In the case of monoamine
transmitters, two closely related vesicular transport proteins have
been identified (VMAT1 and
VMAT2)1 (1, 2). VMAT1 is
expressed in adrenal medulla and other nonneuronal cells, whereas VMAT2
appears in multiple monoamine populations of brain as well as
sympathetic neurons, mast cells, and histamine-containing cells in the
gut (3, 4). In addition to their differences in distribution, the two
VMATs differ in ligand recognition and pharmacology. VMAT2 has a higher
apparent affinity for dopamine, norepinephrine, and serotonin than
VMAT1 (5, 6). Furthermore, VMAT2 transports histamine, whereas VMAT1
does not, presumably accounting for the expression of VMAT2 but not
VMAT1 in histamine-containing cells of the gut as well as in mast cells
(7). VMAT2 also shows 10-fold greater sensitivity to the classical
inhibitor tetrabenazine than VMAT1 (5, 6).
The existence of two closely related proteins that differ substantially
in their substrate affinity and pharmacology provides an opportunity to
dissect the structural basis for these differences. To identify domains
responsible for these differences, we first expressed and analyzed a
series of chimeric transport proteins (8). These chimeras retained
function, and analysis showed that two major regions from VMAT2, one
extending from transmembrane domain (TMD) 5 to the beginning of TMD8
(TMD5-8) and the other from the end of TMD9 through TMD12 (TMD9-12),
are apparently both required for the high affinity interactions
characteristic of VMAT2 (8). Replacement of individual VMAT2 residues
from either region with corresponding VMAT1 residues may therefore
reduce the affinity of VMAT2 interactions to those characteristic of VMAT1, whereas multiple replacements in both TMD5-8 and TMD9-12 of
VMAT1 would be required to increase the affinity of the interactions toward that of VMAT2. Focusing first on TMD9-12, we introduced mutations that convert the VMAT2 sequence into VMAT1 and found that
many residues in this region have no effect on recognition (9).
However, substitution of Tyr-434 with Phe (mutant Y434F) and Asp-461
with Asn (D461N) significantly reduced the affinity of VMAT2 for
tetrabenazine, histamine, and serotonin. Replacement of Lys-446 with
Gln (K446Q) reduced the affinity of VMAT2 for tetrabenazine and
serotonin but not histamine, whereas substitution of Phe-464 with Tyr
(F464Y) reduced serotonin affinity but not tetrabenazine or histamine
recognition. The analysis of multiple mutants indicated that Tyr-434
and Asp-461 account for essentially all of the difference in histamine
recognition between the two VMATs attributable to TMD9-12. In
addition, Tyr-434, Lys-446, and Asp-461 account for essentially all of
the differences in serotonin and tetrabenazine recognition attributable
to this region. To identify the features of the ligand recognized by
these residues, we also examined the interaction of these mutants with
structurally related compounds.
In contrast to other ligands recognized with higher affinity by VMAT2
than VMAT1, tryptamine inhibits transport of [3H]serotonin by VMAT1
much more potently than VMAT2 (9). Replacement of Tyr-434 with Phe
(Y434F) alone increases the affinity of VMAT2 for tryptamine to that of
VMAT1 (9). Thus, the same residue that contributes to the high affinity
interactions of VMAT2 with tetrabenazine, histamine, and serotonin
accounts for the lower affinity interaction of VMAT2 with tryptamine,
suggesting that this residue discriminates between serotonin and
tryptamine.
The analysis of chimeras has suggested that TMD5-8 is required along
with TMD9-12 for most of the high affinity interactions characteristic
of VMAT2. In the present study, we have substituted amino acids in
TMD5-8 of VMAT2 with the equivalent residues from VMAT1. Functional
analysis of these mutants implicates specific residues in the
interaction of VMAT2 with serotonin, histamine, tetrabenazine, and
tryptamine. The comparison of single and multiple mutants also
indicates that these residues interact differently with the
ligands from residues in TMD9-12.
 |
EXPERIMENTAL PROCEDURES |
Mutagenesis--
Mutations were introduced into wild-type VMAT2
using single-stranded, uracil-containing DNA as described previously
(9, 10). VMAT2 double, triple, and quadruple mutant combinations were
produced through the sequential subcloning of single mutants. In
contrast, VMAT1 triple and quadruple mutants were each produced in a
single reaction containing multiple mutagenic oligonucleotide primers.
In all cases, a restriction fragment containing the desired mutation(s)
was sequenced in its entirety by the dideoxy method both to confirm the
desired mutation and to exclude unwanted mutations (11). After sequence
analysis, the fragments were subcloned back into appropriate wild-type
cDNAs.
Transient Expression and Membrane Preparation--
Plasmid DNA
was prepared, COS1 cells were transfected, and membranes were isolated
for analysis as described previously (9). Briefly, transfected cells
were collected by centrifugation and resuspended in cold 0.32 M sucrose, 10 mM HEPES-KOH, pH 7.4 (SH buffer)
containing 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml E-64, 1 µg/ml pepstatin A, and 0.2 mM diisopropyl
fluorophosphate. Cells are then lysed by sonication, and cell debris
were removed by centrifugation at 16,000 × g for 5 min. The resulting supernatant was divided into aliquots and stored at
80 °C.
Transport Assay--
The transport assay was performed as
described previously (9). Briefly, a frozen aliquot of membranes was
thawed on ice, and 10 µl (50-100 µg of protein) was added to 200 µl of SH buffer at 29 °C containing 4 mM KCl, 2 mM MgSO4, 2.5 mM ATP, and either 20 nM [1,2-3H]serotonin (NEN Life Science
Products), 20 nM [2,5,6-3H]dopamine (NEN Life
Science Products ), or 29 nM
[2,5-3H]histamine (Amersham Life Science, Inc.). To
determine the Km for dopamine and serotonin, a range
of nonradioactive substrate concentrations was added to the reaction
solution, and transport was measured after incubation for 1 min at
29 °C. To determine the concentration of compounds needed to inhibit
serotonin transport by 50% (IC50), increasing
concentrations of tetrabenazine (Fluka), histamine (Sigma), and
tryptamine (Sigma) were added to the reaction solution. After
incubation for 1 min at 29 °C, the transport reaction was terminated
by rapid dilution with 3 ml of cold SH buffer and filtration through
0.2-µm Supor 200 membranes (Gelman Instrument Co.). The filters were
then dried, and the bound radioactivity was quantitated by
scintillation counting in Cytoscint (ICN). For each mutant, transport
activity was measured in duplicate on a total of three separate
occasions using membranes prepared from three separate transfections.
The protein concentration of each membrane sample was measured using a
Bradford assay (Bio-Rad), and the unpaired two-tailed t test
was used for all statistical comparisons.
 |
RESULTS AND DISCUSSION |
The analysis of chimeric transporters has suggested that replacing
individual residues in VMAT2 with the equivalent residues from VMAT1
may suffice to reduce the affinity with which VMAT2 interacts with
multiple ligands (8). Indeed, TMD9-12 of VMAT2 contains several
residues that when replaced with the equivalent residues from VMAT1
reduce the high affinity of VMAT2 interactions toward those of VMAT1
(9). In the present study, we have extended the analysis to TMD5-8 in
VMAT2. Of 27 residues in TMD5-8 that differ between the two VMATs, we
focused on 18 that show nonconservative differences (Fig.
1).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Predicted secondary structure and location of
residues divergent between VMAT1 and VMAT2 in the region spanning
transmembrane domains 5-8. A, amino acid residues 215 through 355 of rat VMAT2 containing predicted TMD 5 through 8 are
shown. The circles represent residues of rat VMAT2 as
labeled in Fig. 1. The TMD5-8 region extends from amino acid residue
223 through 332. The 18 residues studied here are labeled with the
amino acid present in VMAT2. B, compilation of sequences
from human (h) VMAT1 (6), human VMAT2 (12-14), rat
(r) VMAT1 (1), rat VMAT2 (1, 2), and bovine (b)
VMAT2 (15) is shown. The number refers to the sequence of
rat VMAT2, and the predicted TMDs are underlined. Bold
print indicates the residues studied here.
|
|
Tetrabenazine Sensitivity--
The majority of VMAT1 substitutions
introduced into TMD5-8 of VMAT2 do not influence the sensitivity to
tetrabenazine as measured by the IC50 for inhibition of
[3H]serotonin transport (Table
I). However, replacement of Ala-315 from
VMAT2 with Thr from VMAT1 (A315T) significantly reduces tetrabenazine sensitivity. The addition of A315T to the triple mutant
Y434F/K446Q/D461N previously shown to account for all of the reduced
tetrabenazine sensitivity attributable to TMD9-12 (9) further reduces
tetrabenazine sensitivity to a level similar to that of wild-type VMAT1
(Table II). Taken together with the
previous results (9), Ala-315, Tyr-434, Lys-446, and Asp-461 thus each
contribute independently to the high affinity recognition of
tetrabenazine by VMAT2 (Fig. 2). These
residues may interact directly with tetrabenazine, but they may also
affect ligand recognition indirectly through changes in protein
conformation.
View this table:
[in this window]
[in a new window]
|
Table I
Individual residues in TMD5-8 influence the recognition of
tetrabenazine, histamine, and serotonin by VMATs
Individual residues from VMAT2 were converted into the corresponding
VMAT1 residue using site-directed mutagenesis. Membranes were prepared
from COS1 cells transfected with the indicated cDNAs and incubated
at 29°C for 1 min in [3H]serotonin. The reaction was
terminated by rapid dilution followed by filtration, and the bound
radioactivity was measured by scintillation counting. Tetrabenazine
sensitivity and histamine recognition were assessed by measuring the
amount of ligand required to inhibit serotonin transport by 50%
(IC50). A range of serotonin concentrations was used to
determine the Km values for serotonin from Lineweaver-Burk analysis. Uptake for zero time at 0°C was subtracted as background. The values shown were calculated from three separate experiments and are expressed as mean ± S.D. All value were
compared to the value obtained from wild-type VMAT2 using the unpaired two-tailed t test.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
The combination of mutations from TMD5-8 and TMD9-12 influence the
recognition of tetrabenazine, histamine, and serotonin by VMATs
Membranes were prepared, transport was assayed, and the results were
displayed as described in Table I. Each value was compared to wild-type
VMAT2 using the unpaired two-tailed t test.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Multiple residues in TMD5-8 and TMD9-12 of
VMAT2 influence tetrabenazine, histamine, and serotonin
recognition. The residues found crucial for the differences in
ligand recognition between VMAT1 and 2 are shown, along with the
predicted secondary structure. The circles represent amino
acid residues as described in Fig. 1, and the predicted transmembrane
helices are indicated above in roman numerals. The crucial
residues for each ligand are shown below their respective transmembrane
helices. All the residues indicated contribute to the high affinity
interactions characteristic of VMAT2 except for multiple residues in
TMD5-8 (not shown in the diagram) that contribute to the high affinity recognition of serotonin (and tryptamine) by VMAT1 (VMAT1 > VMAT2).
|
|
Histamine Recognition--
To determine whether specific residues
from TMD5-8 of VMAT2 contribute to the high affinity recognition of
histamine, we measured the effect of VMAT1 substitutions in this region
on the ability of histamine to inhibit [3H]serotonin
transport. Similar to the analysis of tetrabenazine sensitivity, most
of these mutations, including A315T which influences tetrabenazine
sensitivity, do not affect histamine recognition. However, the
substitution of Pro-237 from VMAT2 with Ala from VMAT1 (P237A)
significantly reduces histamine recognition (Table I). Thus, different
residues in TMD5-8 account for the differences between the two VMATs
in tetrabenazine sensitivity and histamine recognition, consistent
with previous results from the analysis of mutants in TMD9-12
that also suggest distinct recognition sites (9). In addition, the
involvement of a proline in histamine recognition suggests an indirect
effect of this residue on protein structure rather than a direct
interaction with the ligand.
Since Tyr-434 and Asp-461 were the only residues in TMD9-12 of VMAT2
previously found to influence histamine recognition (9), we combined
the P237A mutation from TMD5-8 with Y434F and D461N (Table II).
Analysis of this triple mutant (P237A/Y434F/D461N) showed that the
combination of mutations still does not suffice to completely convert
the histamine recognition of VMAT2 into that of VMAT1. Thus, other
residues in VMAT2 may also contribute to the high affinity recognition
of histamine. Further, P237A/Y434F/D461N has an IC50 for
histamine identical to that of either P237A or Y434F/D461N alone (Table
II), indicating that these residues do not function independently and
hence contribute to a single site of interaction with histamine (Fig.
2). As a control for these multiple mutations, we also combined K328E
in TMD5-8, which alone has no effect on histamine recognition, with
the Y434F/K446Q/D461N triple mutation in TMD9-12 previously shown to
reduce histamine recognition (K328E/Y434F/K446Q/D461N). Surprisingly,
this combination restores the affinity of the triple mutant to that of
wild-type VMAT2, suggesting that the regions spanning TMD5-8 and
TMD9-12 interact to confer high affinity histamine recognition.
Serotonin Affinity--
The recognition of serotonin by VMAT2
appears more complex than the recognition of tetrabenazine and
histamine. The analysis of chimeras has shown that TMD9-12 of VMAT1
reduces serotonin affinity when introduced into VMAT2; however, TMD5-8
of VMAT1 increases serotonin affinity in the context of C-terminal
VMAT1 sequences (8). Thus, although TMD5-8 of VMAT1 reduces the
affinity of VMAT2 for tetrabenazine and histamine, this same region
reverses the loss of affinity for serotonin produced by TMD9-12 from
VMAT1. To determine whether the same residues in TMD5-8 of VMAT1
responsible for low affinity interactions with tetrabenazine and
histamine contribute to the high affinity for serotonin, we analyzed
the VMAT1 substitutions into VMAT2 described above. Surprisingly, many
of the VMAT1 substitutions into TMD5-8 of VMAT2, including mutations
A315T and P237A that, respectively, impair tetrabenazine and histamine
recognition, increase apparent affinity for serotonin (Table I).
The analysis of chimeras suggested that TMD5-8 of VMAT1 has a dominant
effect on serotonin recognition over TMD9-12 from VMAT1 (8). To
determine whether specific residues in this region also have a dominant
effect, we have focused on mutation A315T (because it influences
tetrabenazine and serotonin recognition), P237A (because it influences
histamine and serotonin recognition), and K328E (because it does not
substantially influence any of these interactions). The introduction of
A315T into Y434F/K446Q/D461N (A315T/Y434F/K446Q/ D461N) and
P237A into Y434F/D461N (P237A/Y434F/D461N), both, increase the apparent
affinity for serotonin (Table II). Interestingly, the K328E mutation
that does not alone substantially affect serotonin recognition does
increase the apparent affinity for serotonin when combined with
Y434F/K446Q/D461N (K328E/Y434F/K446Q/D461N) and with F464Y
(K328E/F464Y) (Table II). Taken together, these results indicate that
residues from TMD5-8 of VMAT1 have a dominant effect on serotonin
affinity over VMAT1 residues from TMD9-12. In addition, the
Vmax for serotonin transport by all the
individual and multiple mutants is equivalent to that of the wild-type
VMATs (100-200 pmol/min/mg; data not shown), suggesting comparable
levels of expression and turnover. Importantly, the combination of
P237A with A440T, a mutation from TMD9-12 that increases serotonin
affinity, further enhances serotonin recognition to produce a transport protein with an apparent affinity for serotonin (Km ~ 40 nM) seven times greater than that of wild-type VMAT2
(Km ~ 270 nM), suggesting that the two
residues independently contribute to substrate recognition (Fig. 2).
However, the ability of multiple residues in TMD5-8 of VMAT1 to
increase substrate recognition makes it more likely that these
substitutions act indirectly through changes in protein conformation
rather than through direct interactions with the ligand.
Tryptamine Recognition--
Previous analysis of residues in
TMD9-12 has shown that the replacement of Tyr-434 in VMAT2 with Phe
from VMAT1 increases the recognition of tryptamine, and this single
residue accounts for all of the difference in tryptamine recognition
between the two VMATs (9). Since Pro-237 and Tyr-434 both contribute to histamine recognition, we considered that Pro-237 may also influence tryptamine recognition. Indeed, like Y434F, mutation P237A completely converts VMAT2 into VMAT1 with respect to the recognition of tryptamine (Table III). Since P237A increases the
apparent affinity for serotonin and tryptamine differs from serotonin
only in the absence of the 5-hydroxyl group, alanine at position 237 appears to promote the recognition of some feature in common between
these two ligands. Other residues that increase apparent affinity for
serotonin may therefore also promote tryptamine recognition. Indeed,
the A315T mutation significantly increases tryptamine as well as
serotonin recognition. Furthermore, if these substitutions recognize a
feature common to both serotonin and tryptamine and Tyr-434 recognizes the 5-hydroxyl group present on serotonin but absent from tryptamine as
suggested in previous studies (9), then the combination of either P237A
or A315T with Y434F should further increase tryptamine recognition.
Indeed, the recognition of tryptamine by triple mutant P237A/Y434F/D461N (IC50, 71 nM) significantly
exceeds that of P237A (IC50, 470 nM) and Y434F
(IC50, 350 nM) alone (p < 0.01). Triple mutant A315T/Y434F/K446Q/D461N also recognizes tryptamine with higher affinity than A315T and Y434F alone (p < 0.05). Thus, VMAT1 residues in TMD5-8 interact with a feature of the
indoleamine ligand distinct from that recognized by the phenylalanine
residue in TMD11 of VMAT1. Conversely, Pro-237 of VMAT2 appears to
hinder the interaction with a feature of the ligand common to both
serotonin and tryptamine, whereas Tyr-434 hinders the recognition of
tryptamine but not serotonin. Tyr-434 presumably interacts with the
hydroxyl group on serotonin and the residues in TMD5-8 with some other feature common to tryptamine and serotonin (Fig. 2).
View this table:
[in this window]
[in a new window]
|
Table III
Multiple residues influence the recognition of tryptamine by VMATs
Membranes were prepared from transfected COS1 cells, transport was
assayed, and the results were displayed as described in Table I. The
values for each single mutant were compared to wild-type VMAT2 using
the unpaired two-tailed t test. In addition, the values for
P237A/Y434F/D461N and A315T/Y434F/K446Q/D461N were compared to P237A
and A315T, respectively, as well as Y434F and Y434F/K446Q/D461N. The
values for the VMAT1 mutants F434Y/Q446K/N461D and
A237P/F434Y/Q446K/N461D were compared to wild-type VMAT1. For
convenience, the VMAT1 mutation numbers correspond to the VMAT2
sequence, not the VMAT1 sequence.
|
|
VMAT1 Mutations--
To determine whether the residues required
for the high affinity interactions characteristic of VMAT2 also suffice
to confer high affinity, we introduced these residues from VMAT2 into
the equivalent positions of VMAT1. We first constructed the triple mutant F434Y/Q446K/N461D that contains three major residues from TMD9-12 of VMAT2. (For convenience, the numbers refer to the VMAT2 sequence). In addition, we constructed a quadruple VMAT1 mutant (A237P/F434Y/Q446K/N461D) that includes Pro-237 from TMD5-8 of VMAT2,
since this residue influences histamine, serotonin, and tryptamine
recognition. Analysis of these triple and quadruple mutants shows that
these replacements do not confer high affinity interactions with
tetrabenazine, histamine, or serotonin (Table IV). Indeed, both VMAT1 mutants recognize
these ligands very poorly, suggesting that, rather than simply failing
to restore the VMAT2 phenotype, the mutations have interfered with some
aspect of protein structure. Further, neither VMAT1 mutant transports
[3H]histamine, and the apparent affinity of these mutants
for dopamine is also greatly reduced (data not shown), suggesting a
general defect in ligand recognition. Nonetheless, the mutant
A237P/F434Y/Q446K/N461D, which contains the A237P substitution from
VMAT2, has a lower affinity for serotonin than the F434Y/Q446K/N461D
mutant, which lacks A237P (Table IV), supporting a dominant role for
TMD5-8 of VMAT1 in substrate recognition. In addition,
A237P/F434Y/Q446K/N461D recognizes tryptamine very poorly, whereas
the recognition of tryptamine by F434Y/Q446K/N461D resembles wild-type
VMAT2 (Table III), consistent with a role for residues in TMD5-8 and
specifically, the alanine (in TMD5) in tryptamine recognition.
View this table:
[in this window]
[in a new window]
|
Table IV
VMAT2 substitutions into VMAT1 do not confer high affinity ligand
recognition
The assays were performed, and the results are displayed as described
in Table I. The values for wild-type VMAT2 mutants (Y434F/K446Q/D461N
and P237A/Y434F/D461N) were compared to wild-type VMAT2 using the
unpaired two-tailed t test, whereas the values for the
VMAT1 mutants (F434Y/Q446K/N461 and A237P/F434Y/Q446K/ N461D)
were compared to wild-type VMAT1.
|
|
Conclusion--
In summary, multiple residues contribute to the
high affinity interaction of VMAT2 with ligands. The increased
sensitivity to tetrabenazine appears to derive independently from
Ala-315 in TMD7 and Tyr-434, Lys-446, and Asp-461 in TMD9-12. The
ability of VMAT2 to recognize histamine requires Pro-237 in TMD5 as
well as Tyr-434 and Asp-461 in TMD9-12, and the comparison of single and multiple mutants suggests that although not all the residues involved in histamine recognition have been identified, Pro-237 and
TMD9-12 contribute to a single recognition site. In contrast, essentially all of the residues from TMD5-8 of VMAT1 increase serotonin affinity relative to VMAT2, and this effect dominates over
mutations in TMD9-12, strongly supporting distinct roles for the two
regions in substrate recognition. Similar to Tyr-434 in TMD9-12,
replacement of Pro-237 and other residues in TMD5-8 of VMAT2 with
equivalent residues from VMAT1 improves tryptamine recognition.
However, unlike VMAT1 substitutions into TMD5-8 of VMAT2 that increase
serotonin recognition, Y434F impairs the apparent affinity for
serotonin, further indicating that the two sites interact with distinct
features of the ligand. Thus, although the recognition of histamine
appears to involve a single recognition site contributed by both major
domains, VMAT2 recognizes the other ligands through multiple
independent interactions.