(Received for publication, September 8, 1995; and in revised form, November 8, 1995)
From the
The vesicular monoamine transporters (VMATs) 1 and 2 show close sequence similarity but substantial differences in apparent substrate affinity and drug sensitivity. To identify structural domains that determine these functional characteristics, chimeric transporters were constructed and their properties were analyzed in a heterologous expression system. The results implicate multiple regions in the recognition of serotonin and histamine and the sensitivity to tetrabenazine. Two domains of VMAT2, one extending from transmembrane domain (TMD) 5 to the beginning of TMD8 and the other from the end of TMD9 through TMD12, increase the affinity for serotonin and histamine as well as the sensitivity to tetrabenazine but only in the context of more C-terminal and more N-terminal VMAT2 sequences, respectively. In addition, the extreme N terminus of VMAT2 alone suffices to confer a partial increase in substrate affinity and tetrabenazine sensitivity. Despite these similarities among the interactions with serotonin, histamine, and tetrabenazine, the region of VMAT2 from TMD3 through TMD4 increases serotonin affinity but not histamine affinity or tetrabenazine sensitivity, and whereas the region from TMD5 to TMD8 of VMAT2 increases serotonin affinity in the context of more C-terminal VMAT2 sequences, the region encompassing TMD5 through TMD7 reduces serotonin but not histamine affinity or tetrabenazine sensitivity in the context of more N-terminal VMAT2 sequences. Thus, the chimeric analysis also reveals differences between serotonin recognition and the recognition of both histamine and tetrabenazine that may account for the observed differences in their interaction with the transport protein.
Neurons communicate through the regulated exocytotic release of
vesicles containing neurotransmitter. Because classical
neurotransmitters are synthesized in the cytoplasm, they require
packaging into vesicles. Four distinct types of vesicular
neurotransmitter transport activity have been described: one for
monoamines, another for acetylcholine, a third for glutamate, and a
fourth for -aminobutyric acid and
glycine(1, 2, 3) . The bioenergetics of
vesicular amine transport have been characterized using granules
isolated from chromaffin cells of bovine adrenal medulla. These studies
have shown that amine uptake depends on a proton gradient generated by
a vacuolar H
-ATPase(4, 5) , and
involves the exchange of two lumenal protons for a cytoplasmic
amine(6, 7, 8) . In terms of substrate
recognition, the vesicular amine transporter recognizes a large,
structurally diverse group of compounds, including the catecholamines
and the indolamines, such as serotonin and histamine, as well as
several
toxins(9, 10, 11, 12, 13) .
Previous studies have characterized two inhibitors of vesicular
amine transport, reserpine and tetrabenazine. Reserpine and
tetrabenazine both inhibit the activity of the transporter but appear
to interact differently with the protein. Studies using bovine
chromaffin granules show that substrates compete with reserpine for
binding to the transporter at concentrations close to their K for transport(14) , whereas
only very high concentrations inhibit tetrabenazine
binding(15) . This suggests that reserpine binds near the site
of amine recognition and that tetrabenazine binds at a site distinct
from reserpine and substrates. In addition, the presence of a pH
gradient across the vesicle membrane accelerates the binding of
reserpine but not tetrabenazine(15) . Interestingly,
tetrabenazine inhibits reserpine binding to the transporter, suggesting
that the sites may interact in an allosteric manner(16) . These
observations have been used to construct a model of the transport
cycle. After translocation of the loaded carrier to the interior of the
vesicle and delivery of the substrate, protonation may promote movement
of the unloaded carrier back to the cytoplasmic surface. Reserpine
presumably binds to the unloaded cytoplasmically oriented protein. The
site for tetrabenazine action, however, remains unclear. Thus,
reserpine and tetrabenazine may be used as tools to dissect
conformational changes of the protein during the transport cycle. The
cDNAs that encode the proteins responsible for vesicular
neurotransmitter transport have, however, remained elusive until quite
recently.
Selection of transfected cells in the neurotoxin
MPP has led to the identification of a novel gene
family that includes two vesicular monoamine transporters, VMAT1 (
)and VMAT2(12, 17) . The nucleotide
sequence of the two cDNAs predicts proteins with a high degree of
similarity, but these proteins show important differences in tissue
distribution and in biochemical function. VMAT1, originally isolated
from the rat PC12 cell line, is expressed primarily in the adrenal
gland (accounting for the original name of chromaffin granule amine
transporter or CGAT), whereas VMAT2 is expressed in the central nervous
system (accounting for its original name of synaptic vesicle amine
transporter or SVAT)(18) . To determine whether VMAT1 differs
from VMAT2 in substrate affinity or in interactions with well known
inhibitors such as reserpine and tetrabenazine, we have used
heterologous expression of the two cloned cDNAs(19) . In terms
of substrate affinity, VMAT2 has a substantially higher apparent
affinity than VMAT1 for all amine transmitters. Most strikingly, VMAT2
has a 10-100-fold higher apparent affinity for histamine than
VMAT1. This functional characteristic correlates with the expression of
VMAT2 and not VMAT1 by histamine-containing cells(18) . In
terms of pharmacology, reserpine inhibits transport by VMAT1 and VMAT2
with equal potency. Tetrabenazine, however, inhibits transport by VMAT2
with 10-fold greater potency than transport by VMAT1. Furthermore, at
low concentrations of the drug,
[
H]dihydrotetrabenazine does not appear to bind
to VMAT1, indicating that sensitivity to the drug relates directly to
differences in binding(19) .
The high degree of sequence similarity between VMAT1 and VMAT2 (62% identity) suggests the possibility of constructing chimeras that will retain function. These functional chimeras can then be used to identify domains responsible for the observed physiologic and pharmacologic differences between VMAT1 and VMAT2.
The VMAT1-VMAT2 class of chimeras contained six
functional transporters (Fig. 1). We denote this class of
chimeras CXS, where C represents VMAT1 (previously known as
chromaffin granule amine transporter or CGAT), S represents VMAT2
(previously known as synaptic vesicle amine transporter or SVAT), and X represents the residue in VMAT1 at which the junction
occurs. Most of the chimeras contain junctions within hydrophilic loops
or at the border of predicted transmembrane domains (TMDs). One of the
chimeras contains a junction in predicted TMD12 (C456S). Another of the
chimeras (C493S) contains a 14-amino acid deletion at the C terminus of
the protein, apparently due to recombination at a repeated codon for
proline. All of these chimeras demonstrate substantial serotonin
transport activity when measured at a concentration of
[H]serotonin (20 nM) well below the K
(data not shown). However, the V
of these chimeras varies considerably (Table 1). In addition, VMAT1-VMAT2 chimeras with junctions in
TMD3 (C168S) and TMD11 (C435S) show no activity, and two others with
junctions in the loops between TMD7 and TMD8 (C318S) and TMD10 and
TMD11 (C419S) show minimal if any activity (data not shown).
Interestingly, the chimera with a C-terminal deletion shows substantial
activity, indicating that it retains the signals necessary for
internalization to an acidic compartment that can provide the driving
force for transport.
Figure 1: Functional chimeric vesicular monoamine transporters. A, VMAT1-VMAT2 chimeric transporters that show significant activity in a standard transport assay using membranes from transfected COS cells. The white circles indicate residues derived from VMAT1, and the black circles indicate residues derived from VMAT2. In the nomenclature CXS, C refers to residues derived from VMAT1 (formerly known as chromaffin granule amine transporter or CGAT), X refers to the last amino acid in the chimera derived from VMAT1, and S refers to VMAT2 (formerly known as synaptic vesicle amine transporter or SVAT). The arrows pointing at residues in the C-terminal tail of chimera C493S indicate a deletion of 14 amino acids presumably due to a recombination event at a repeated codon for proline. B, VMAT2-VMAT1 chimeric transporters. In the nomenclature SXC, S refers to VMAT2 (SVAT), X refers to the last amino acid in the chimera derived from VMAT2, and C refers to VMAT1 (CGAT). The two horizontal lines indicate the vesicle membrane, with the lumen above and the cytoplasm below.
The VMAT2-VMAT1 class of chimeras contained
seven distinct cDNAs that conferred transport activity (Fig. 1).
As above, we denote these chimeras SXC, where S is VMAT2
(SVAT), C is VMAT1 (CGAT), and X refers to the residues of
VMAT2 at which the junction occurs. For most of the VMAT2-VMAT1
chimeras, junctions occur within hydrophilic loops or at the border of
predicted transmembrane domains, except for chimera S372C, which
contains a junction in the middle of TMD9. Similar to the VMAT1-VMAT2
chimeras, all of the functional chimeras show substantial transport
activity, but the V varies considerably (Table 2). One VMAT2-VMAT1 chimera (S321C) showed no activity and
had a junction in the loop between TMD7 and TMD8 (data not shown).
Because the study of chimeras has the advantage of studying functional
proteins, we have not further analyzed the nonfunctional chimeric
transporters.
To identify domains of the VMATs that confer differences in apparent affinity, the chimeric VMAT transporters described above (Fig. 1) were analyzed in terms of their interaction with serotonin and histamine. Analysis of the VMAT1-VMAT2 chimeras demonstrates that replacement of VMAT2 with VMAT1 sequences from the N terminus through TMD1 (C38S) to the beginning of TMD5 (C227S) display a high apparent affinity for serotonin similar to VMAT2 (Table 1). When substitution with VMAT1 sequences extends past TMD5 to TMD8 (C334S) and beyond, the apparent affinity for serotonin falls to the level characteristic of VMAT1.
The analysis of apparent substrate affinity using the VMAT2-VMAT1 chimeras shows a more complex pattern (Table 2). First, chimera S37C, which replaces VMAT1 sequences with VMAT2 from the N terminus through TMD1, has an intermediate apparent affinity for serotonin. The next chimera (S159C), which includes additional VMAT2 sequences from the end of TMD1 through TMD3, exhibits a low affinity for serotonin, similar to VMAT1. The addition of VMAT2 sequences from the start of TMD3 to the beginning of TMD5 (S210C and S222C), however, confers high affinity for serotonin, similar to VMAT2. Surprisingly, extension of VMAT2 sequences from the beginning of TMD5 through TMD7 to TMD9 (S310C and S372C) reduces the affinity for serotonin. Nonetheless, the addition of VMAT2 sequences from the end of TMD9 to just past TMD12 (S468C) restores high affinity serotonin transport. These results indicate a major role for three regions in the apparent affinity for serotonin. TMD3 through TMD4 and TMD9 through TMD12 of VMAT2 increase the apparent affinity, but TMD9 through TMD12 requires N-terminal VMAT2 sequences; curiously, TMD5 through TMD8 of VMAT2 increases affinity in the presence of C-terminal VMAT2 sequences but reduces the affinity in the presence of N-terminal VMAT2 sequences.
Analysis of the VMAT2-VMAT1 chimeras indicates that replacement of VMAT1 with increasing amounts of VMAT2 from the N terminus of the protein up to the end of TMD9 (S372C) does not increase the low affinity for histamine characteristic of VMAT1 (Table 2). Extension of VMAT2 sequences from the end of TMD9 to the end of TMD12 (S468C), however, increases the affinity to that characteristic of VMAT2. Thus, as observed in the chimeric analysis of serotonin transport, the results indicate that high affinity interaction with histamine requires two distinct regions of VMAT2, TMD5 through TMD8 and TMD9 through TMD12. In addition, the extreme N terminus of VMAT2 produces a partial VMAT2 phenotype (S37C) with regard to the affinity for histamine as well as serotonin (Table 2).
To determine the sensitivity of each
chimera to tetrabenazine, we measured
[H]serotonin transport in the presence of
different concentrations of the drug and used this dose-response
analysis to estimate the concentration required to inhibit transport by
50% (IC
). Similar to the observations regarding substrate
affinity, analysis of the VMAT1-VMAT2 series of chimeras shows that
replacement of VMAT2 with VMAT1 from the N terminus up to the beginning
of TMD5 (C227S) does not reduce the high sensitivity to tetrabenazine
characteristic of VMAT2 (Table 1). Extension of the VMAT1
sequences to TMD8 (C334S) and beyond reduces the tetrabenazine
sensitivity to that observed for VMAT1, as also reported for the
analysis of substrate affinity.
Analysis of the VMAT2-VMAT1 chimeric transporters shows that replacement of VMAT1 sequences with VMAT2 from the N terminus of the protein to the end of TMD9 (S372C) does not alter the low sensitivity to tetrabenazine characteristic of wild-type VMAT1 (Table 2). Extension of VMAT2 sequences from the end of TMD9 to the end of TMD12 (S468C), however, leads to a dramatic increase in sensitivity to tetrabenazine. Interestingly, the same chimera that contains VMAT2 from the N terminus to just past TMD1 (S37C) and exhibits an intermediate affinity for both serotonin and histamine also exhibits a sensitivity to tetrabenazine intermediate between VMAT1 and VMAT2. Taken together, the results show that, like high substrate affinity, two distinct regions of VMAT2 are necessary for high sensitivity to tetrabenazine, one from the beginning of TMD5 through TMD8 and the other from TMD9 through TMD12. Although both of these regions are important for the high sensitivity of VMAT2 to tetrabenazine, neither region alone suffices to confer the high sensitivity phenotype. Rather, TMD5 through TMD8 appear to act in the context of more C-terminal VMAT2 sequences (possibly TMD9 through TMD12) to confer high tetrabenazine sensitivity, and TMD9 through TMD12 appear to act in the context of more N-terminal VMAT1 sequences (possibly TMD5 through TMD8), similar to the observations with regard to substrate affinity.
Site-directed mutagenesis has the potential to identify the role of specific amino acid residues in the biological activity of a protein, but this method has several limitations. First, mutagenesis at a specific site may have the undesired side effect of perturbing the overall structure of the protein, making it difficult to interpret mutations that impair function. Second, site-directed mutagenesis relies on additional information to guide the analysis toward the few critical residues. The use of chimeras between two related proteins that differ in function circumvents these problems. The study of chimeras has the advantage that it derives information from functional proteins rather than nonfunctional point mutants. In addition, it does not depend on other information to guide mutagenesis but rather considers a wide range of possibilities and systematically identifies the domains responsible for particular functional characteristics. The use of chimeras, however, depends on their ability to retain function. Chimeric rat and human plasma membrane serotonin transporters retain function(24) , but the two parental proteins show a high degree of identity (92%). Although plasma membrane transporters for dopamine and norepinephrine show less similarity (78% identity) than the two serotonin transporters, dopamine/norepinephrine chimeras retain activity(21, 25) , suggesting that VMAT chimeras, with 62% identity between the parental proteins, might also function.
The present study shows that chimeras between VMAT1 and VMAT2 generally retain function. Interestingly, whereas chimeras between plasma membrane dopamine and norepinephrine transporters that contain junctions toward the central region of the protein (between TMD5 and TMD8) show greatly reduced activity (21) and chimeric plasma membrane norepinephrine and serotonin transporters show no activity except with junctions close to the N terminus(20) , VMAT chimeras with junctions toward the center of the protein retain substantial transport activity. Rather, the few VMAT1-VMAT2 chimeras that show no activity have junctions in TMD3, TMD11, and the loops between TMDs 7 and 8 and TMDs 10 and 11. Interestingly, the functional chimeras in this series have junctions near TMDs 5 (C227S), 8 (C334S), and 11 (C439S), suggesting that the loss of activity results from interruption of a specific sequence not the overall proportion of the two sequences. Similarly, the junction of the nonfunctional VMAT2-VMAT1 chimera S321C occurs in the loop between TMDs 7 and 8, near the junction of the functional chimera S310C at the end of TMD7. In addition, the method used here to construct the chimeras has yielded two VMAT1-VMAT2 chimeras (C38S and C227S) that have almost exactly reciprocal VMAT2-VMAT1 partners (S37C and S222C), suggesting that although the method has the potential to generate chimeras with random junctions between the two VMATs, sequence similarity in particular regions can influence the site of homologous recombination.
Figure 2: Domains of VMAT2 responsible for high affinity substrate recognition and sensitivity to tetrabenazine. The analysis of chimeric vesicular monoamine transport proteins has implicated three major regions in substrate affinity and tetrabenazine sensitivity. Region A of VMAT2 includes TMD3 and TMD4 and confers a higher apparent affinity for serotonin but not a higher affinity for histamine or higher sensitivity to tetrabenazine. Region B of VMAT2 extends from TMD5 to TMD8 and confers higher affinity for serotonin and histamine and higher sensitivity to tetrabenazine but only in the context of more C-terminal VMAT2 sequences. Within this region, TMD5 through TMD7 actually reduce the apparent affinity for serotonin when in the context of more N-terminal rather than C-terminal VMAT2 sequences. Region C of VMAT2 extends from the end of TMD8 through TMD12 and also increases substrate affinity and tetrabenazine sensitivity but only in the context of more N-terminal VMAT2 sequences. Thus, regions B and C require other regions (possibly each other) to confer the VMAT2 phenotype, whereas region A alone suffices. The extreme N terminus of VMAT2 extending into TMD1 also suffices to confer a partial VMAT2 phenotype in terms of substrate affinity and tetrabenazine sensitivity and is not shown here. The circles indicate amino acid residues of VMAT2, with black indicating those residues identical to VMAT1, gray indicating those residues similar to VMAT1, and white indicating those residues not conserved with VMAT1. Acidic amino acid residues are indicated with a minus sign and basic residues with a plus sign. The two black horizontal lines indicate the vesicle membrane, with the lumen above and the cytoplasm below.
The multiple domains that influence apparent affinity
for serotonin both positively and negatively raise the question of
whether the observed changes result from local disturbances in the
structure of the chimeric transporters. This seems unlikely for several
reasons. First, the values determined all fall within the range between
those previously reported for wild-type VMAT1 and VMAT2 and hence do
not indicate a loss of function from the parental transporters. Second,
the chimeras all show substantial transport activity. Although the V values vary widely, they do not correlate with
the changes in K
. For example, chimeras C334S,
C329S, C456S, and C493S with a relatively low apparent affinity for
serotonin all have a higher V
(1.4-5.5
pmol/min) than the C227S chimera (V
,
0.7)
with a relatively high apparent affinity. Similarly, the VMAT2-VMAT1
chimera S159C has a relatively low apparent affinity for serotonin and
a V
of
3.5 pmol/min, whereas the higher
affinity chimeras S37C (V
,
3.4) and S210C (V
,
2.3) have the same or lower V
. Although V
reflects
both the level of expression and the intrinsic properties of the
transport protein, the V
measurements provide
evidence that general structural features of the chimeras do not
account for the variations in substrate recognition. Third, the
interaction with histamine and tetrabenazine provide additional
controls for a subset of the observed effects on apparent affinity for
serotonin.
In addition to the usual monoamine transmitters,
histamine is known to be a substrate for the amine transporter in
bovine chromaffin granules but undergoes accumulation at a much slower
rate(26) . Expressed in a heterologous system, rat VMAT2 also
recognizes histamine as a substrate (23) . Rat VMAT1, however,
shows a much lower affinity than VMAT2(19) . Using the
inhibition of [H]serotonin transport as a measure
of the interaction, histamine inhibits serotonin transport by VMAT2
with 10-fold greater potency than transport by VMAT1, enabling the use
of the chimeras to identify domains that influence the affinity of this
interaction. In the VMAT1-VMAT2 series of chimeras, extension of VMAT2
sequences from TMD8 to TMD5 increases the affinity for histamine from
that characteristic of VMAT1 to that characteristic of VMAT2. However,
VMAT2-VMAT1 chimeras containing VMAT2 from the N terminus to the end of
TMD9 have a low affinity for histamine, even though the S372C chimera
includes TMD5 through TMD8 implicated in higher histamine affinity by
the VMAT1-VMAT2 chimeras. Thus, TMD5 through TMD8 of VMAT2 confers a
high affinity for histamine but only in the presence of more C-terminal
VMAT2 sequences. Conversely, the addition of a domain from TMD9 through
TMD12 of VMAT2 increases the affinity for histamine in the VMAT2-VMAT1
chimeras, but C336S includes this domain and shows a low affinity for
histamine, indicating that it also appears to have an effect only
within the context of more N-terminal VMAT2 sequences. Presumably these
N-terminal sequences occur within TMD5 through TMD8 implicated by the
VMAT1-VMAT2 chimeras, but this remains unknown (Fig. 2).
Monoamines require hydroxyl groups on the aromatic ring for recognition as substrates by the VMATs(8) , but histamine is a substrate for VMAT2 and lacks hydroxyl groups, suggesting different interactions with the transport protein. The results presented here illuminate both the similarities and differences between serotonin and histamine recognition. In terms of similarities, sequences within TMD5 through TMD8 and TMD9 through TMD12 influence the affinity for both serotonin and histamine in similar directions. However, TMD5 through TMD7 of VMAT2 reduces serotonin affinity in the presence of N-terminal VMAT2 sequences, whereas it has no effect on histamine affinity in the absence of C-terminal VMAT2 sequences. In further contrast, TMD3 through TMD4 (in the context of more N-terminal VMAT2 sequences) suffices for high affinity serotonin but not histamine recognition. Recent data also show that histamine does not inhibit reserpine binding to the transporter, further suggesting the interaction of histamine at a site distinct from that involved in serotonin and catecholamine recognition(23) . Interestingly, serine residues of VMAT2 implicated in recognition of hydroxyl groups on the aromatic ring of the ligand occur within TMD3(27) . In summary, TMD5 through TMD8 and TMD9 through TMD12 in the context of other VMAT2 sequences (presumably each other) influence the affinities for both serotonin and histamine, but TMD5 through TMD8 have opposite effects on serotonin transport depending on the presence of other sequences, and TMDs 3 and 4 influence only serotonin recognition, perhaps accounting for the observed difference in interaction between these two substrates. Analysis of the chimeras also indicates that the same domains affecting histamine recognition similarly influence sensitivity to tetrabenazine.
Inhibition by reserpine provides an additional control for the role of TMD5 through TMD8 and TMD9 through TMD12 in both substrate affinity and tetrabenazine sensitivity. Reserpine inhibits serotonin transport by VMAT1 with at least as much potency as transport by VMAT2. We have therefore determined the sensitivity to reserpine of two chimeras containing either TMD5 through TMD8 or TMD5 through TMD8 plus TMD9 through TMD12 from VMAT1 and found at least as much sensitivity to reserpine as the parental proteins. Thus, reserpine sensitivity does not vary in parallel with substrate affinity and tetrabenazine sensitivity among the different chimeras, suggesting that TMD5 through TMD8 and TMD9 through TMD12 from VMAT1 account for differences in specific interactions by the chimeric transporters rather than a general perturbation of protein structure.