Mutagenesis and Derivatization of Human Vesicle Monoamine
Transporter 2 (VMAT2) Cysteines Identifies Transporter Domains Involved
in Tetrabenazine Binding and Substrate Transport*
David S.
Thiriot
and
Arnold E.
Ruoho§
From the Department of Pharmacology, University of
Wisconsin-Madison Medical School, Madison, Wisconsin 53706-1532
Received for publication, May 2, 2001
 |
ABSTRACT |
The vesicle monoamine transporter (VMAT2)
concentrates monoamine neurotransmitter into synaptic vesicles.
Photoaffinity labeling, chimera analysis, and mutagenesis have
identified functionally important amino acids and provided some
information regarding structure and ligand binding sites. To extend
these studies, we engineered functional human VMAT2 constructs with
reduced numbers of cysteines. Subsets of cysteines were discovered,
which restore function to an inactive cysteine-less human VMAT2.
Replacement of three transmembrane (TM) cysteines together (net
removal/replacement of three atoms) significantly enhanced monoamine
transport. Cysteine modification studies involving single and
combination cysteine mutants with methanethiosulfonate ethylamine
revealed that [3H]dihydrotetrabenazine binding is
>90% inhibited by modification of two sets of cysteines. The primary
target (responsible for ~80% of inhibition) is Cys439 in
TM 11. The secondary target (responsible for ~20% of inhibition) is
one or more of the four non-TM cysteines.
[3H]Dihydrotetrabenazine protects against modification of
Cys439 by a 10,000-fold molar excess of
methanethiosulfonate ethylamine, demonstrating that Cys439
is either at the tetrabenazine binding site, or conformationally linked
to tetrabenazine binding. Supporting a direct effect, the position of
tetrabenazine-protectable Cys 439 is consistent with previous
mutagenesis, chimera, and photoaffinity labeling data, demonstrating
involvement of TM 10-12 in a tetrabenazine binding domain.
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INTRODUCTION |
The vesicle monoamine transporter
(VMAT2)1 is a
proton-monoamine antiporter, which concentrates monoamine
neurotransmitters into synaptic vesicles. VMAT2 transports a wide range
of substrates including serotonin, dopamine, and norepinephrine and
is inhibited by a number of drugs including reserpine, tetrabenazine
(TBZ), and ketanserin. Aspects of VMAT2 biology and physiology have
been recently reviewed (1-3). Briefly, bovine VMAT2 has been
biochemically purified (4), and the initial cDNA clones of a VMAT
were from rat PC12 cells (5) and a rat cDNA library (6).
Recombinant rat VMAT2 can be expressed at high levels in Sf9
cells and purified (7). The gene structure and promoter regions of both
human (8) and mouse (9) VMAT2 have been identified, and knockout mice
have been generated. VMAT2 knockout mice are born, but homozygotes die
within a few days after birth (10). Heterozygotes live into adulthood,
but display altered sensitivity to amphetamine, cocaine, and the
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (11,
12). A percentage of heterozygotes die suddenly from what appears to be
cardiac arrythmias due to prolonged QT intervals (11, 13).
Based on amino acid sequence, the prediction is that VMAT2 has 12 transmembrane (TM) helices, a structural commonality with a number of
other transporters including plasma membrane monoamine reuptake
transporters and P-glycoprotein. A number of mutations of rat VMAT1 and
VMAT2 have been tested for their effects on function (14, 15),
including chimera experiments to probe the domains and residues
responsible for binding affinity differences between VMAT2 and the
related isoform VMAT1 (16-19). These studies have suggested important
residues that may contribute to ligand binding and monoamine transport.
In addition, two significant observations that have contributed VMAT2
structural information are the discovery that a lysine in predicted TM
2 and an aspartate in predicted TM 11 interact functionally as an ion
pair (20), and the identification in rat VMAT2 of peptide domains near
or at the ketanserin and tetrabenazine binding sites, and an amino acid
residue (Lys20) near or at the ketanserin binding site,
from photoaffinity labeling studies performed in our laboratory (7,
21). Although the reasonable inferences from molecular biology and the
biochemical structural information from photoaffinity labeling have
provided significant and novel information, a more general approach
that can be applied to obtain structural information from the entire molecule and to map ligand binding sites has been sought.
With a few notable exceptions, large membrane proteins like VMAT2 have
represented an enormous challenge to structural biology, and in most
cases still pose serious technical difficulties to analysis by
crystallography or NMR spectroscopy. Recently, a creative combination
of protein engineering and biochemical methods has been applied to
large membrane proteins including receptors, transporters, and ion
channels, to obtain information about membrane protein structure,
function, and ligand binding sites. Many of these methods utilize
native or engineered cysteines as sites of unique chemical reactivity
within the membrane protein, and require either a molecule that is
devoid of native cysteine residues or a molecule with a non-reactive
background of remaining cysteines for the assays being applied
(22-28). Cysteine-based assays, including site-directed labeling with
chemical probes (i.e. sulfhydryl-reactive
methanethiosulfonate reagents) and intramolecular cysteine
cross-linking to determine helix proximity and orientation, have proven
useful and general approaches. A few of the most notable examples of
transporters studied by these engineered cysteine-based methods include
lactose permease (22) and other bacterial transporters (23, 24), monoamine transporters (25-27), and P-glycoprotein (28).
Inspired by the example of a number of functional cysteine-less
membrane proteins and the flexibility and utility of cysteine-based methods, we sought to apply such methods to the study of human VMAT2.
In this paper, we report a study of the role of the 10 native cysteines
in human VMAT2 expression, ligand binding, and monoamine transport, and
demonstrate by cysteine replacement, derivatization, and protection
experiments that certain cysteines are in important positions for human
VMAT2 [3H]TBZOH binding and [3H]serotonin transport.
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EXPERIMENTAL PROCEDURES |
Materials--
The following materials used in this research
were from the indicated sources (in parentheses): plasmids pCDNA
3.1- and pBlueBac 4.5 (Invitrogen, Carlsbad, CA); Miniprep and large
scale plasmid DNA preparation kits (Qiagen, Valencia, CA; Promega,
Madison, WI; Bio-Rad); Amplitaq FS and Big Dye DNA sequencing
reagents (PerkinElmer Life Sciences, through University of Wisconsin
Biotechnology Center); COS-7 or -7L cells (ATCC, Manassas, VA; Life
Technologies, Inc.); QuickChange mutagenesis kit (Stratagene, La Jolla,
CA); gene-editor mutagenesis kit (Promega); restriction enzymes (New England Biolabs, Beverly, MA; Promega); Pfu and
Taq precision DNA polymerase (Stratagene); DMEM,
penicillin/streptomycin/glutamine, Fungizone, cell culture trypsin, and
PBS (Life Technologies, Inc.); [3H]sodium borohydride,
and [3H] serotonin (PerkinElmer Life Sciences);
tetrabenazine (Fluka, Milwaukee, WI); methanethiosulfonate reagents
(Toronto Research Chemicals, North York, Ontario, Canada) ethylamine
(MTSEA), ethyltrimethylammonium (MTSET), and 2-sulfonatoethyl (MTSES, a
gift from Dr. Cynthia Czajkowski, University of Wisconsin-Madison);
anti-HA antibody (Covance Research Products, Denver, PA); cysteamine,
polyethyleneimine, ATP, 2-mercaptoethanol, and anti-Flag M2 antibody
(Sigma); goat anti-mouse horseradish peroxidase antibody,
electroporation cuvettes, precast electrophoresis ready gels, SDS
sample buffer, and prestained molecular weight markers (Bio-Rad);
Supersignal West pico chemiluminescent substrate and Superblock PBS
blocking buffer (Pierce); Hyperfilm ECL (Amersham Pharmacia Biotech);
GF/B filters (Whatman via Fisher; or Brandel, Gaithersburg, MD); 0.22- and 0.45-µm filters (Millipore via Fisher); transfection-competent
Escherichia coli (Stratagene; Novagen, Madison, WI);
emulsifier Safe scintillation mixture (Packard, Meriden, CT). Other
reagents (sucrose, HEPES, KCl, MgSO4, NaOH, KOH, Tris, DTT,
additional restriction enzymes, etc.) were from reputable and readily
available sources.
Construction of Human VMAT2 Mutants--
A cDNA encoding
human VMAT2 was the kind gift of Dr. Robert H. Edwards (University of
California San Francisco School of Medicine, San Francisco, CA).
5'-Forward and 3'-reverse oligonucleotides with desired restriction
sites (5' BglII, 3' HindIII) were used to amplify
the cDNA by PCR, the restriction-cleaved PCR product was ligated
into the plasmid pBlueBac 4.5, and the entire VMAT2 coding region was
checked for mutations by DNA sequencing. The 3' reverse PCR primer
incorporated a FLAG epitope, enterokinase cleavage site, and a
6-histidine epitope at the C terminus of the protein, adding the amino
acids -Y-K-D-D-D-D-K-H-H-H-H-H-H. The correct FLAG- and
6-histidine-tagged human VMAT2 cDNA was then subcloned from
pBlueBac 4.5 into the mammalian cell expression plasmid pCDNA 3.1- at the XhoI and HindIII restriction sites. An
N-terminal FLAG-tagged construct was compared with the C-terminal tagged construct in a single high concentration [3H]TBZOH
binding experiment and found to have less overall ligand binding (data
not shown). Therefore, all human VMAT2 constructs used in this study
were derived from the initial C-terminal FLAG- and 6-histidine tagged
construct, which hereafter will be referred to as "WT." Except for
the C-terminal epitope tags, the sequence of this base construct is
identical to the sequences for human VMAT2 deposited in GenBankTM
under accession nos. L14269 and L23205.
Each of the 10 individual cysteine residues in human VMAT2 were
replaced by one of several site-directed mutagenesis methods. In the
incremental process of creating a cysteine-less human VMAT2, all of the
cysteines were changed to serines. However in generating 10 separate
single cysteine mutants, some cysteine replacements were to serine and
some to alanine (as indicated in Fig. 1C). The methods
employed in the replacement of cysteines included the GeneEditor
(Promega) and QuickChange (Stratagene) methods, a PCR-based method, and
a mutually priming oligo extension method. In every case, the resulting
constructs were thoroughly sequenced to ensure the absence of any
unwanted mutations in the human VMAT2 protein. To facilitate assessment
of relative expression levels by Western blot, each of the four
consensus glycosylation sites were mutated in the third position, from
N-X-(S/T) to N-X-(V/A), and an HA epitope was
added (Y-P-Y-D-V-P-D-Y-A) after arginine 94 in the large lumenal loop
(see Fig. 2B). (The four mutations were Thr58
Ala, Thr86 Val, Thr93 Val, and
Ser111 Ala.) Constructs which had this set of mutations
and the HA insertion were designated "G
HA," for
"glycosylation-negative, HA epitope" Additional desired mutations
introduced by one of the above methods included "silent" mutations
(which did not change the encoded protein sequence) to introduce
restriction sites. Among others, these included a particularly useful
KpnI restriction site between predicted TM segments 2 and 3, and sites added with the replacement of many of the cysteine residues
to facilitate screening by restriction digests.
Transient Expression and Harvesting from COS-7 Cells--
The
large quantities of DNA needed for transfection and assay of human
VMAT2 constructs in mammalian cells were obtained by growth in LB
medium of transformed E. coli followed by DNA purification on Qiagen DNA Prep columns. COS-7 cells were grown on 150-mm plates at
37 °C in DMEM (Life Technologies, Inc. catalog no. 11965-084) with
9% cosmic calf serum (HyClone catalog no. SH30087.02),
penicillin/streptomycin/glutamine (10 ml/liter; Life Technologies
catalog no. 10378-016), in a 5% CO2 atmosphere. On some
occasions the medium was also supplemented with less than 2 mg/liter
anti-mycotic fungizone (amphotericin B), which at the concentration
used did not seem to have any noticeable effect on the growth or
appearance of the COS cells, or on expressed human VMAT2 function. For
most experiments, three 150-mm plates of COS-7 cells were transfected
for each VMAT construct being tested. As an internal control, WT
(C-FLAG/His) human VMAT2 was transfected and assayed on each occasion
of COS cell expression. Transfection was performed by electroporation
of COS cells in PBS with 15-50 µg of DNA (equal for all constructs
in a given experiment) in 0.4-cm gap width cuvettes with the voltage of
the Bio-Rad GenePulser/capacitance extender unit set at 0.226 mV and the capacitance set at 0.950 microfarads. Cells were harvested 1.5-4
days after transfection, the cell culture medium usually being changed
once within that time period. Cells were detached with trypsin, washed
with 300 mM sucrose, 10 mM Hepes (pH 7.2), protease inhibitor-containing buffer (generally 20 µg/ml leupeptin, 100 µM phenylmethylsulfonyl fluoride, 100 µM benzamidine, 10 µg/ml soybean trypsin inhibitor),
and homogenized with a custom-built steel ball homogenizer, or "cell
cracker" (29). After 30-50 passages through the cell cracker, the
homogenate was centrifuged for 5 min at 735 × g, and
the vesicle containing supernatant was collected for use in assays.
Protein concentrations for COS vesicle preparations were assessed by
the Bradford method using the Bio-Rad protein assay reagent, and were
generally in the range of 2-5 mg/ml. Thus, assays that used 50 µl of
COS homogenate generally contained between 0.1 and 0.25 mg of total
protein. Assays were performed on freshly prepared samples (not frozen)
of COS cell homogenates, and results were normalized per milligram of
COS homogenate protein.
Radioligand Synthesis--
The radioligand used in binding
assays was [3H]dihydrotetrabenazine
([3H]TBZOH). This was synthesized by reaction at room
temperature of equal volumes (0.5-1 ml) of a 1.5 mg/ml solution of TBZ
in 100% methanol with 5-25 mCi of [3H]sodium
borohydride (50-75 Ci/mmol, in 0.1 M NaOH). Reduction occurs at the ketone, incorporating the 3H label. After >3
h, the reaction mixture was concentrated under a stream of nitrogen,
diluted with 100% methanol, reconcentrated a number of times to remove
water, and then streaked onto a silica gel TLC plate and developed with
a solvent mixture of 5 parts ethyl acetate to 1 part methanol. The
major [3H]TBZOH product, in a chemical amount visible
upon illumination with a hand-held UV light, migrated with an
approximate RF of 0.75, and was confirmed by
comparison with the nonradioactive standard compound.
The [3H]TBZOH was extracted from the silica gel with
five 1-ml aliquots of methanol and quantitated by scintillation
counting. The resulting [3H]TBZOH was chemically stable
for use during at least 2 years when stored at 20 °C.
[3H]TBZOH Binding and [3H]Serotonin
Uptake Assays--
Ligand binding curves were performed by incubation
of 50 µl of COS homogenate (generally 2-5 µg/µl protein by the
Bradford protein assay) with 200 µl of 300 mM sucrose, 10 mM Hepes (pH 7.2) buffer containing the
[3H]TBZOH. Curves were derived from binding measurements
at four to seven different concentrations of [3H]TBZOH,
generally in the range of 1-50 nM. Specific binding was assessed by addition of excess non-radioactive TBZ (~50
µM). In addition to some nonspecific or
non-TBZ-protectable binding, it was found that non-transfected COS
cells themselves have a low level of "specific," or
TBZ-protectable, [3H]TBZOH binding. This was controlled
for by performing parallel analysis of non-transfected or
mock-transfected COS cells. In instances where this was not done (some
of the many binding curves summarized in Fig. 1C), a
calculated adjustment was made by subtracting an average COS cell
specific binding background value (derived from multiple experiments)
at each ligand concentration. Ligand binding at 30 °C reached a
maximum level by ~10 min (data not shown), but was generally allowed
to proceed for >20 min. Monoamine uptake experiments were performed by
incubation at 30 °C of 50 µl of COS homogenate in the presence or
absence of ~50 µM TBZ (5 µM proton
ionophore FCCP inhibited uptake to the same extent, data not shown)
with 200 µl of buffer (sucrose/HEPES/protease inhibitors/ATP and
salts) for final concentrations of 50 nM
[3H]serotonin, 5 mM ATP, 5 mM
MgSO4, 4 mM KCl. From time-course studies, 3 min was considered to be in the initial linear range, and steady state
accumulation was reached by times shortly after 5 min. Human
VMAT2-containing vesicles with bound [3H]TBZOH or
accumulated [3H]serotonin were collected on filters
(usually Whatman GF/B, occasionally Millipore nitrocellulose/cellulose
acetate) by vacuum filtration on Millipore 12-well filtration manifolds
or on a Brandel M-48 BIT cell harvester. Ligand binding and uptake
assays, which were vacuum-filtered on the Millipore manifolds, were
performed in glass test tubes, and the test tubes were generally rinsed
with 2 × 4 ml of buffer (which was also vacuum-filtered). Assays
filtered using the Brandel cell harvester were performed in 1.2-ml well volume polypropylene 96-well plates, and the wells were rinsed with
5 × 1 ml of buffer. Radioactivity was quantitated by
scintillation counting in a Packard model 1600CA or 2000CA
scintillation counter.
Assessment of Relative Protein Expression Levels by Western
Blot--
Bradford protein assays were used to quantitate protein
concentrations to ensure equal loading of COS homogenate for each construct in Western blots. Protein samples were dissolved in SDS
sample buffer at a protein concentration of 0.1-0.5 µg/µl, and
electrophoresed on SDS-polyacrylamide gel electrophoresis minigels
(12%). Proteins were transferred to a 0.2-µm nitrocellulose membrane, and nonspecific antibody binding sites were blocked by
incubating the blot 1 h or longer in Superblock PBS (Pierce) + 0.05% Tween 20. Anti-HA antibody was diluted 1:850-1:1000 in 15 ml of
Superblock + 0.05% Tween and allowed to bind for 1 h or longer,
followed by at least six washes of 5 min or longer with Tris-buffered
saline, 0.05% Tween 20. The secondary antibody conjugate was goat
anti-mouse horseradish peroxidase (Bio-Rad), diluted 1:50,000-1:65,000
in 15 ml of Superblock, 0.05% Tween, and was allowed to bind for at
least 1 h, followed by a second set of six 5-min washes in
Tris-buffered saline, 0.05% Tween. Pierce ECL reagents were used
following the manufacturer's recommendations, and then the blots were
imaged using Hyperfilm ECL (Amersham Pharmacia Biotech).
Assessment of the Effect of Cysteine Derivatization on
[3H]TBZOH Binding--
Sulfhydryl modifying MTS reagents
or N-ethylmaleimide (NEM) (added from concentrated solution
stocks) were allowed to react with transfected COS cell vesicles
(generally 0.10-0.25 mg in a 50-µl volume) containing WT human VMAT2
and cysteine replacement constructs at sulfhydryl-reagent
concentrations of 0.5-5 mM, as indicated in the figures.
Parallel reactions were performed on mock-transfected COS cells and
were subtracted as background. Reaction occurred for 10 min at
30 °C, in a volume of 50 µl, after which the reaction was diluted
10- or 20-fold (to a volume of 500 or 1000 µl) by addition of
[3H]TBZOH-containing sucrose/HEPES buffer to terminate
(or greatly reduce the rate of) any further cysteine modification
reaction. Supporting this role for the dilution step, treatment with
low concentrations of MTSEA (0.2 mM) for 10-20 min at
30 °C has essentially no effect on [3H]TBZOH binding
by WT human VMAT2 (data not shown). In an experiment assessing the
ability of NEM to block reaction at the MTSEA primary site,
pretreatment was performed sequentially with the two cysteine-modifying reagents (i.e. first 1.5 mM NEM, then 3 mM MTSEA) for 10 min each prior to dilution. Dilution with
[3H]TBZOH-containing buffer initiated the ligand binding
portion of the experiment, which was for >15 min at 30 °C, and was
followed by filtration and scintillation counting. In experiments
assessing the ability of 100 mM DTT, 
-mercaptoethanol,
or cysteamine to reverse the effects of MTSEA derivatization, 100 mM reducing agent was added to the COS vesicles with the
[3H]TBZOH-containing dilution buffer (after 10-min
pretreatment of the COS vesicles with 2 mM MTSEA) and was
present throughout the ligand binding incubation period. For the
experiment demonstrating the requirement for sulfhydryl reactivity for
MTSEA efficacy, the concentrated stock solution of MTSEA (25 mM) was incubated for 10 min at 30 °C with an 8-fold
molar excess of -mercaptoethanol (200 mM) prior to
addition to the homogenized COS vesicles. During MTSEA reaction with
human VMAT2-containing COS vesicles, the MTSEA concentration was 2 mM, and the -mercaptoethanol concentration was 16 mM. Subsequent 10-fold dilution with
[3H]TBZOH-containing sucrose/HEPES buffer to initiate the
[3H]TBZOH binding portion of the experiment resulted in
final concentrations of 0.2 mM MTSEA, 1.6 mM
-mercaptoethanol, and 50 nM [3H]TBZOH.
[3H]TBZOH Protection against MTSEA Inhibition of
Ligand Binding--
[3H]TBZOH protection against MTSEA
inhibition of ligand binding was demonstrated by an assay that utilized
[3H]TBZOH both to protect the binding site during the
MTSEA reaction and as radioligand during the ligand binding experiment.
Parallel MTSEA inhibition curve experiments were performed with the
same concentrations of MTSEA, with [3H]TBZOH added either
before or after treatment with MTSEA. For all conditions, a control
experiment was performed with mock-transfected COS cells and subtracted
as background. As with the other assays, results were normalized for
the amount of COS homogenate protein, which for this set of experiments
varied slightly between constructs in the range of 0.11-0.16 mg/assay.
For the "unprotected" condition, MTSEA was preincubated with COS
cell homogenate in a volume of 50 µl, and after 10 min a 20-fold
dilution was made with sucrose/HEPES buffer containing
[3H]TBZOH (final concentration of [3H]TBZOH
~50 nM). For the "protected" condition,
[3H]TBZOH was added 5 min prior to addition of MTSEA (the
[3H]TBZOH concentration at the time of MTSEA incubation
was 500 nM), and after the 10-min MTSEA incubation, 20-fold
dilution was made without adding any additional radioligand, for a
final [3H]TBZOH concentration of 25 nM.
Incubation was performed for >20 min at 30 °C, followed by
filtration and scintillation counting. Specific binding was assessed by
addition of excess non-radioactive TBZ to a final concentration of
~50 µM, and specific binding was easily measurable with
similar results whether the non-radioactive TBZ (50 µM)
was added to the "protected" condition before or after addition of
the 500 nM [3H]TBZOH.
Statistical Analysis--
Graphing, non-linear curve fitting
(Kd and Bmax calculations),
linear regressions for protein assays, and statistical analysis were
performed using the computer program Prism (GraphPad, San Diego, CA).
Statistical significance of observed effects was assessed based on
scintillation counting data using this program by a two-tailed, paired
t test and is indicated in the figures as follows: *,
p < 0.1; **, p < 0.01; ***,
p 
0.001.
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RESULTS |
Replacement of Individual Cysteines, or All Non-TM Cysteines, Has
Little Effect on [3H]TBZOH Binding--
The positions of
the 10 native cysteines in human VMAT2 are indicated on a diagram of
the proposed secondary structure (Fig. 1A). As shorthand throughout
this paper, the 10 cysteines are distinguished with numbers
and bold text
(Cys1-Cys10) based on
the order in which they appear in the protein sequence from the N
terminus to C terminus. Their actual positions in the human VMAT2
primary amino acid sequence are (indicated in parentheses): Cys1 (residue 126), Cys2
(176), Cys3 (207), Cys4
(311), Cys5 (333), Cys6 (369),
Cys7 (383), Cys8 (439),
Cys9 (476), and Cys10
(497). The effect of mutation of each individual cysteine on
[3H]TBZOH binding was assessed (Fig. 1C). To
control for biological and other sources of variability between sets of
binding curves, a complete binding curve for WT was measured on each
and every occasion. Compilation of values from all WT curves indicates
a Kd of 12.5 ± 2.3 nM (or
±18.4%, 95% confidence interval). Comparison was made between
Kd values for cysteine replacement constructs and WT
human VMAT2 within each set of experiments. As seen in Fig.
2B, replacement of any
individual cysteine, or replacement of all non-TM cysteines together
(Cys1, Cys5,
Cys9, and Cys10 to Ser
(C1.5.9.10S)), had no more than a modest effect on binding affinity.
Beyond this general and qualitative conclusion, it is interesting
to note the cysteine that, when replaced with alanine,
consistently resulted in the largest decrease in binding affinity
(2.5-fold increase over WT Kd) was
Cys3 in putative TM 4.
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Fig. 1.
Positions of human VMAT2 cysteines and the
effect of their replacement on [3H]TBZOH binding
affinity. A, human VMAT2 is predicted to have 12 membrane-spanning domains, with the N and C termini on the cytoplasmic
face of the vesicle membrane. All human VMAT2 constructs used in this
study had C-terminal FLAG and 6-histidine epitope tags. This common
epitope-tagged background without any additional amino acid changes is
referred to throughout this paper as WT, for wild type. B,
structure of tetrabenazine. C, Kd values
were obtained from ligand binding curves (as described under
"Experimental Procedures"), and are plotted as percentage of the WT
human VMAT2 Kd from a control binding curve in every
set of experiments (>20). Error bars show the
S.E. of values from multiple binding curves (indicated in
parentheses). Double cysteine to serine replacements
included C1.5S and C9.10S, and C1.5.9.10S was a quadruple cysteine to
serine replacement mutant. The cysteine replacement that reproducibly
showed the largest decrease in relative [3H]TBZOH binding
affinity (2.5-fold) was Cys3 to alanine in
predicted TM 4.
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Fig. 2.
A cysteine-less human VMAT2 is expressed but
not functional. A, [3H]TBZOH ligand
binding curves for WT human VMAT2, Cys-less VMAT2 (a construct in which
all 10 cysteines were replaced by serines), and mock-transfected COS
cells. The mock-transfected condition was treated the same as the other
two conditions, including COS cell electroporation, but purified
plasmid DNA was not added. Error bars show the
S.E. of multiple measurements at each [3H]TBZOH
concentration. WT, solid square; Cys-less,
solid diamond; mock, solid
triangle. Cys-less human VMAT2 does not show
[3H]TBZOH binding or [3H]serotonin
transport (data not shown) above the level of mock-transfected COS-7
cells. B, to facilitate detection by Western blot, the four
consensus glycosylation sequences were mutated and an HA epitope tag
was added, as described under "Experimental Procedures." This set
of mutations was designated GHA. C, Western blots of cell
homogenates from COS cells transfected with the WT GHA and Cys-less
GHA constructs showed equivalent levels of human VMAT2 GHA
immunoreactivity. An equal amount of transfected COS homogenate (2 µg, assessed by the Bradford protein assay) was loaded for each lane.
These data demonstrated that the total lack of ligand binding or
transport by Cys-less human VMAT2 was not due to significant
differences in protein expression. Wild type COS cells showed no
immunoreactivity at the position of GHA human VMAT2, and very little
HA immunoreactivity overall (data not shown).
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A Cysteine-less Human VMAT2 Is Expressed but Not
Functional--
Although no individual cysteine was found critical for
[3H]TBZOH binding, a cysteine-less human VMAT2 (in which
all the cysteines were replaced by serines) does not bind
[3H]TBZOH (Fig. 2A) or transport
[3H]serotonin (data not shown) above the low level of
mock-transfected COS-7 cells. Three possible explanations for this
finding are: 1) that there is a lack of protein expression, 2) that
there is a requirement for a subset of cysteines in combination, or 3) that serine is a poor replacement at one or more positions, and that a
cysteine-less construct with different cysteine replacements might be
functional. To address the question of whether total lack of ligand
binding and monoamine uptake was a result of lack of protein
expression, we prepared human VMAT2 constructs with epitope tags for
Western blotting. The four consensus glycosylation sequences present in
the large lumenal loop were mutated, and an HA epitope tag was inserted
into human VMAT2 (described under "Experimental Procedures" and
Fig. 2B) at a site closely corresponding to the position of
a readily detectable HA tag that had been reported previously (30).
This set of mutations we designated "GHA," for
"glycosylation-negative, HA epitope." (For human VMAT2 Western blotting, we found the anti-HA antibody/lumenal HA epitope combination much superior to the M2 anti-FLAG antibody/C-terminal FLAG epitope combination.) Comparison of WT with WT GHA for
[3H]TBZOH binding demonstrated a slight decrease in
Bmax, but little or no change in
Kd (data not shown), consistent with the reported
effect of deglycosylation of rat VMAT1 (31). Western blots of cell
homogenates from COS cells transfected with the WT GHA and Cys-less
GHA constructs (Fig. 2C) show equivalent levels of
immunoreactivity. These data demonstrate that the total lack of ligand
binding or transport by Cys-less human VMAT2 is not due to significant
differences in protein expression. (Wild type COS cells showed no
immunoreactivity at the position of GHA human VMAT2 and very little
HA immunoreactivity overall.) Therefore, having ruled out protein
expression as responsible for lack of detectable function, we focused
on identifying cysteine subsets that would restore the function that
was lost in the cysteine-less VMAT2.
Subsets of Cysteines That Support Human VMAT2
[3H]TBZOH Binding--
As noted in Fig. 1C,
replacement of all the non-TM cysteines with serines did not
significantly affect Kd for [3H]TBZOH
binding. This construct (designated C1.5.9.10S), also had essentially
WT Bmax (Fig.
3B) and expression (Western
blot, Fig. 3C). At this time, useful information regarding
TM cysteines came from the sequence of a recently discovered
Caenorhabditis elegans VMAT containing only three cysteines,
all in predicted TM segments (32). The number and position of the
C. elegans VMAT cysteines suggested a cysteine combination
that might restore function to the cysteine-less human VMAT2
background. As seen in Fig. 3A, amino acid replacements in
C. elegans VMAT at the corresponding positions of cysteines
in human VMAT2 include a threonine (Cys4), a
glycine (Cys5), a methionine
(Cys9), and four serines
(Cys1, Cys6,
Cys7, and Cys10). When
the human VMAT2 construct (designated +2.3.8, see Fig. 3A)
which has only these three "C. elegans" cysteines was assayed for functional activity, significant [3H]TBZOH
binding and monoamine uptake activity was observed (Figs. 3B
and 4A). From multiple ligand
binding curves and uptake experiments, +2.3.8 bound
[3H]TBZOH with essentially wild type affinity
(Kd 5-20 nM), had an approximate 75%
reduction in [3H]TBZOH Bmax, and
accumulated serotonin at the same rate as WT (when normalized for the
number of [3H]TBZOH binding sites, see Fig. 4
(A and B)). Like cysteine-less (GHA) and
C1.5.9.10S (GHA) human VMAT2, +2.3.8 (GHA) was expressed at a level
equivalent to WT as assessed by Western blot (Fig. 3C),
leading to the general conclusion that replacement of 7 or all 10 cysteines in VMAT2 had little effect on total expression levels. One
possible explanation for how +2.3.8 could have a WT Kd, with a decrease in Bmax
but wild type levels of Western blot immunoreactivity, is a change in
the ratio of expression of native transporters versus
non-native transporters (which do not bind [3H]TBZOH with
high affinity).
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Fig. 3.
Subsets of cysteines that restore human VMAT2
[3H]TBZOH binding. A, the positions of
the 10 wild type human VMAT2 cysteines are shown diagrammatically,
together with the identity of the replacement at a given cysteine
position in four human VMAT2 mutants and C. elegans VMAT.
B, [3H]TBZOH ligand binding curves for WT,
C1.5.9.10S, +2.3.8, and +1.4.5.6.7.9.10 human VMAT2 constructs.
Error bars show the S.E. of multiple measurements
at each [3H]TBZOH concentration. Symbols and
lines are as follows: WT, solid line
and solid square; C1.5.9.10, large
dashed line with solid
diamond; +2.3.8, small dashed line
with open triangle; +1.4.5.6.7.9.10,
alternating large and small
dashed line with cross). (Note that
background binding of the "mock" condition has already been
subtracted, and specific binding shown is above background.)
Kd values from the experiment shown were WT (17 nM), C1.5.9.10S (10 nM), +2.3.8 (20 nM), and +1.4.5.6.7.9.10 (31 nM), with 95%
confidence intervals less than ± 30%. C, Western blot
showing the relative expression of WT GHA (1), C1.5.9.10S GHA (2),
and +2.3.8 GHA (3). The amount of protein loaded in each lane was 1 µg of the resuspended pellet fraction from a 1-h 100,000 × g centrifugation. (GHA modifications are explained under
"Experimental Procedures" and in the legend to Fig.
2B.)
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Fig. 4.
Enhancement of [3H]serotonin
uptake by replacement/removal of three atoms. Statistical
significance of effects was assessed by a paired, two-tailed
t test as described under "Experimental Procedures." *,
p < 0.1; **, p < 0.01; ***,
p 0.001. Error bars represent
the S.E. of multiple measurements at the indicated time point or
condition. A, time course showing 50 µM
TBZ-protectable [3H]serotonin uptake normalized to the
number of [3H]TBZOH binding sites
(Bmax from a ligand binding curve). Symbols and
lines are as follows: WT, solid line and
solid square; +2.3.8, small dashed
line with open triangle;
+1.4.5.6.7.9.10, alternating large and
small dashed line with cross).
Percentage of WT steady state serotonin accumulation per
[3H]TBZOH binding sites (second y axis) was
calculated based on the WT uptake value at 15 min. B,
significant enhancement of serotonin uptake per number of tetrabenazine
binding sites for +1.4.5.6.7.9.10 compared with WT was reproducible, as
shown by the comparison of results from multiple experiments at a 3-min
time point. Experiment A shows uptake normalized to
[3H]TBZOH binding sites which were estimated from the
level of binding of a single, saturating concentration of
[3H]TBZOH (50 nM). Experiment B is normalized
to [3H]TBZOH Bmax from a ligand
binding curve (the 3-min time point from panel
A). C, to investigate whether one of the three
cysteine replacements (Cys2,
Cys3, or Cys8) was
primarily responsible for enhancement of uptake by the +1.4.5.6.7.9.10
construct, the levels of TBZ-protectable [3H]serotonin
uptake/[3H]TBZOH binding sites at 3 min for the single
cysteine mutants C2S, C3A, C8A, and the +2.3.8 and +1.4.5.6.7.9.10
constructs were measured. Comparison was made to WT human VMAT2 and
expressed as percentage of WT.
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To test whether the combination of Cys2,
Cys3, and Cys8 is
necessary as well as sufficient for ligand binding and transport, we
replaced this set of the three C. elegans cysteines in WT
human VMAT2 and tested [3H]TBZOH binding and
[3H]serotonin uptake. Consistent with our initial
observation that no individual cysteine is critical for function, this
combination of three cysteines together is also non-essential for
function. This construct, designated +1.4.5.6.7.9.10 (see Fig.
3A), was found to have a high affinity
[3H]TBZOH binding Kd which was nearly
identical to both WT and +2.3.8 (not more than a modest 2-fold change,
Fig. 3B). Although replacement of the non-TM cysteines does
not affect the number of high affinity binding sites
(Bmax), replacement of either set of three TM
cysteines (Cys4, Cys6,
and Cys7, in +2.3.8, or
Cys2, Cys3, and
Cys8 in +1.4.5.6.7.9.10) leads to a similar 75%
reduction in Bmax (Fig. 3B).
Presumably, an alteration in the ratio of native transporters versus non-native transporters (which do not bind
[3H]TBZOH with high affinity) similar to the situation
for +2.3.8 also occurs for the +1.4.5.6.7.9.10 construct. This effect
may suggest a small but additive role for individual cysteines in TM
segments in preserving the ligand binding competence and structural integrity of the transporter.
Comparison of [3H]Serotonin Uptake between WT and
Cysteine Replacement Mutants--
Serotonin uptake time course
experiments were performed comparing TBZ-protectable
[3H]serotonin-specific uptake for WT, +2.3.8, and
+1.4.5.6.7.9.10 human VMAT2 constructs (Fig. 4A). When
normalized for the number of [3H]TBZOH binding sites (to
control for the reduction in high-affinity ligand binding
transporters), WT and +2.3.8 accumulate serotonin at the same rate.
However, +1.4.5.6.7.9.10 accumulates serotonin at a much faster rate
than WT. In terms of total [3H]serotonin accumulated, WT
and +1.4.5.6.7.9.10 are nearly equal, although +1.4.5.6.7.9.10 has only
one-fourth the number of native transporters (measured as high affinity
[3H]TBZOH sites). Since specific uptake is defined as
TBZ-protectable uptake, if there were to be any VMAT molecules that
transport but that do not bind TBZ, they would not contribute to the
measured values, but would be subtracted with the nonspecific
background. The human VMAT2 construct +1.4.5.6.7.9.10 therefore appears
to transport [3H]serotonin significantly more efficiently
than WT. Uptake for +2.3.8 and +1.4.5.6.7.9.10 relative to WT was
reproducible in multiple experiments (Fig. 4B). To
investigate whether one of the three cysteine replacements
(Cys2, Cys3, or
Cys8) was primarily responsible for enhancement
of uptake by the +1.4.5.6.7.9.10 construct, the levels of
TBZ-protectable [3H]serotonin
uptake/[3H]TBZOH binding sites for the single cysteine
mutants C2S, C3A, and C8A were compared with WT, +2.3.8, and
+1.4.5.6.7.9.10 (Fig. 4C). From this analysis, it appears
that enhancement of uptake is not due primarily to replacement of any
one of these three cysteines, but is more likely an effect of the
combination of replacement of these three cysteines together. The
molecular differences between WT and +1.4.5.6.7.9.10 are only three
atoms; replacement of two sulfur atoms (at Cys2
and Cys3) with oxygen atoms, and the removal of
a third sulfur atom (at Cys8). Although details
regarding the mechanism of this enhancement of
[3H]serotonin transport remain to be elucidated, residues
near these positions may interact functionally in the transport process
and define a portion of the transport channel.
Identification and Probing the Environment of the Primary and
Secondary MTSEA Cysteine Targets--
WT and cysteine replacement
human VMAT2 constructs were tested for the effect of treatment with
cysteine-modifying reagents on their ability to bind
[3H]TBZOH. This led to the discovery of a second
remarkable feature of the +1.4.5.6.7.9.10 human VMAT2 construct,
i.e. profound insensitivity to [3H]TBZOH
binding inhibition by the cysteine-reactive small molecule MTSEA (Fig.
5A). (The structures of MTSEA
and additional cysteine-modifying reagents are presented in Fig.
5B). In MTSEA concentration curve experiments using 50 nM [3H]TBZOH (Fig. 5A), WT,
C1.5.9.10S, and +2.3.8 demonstrate a very significant,
dose-dependent MTSEA inhibition of [3H]TBZOH
binding. In contrast, MTSEA had much less effect on
[3H]TBZOH binding by the +1.4.5.6.7.9.10 construct. Even
at 5 mM MTSEA, ligand binding by this construct was only
inhibited ~20%. [3H]TBZOH binding by WT is more
sensitive (statistically significant) to MTSEA than any of the other
constructs, and +1.4.5.6.7.9.10 is much less sensitive than any of the
other constructs. This suggested that MTSEA sensitivity has two unequal
"components," or sets of cysteine targets. In +1.4.5.6.7.9.10, the
primary MTSEA target (responsible for ~80% of binding inhibition)
appears absent but the secondary MTSEA target (responsible for ~20%
of binding inhibition) is present. In C1.5.9.10S and +2.3.8, the
primary target is present but the secondary target is absent. In WT,
both the primary and secondary cysteine targets are present, conferring maximal sensitivity. From the data in Fig. 5A, it appears
that the primary MTSEA target (with respect to
[3H]TBZOH binding inhibition) is one or more of
Cys2, Cys3, or
Cys8, and that the secondary target is one or
more of the non-transmembrane cysteines Cys1,
Cys5, Cys9, and
Cys10.
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Fig. 5.
Effect of sulfhydryl-modifying reagents on
[3H]TBZOH binding by human VMAT2 cysteine mutants.
Statistical significance of effects was assessed by a paired,
two-tailed t test as described under "Experimental
Procedures." *, p < 0.1; **, p < 0.01; ***, p 0.001. Error bars
represent the S.E. of multiple measurements at the indicated
concentration point or condition. A, comparison of MTSEA
sensitivity between WT human VMAT2 and constructs with different
cysteine replacements. For ease of comparison between constructs (which
have different Bmax values), the percentage of
maximal [3H]TBZOH binding for a given construct is
plotted (y axis) versus MTSEA concentration
(x axis). WT is significantly more sensitive than the other
constructs, and +1.4.5.6.7.9.10 is significantly less sensitive than
the other constructs, suggesting two unequal components of MTSEA
sensitivity. B, structures of cysteine-reactive small
molecules with differing charges, hydrophobicity, and membrane
permeabilities. C, comparison of the effects of
cysteine-modifying reagents on WT human VMAT2 [3H]TBZOH
binding. Pretreatment and binding experiments were performed in a
manner similar to those in panel A, as described
under "Experimental Procedures." For each data set, the control
condition is defined as the condition without treatment with the
indicated sulfhydryl-modifying reagent. Set 1,
treatment with 3 mM MTSEA results in >90%
[3H]TBZOH binding inhibition. Sets
2 and 3, separate experiments demonstrating that
treatment with 5 mM MTSET results in 20-30%
[3H]TBZOH binding inhibition. Set
4, treatment with 2 mM NEM has no effect on
[3H]TBZOH binding. Set 5, treatment
with 1.5 mM has no effect on [3H]TBZOH
binding, nor can pretreatment with 1.5 mM NEM block
modification and inhibition by 3 mM MTSEA. Set
6, treatment with 3 mM MTSES has no effect on
[3H]TBZOH binding. D, assessment of the
ability of MTSEA to inhibit [3H]TBZOH binding by single
cysteine mutants. Pretreatment and binding experiments were performed
in a manner similar to those in panel A, as
described under "Experimental Procedures." Statistically
significant effects, both of MTSEA treatment, and of differences in
response between constructs, are indicated. These data demonstrate that
Cys8 is the primary MTSEA target mediating
inhibition of [3H]TBZOH binding by VMAT2.
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To probe the environment in which these target cysteines reside, the
ability of additional cysteine-modifying reagents of differing
charge and hydrophobicity (Fig. 5B) to inhibit
[3H]TBZOH binding by WT human VMAT2 was examined. Three
methanethiosulfonate derivatives (MTSEA, MTSET, and MTSES) and NEM were
used in this study. MTSEA can be either charged or uncharged, depending
upon pH and environment, and is considered membrane-permeable. MTSET is
bulkier and carries a permanent positive charge, whereas MTSES carries
a permanent negative charge. Both MTSET and MTSES are considered
membrane-impermeable. NEM is uncharged but primarily reacts with
cysteines in an aqueous (non-hydrophobic) environment. As seen for WT
human VMAT2 in Fig. 5C (set 1), MTSEA
reaction at both primary and secondary targets results in >90%
inhibition of [3H]TBZOH binding by 3 mM
MTSEA. MTSET (5 mM) has a much smaller but statistically
significant effect (Fig. 5C, sets 2 and 3), with inhibition at a level consistent with and
suggestive of modification at only the MTSEA secondary target (one or
more of Cys1, Cys5,
Cys9, and Cys10). MTSET
carries a permanent positive charge and is not expected to cross
vesicle membranes. If COS-expressed VMAT2 molecules are correctly
oriented with respect to sealed vesicle membranes (which we believe to
be the case from proteolysis experiments; data not shown), only two of
these cysteines (Cys9 and
Cys10) would be predicted to be accessible to
MTSET, whereas the others would be protected inside the vesicle lumen.
NEM (2 mM) has no effect on [3H]TBZOH binding
(Fig. 5C, set 4), nor can pretreatment
with 1.5 mM NEM block subsequent inhibition of ligand
binding by MTSEA (Fig. 5C, set 5).
MTSES (3 mM) also has no effect on [3H]TBZOH
binding (Fig. 5C, set 6). Taken
together, these data support the conclusion that the primary MTSEA
target is in a hydrophobic and perhaps sterically hindered environment
(i.e. in a transmembrane segment, as has been predicted for
Cys2, Cys3, and
Cys8), that the MTSEA secondary sites are in
hydrophilic regions of the transporter, and that the MTSET target
appears to be a secondary MTSEA target on the cytoplasmic face of the
vesicle membrane (perhaps Cys9 and
Cys10). Since inhibition was seen by MTSEA and
MTSET but not by MTSES or NEM, modification with a positively charged
reagent at a secondary (with respect to MTSEA inhibition) cysteine
target may be important for the observed effect of modest
[3H]TBZOH binding inhibition.
To investigate the relative contributions of
Cys2, Cys3, and
Cys8 as the primary MTSEA targets, MTSEA
pretreatment/[3H]TBZOH binding experiments were performed
on the single cysteine mutants (Fig. 5D). Replacement of
Cys2 with serine or Cys3
with alanine did not remove either the primary or secondary MTSEA
targets, resulting in the WT level of ~90% ligand binding inhibition. In contrast, replacement of Cys8
with alanine (net removal of a single sulfur atom from the entire
transporter) resulted in loss of MTSEA sensitivity and recovery of
~80% of maximal [3H]TBZOH binding at 3 mM
MTSEA. Thus, Cys8 (cysteine 439) is the single
primary target responsible for ~80% of [3H]TBZOH
binding inhibition by MTSEA.
Characteristics of [3H]TBZOH Binding Inhibition by
MTSEA--
To demonstrate the requirement for sulfhydryl reactivity
(through the cysteine-reactive methanethiosulfonate leaving group) for
MTSEA efficacy of inhibition of [3H]TBZOH
binding, a concentrated stock solution of MTSEA was treated with an
8-fold molar excess of -mercaptoethanol prior to reaction with
VMAT2-containing vesicles and a [3H]TBZOH binding
experiment. Prereaction with -mercaptoethanol completely eliminated
the ligand binding-inhibitory effect of MTSEA (Fig.
6A), demonstrating the
requirement for sulfhydryl reactivity for the MTSEA inhibition of
[3H]TBZOH binding.
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Fig. 6.
Inhibition of [3H]TBZOH binding
by MTSEA: requirement for sulfhydryl reactivity, proton-gradient
independence, and apparent irreversibility at the primary target.
Statistical significance of effects was assessed by a paired,
two-tailed t test as described under "Experimental
Procedures." *, p < 0.1; **, p < 0.01; ***, p 0.001. Error bars
show the S.E. of multiple measurements at the indicated concentration
point or condition. A, prereaction of MTSEA (as described
under "Experimental Procedures") with 8-fold molar excess
-mercaptoethanol completely eliminates the ability of MTSEA to
inhibit [3H]TBZOH binding. Bar 1,
control (no -mercaptoethanol, no MTSEA). Bar
2, 2 mM MTSEA, not pretreated with
-mercaptoethanol. Bar 3, pretreatment of 25 mM MTSEA with 200 mM -mercaptoethanol for 10 min at 30 °C (followed by addition to COS vesicles, which diluted
the concentrations to 2 mM MTSEA, 16 mM
-mercaptoethanol) eliminated the efficacy of MTSEA in
[3H]TBZOH binding inhibition, demonstrating the
requirement for the highly sulfhydryl-reactive methanethiosulfonate
leaving group. B, sensitivity of WT human VMAT2
[3H]TBZOH binding to MTSEA inhibition was assessed in the
presence and absence of 5 mM ATP, 5 mM
Mg2+, 4 mM Cl (conditions used
for uptake experiments). MTSEA sensitivity is independent of the
presence of a proton gradient. C, the effect of adding
-mercaptoethanol or DTT on recovery of [3H]TBZOH
binding after inhibition by MTSEA. After treatment of COS vesicles
containing WT human VMAT2 with 2 mM MTSEA (as described
under "Experimental Procedures"), incubation with 100 mM DTT for >15 min at 30 °C had no reversal effect,
whereas 100 mM -mercaptoethanol (under the same
conditions) had a small but statistically significant effect. The small
effect of -mercaptoethanol is consistent with sulfhydryl reversal at
the secondary, but not the primary (Cys8), MTSEA
target site. D, the effect of adding -mercaptoethanol
(separate experiment from panel C) or cysteamine
on recovery of [3H]TBZOH binding after inhibition by
MTSEA. After treatment of COS vesicles containing WT human VMAT2 with 2 mM MTSEA (as described under "Experimental
Procedures"), incubation with 100 mM cysteamine for >15
min at 30 °C had no reversal effect, whereas 100 mM
-mercaptoethanol (under the same conditions) had a small but
statistically significant effect. The small effect of
-mercaptoethanol is consistent with sulfhydryl reversal at the
secondary, but not the primary (Cys8), MTSEA
target site.
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We considered the possibility that a conformational change in VMAT2
structure in the presence of a proton electrochemical gradient might
change the MTSEA reactivity of transporter cysteines (Fig.
6B). COS cell homogenates prepared as described under
"Experimental Procedures" did not contain the levels of
ATP/Mg2+/Cl necessary to generate a proton
electrochemical gradient and transport serotonin (data not shown).
Therefore, to assess whether binding of [3H]TBZOH would
be either more or less sensitive to MTSEA in the presence of a proton
electrochemical gradient (indicative of a conformational change
involving MTSEA target residues), sensitivity to MTSEA inhibition was
assessed in the presence and absence of 5 mM ATP, 5 mM Mg2+, 4 mM Cl
(conditions used for uptake experiments). MTSEA sensitivity was found
to be independent of the presence of a proton gradient; therefore, no
change in cysteine accessibility in the presence of a proton gradient
is indicated from this experiment.
To further probe the environment of the MTSEA primary target cysteine
and secondary target cysteine or cysteines, we performed disulfide
reversal experiments with the reducing agents -mercaptoethanol, dithiothreitol (DTT), and cysteamine (Fig. 6, C and
D). At concentrations as high as 100 mM, these
three reducing agents themselves had little or no effect on
[3H]TBZOH binding. However, none of these reagents at
this concentration reversed the effect of the MTSEA reaction
corresponding to the primary target site (Cys8).
-Mercaptoethanol did show a statistically significant effect of
reversal of MTSEA inhibition at a level consistent with reversal of
modification at the secondary MTSEA target cysteine or cysteines
(i.e. Cys9 and
Cys10). Although the observed lack of disulfide
reversal at the primary MTSEA target is somewhat surprising, the
identity of the primary MTSEA target as Cys8 is
firmly established, and also the requirement for the highly cysteine-reactive methanethiosulfonate leaving group for MTSEA inhibition of [3H]TBZOH binding. The most likely
explanation for the lack of observed reversal is that
Cys8 may be in a very hydrophobic (and perhaps
sterically hindered) environment. This is consistent with the
prediction for a very hydrophobic tetrabenazine binding site from a
correlation study of the apparent affinity and hydrophobicity (over
several orders of magnitude) for a series of tetrabenazine derivatives
(33).
[3H]TBZOH Blocks Modification of the Primary Target,
Cys8 (439), by MTSEA--
To support the
hypothesis that inhibition of [3H]TBZOH binding by MTSEA
reaction at the primary (Cys8) or
secondary (Cys1, Cys5,
Cys9, and Cys10) cysteine
targets was actually occurring at the tetrabenazine binding site, it
was necessary to determine whether tetrabenazine occupancy at its
binding site could protect against inhibition by MTSEA. Recognizing the
significant technical challenges of completely removing a hydrophobic,
high affinity ligand from a cell homogenate, a novel protection assay
was developed. This assay utilized [3H]TBZOH to both
protect the tetrabenazine binding site during the MTSEA reaction, and
as radioligand during the ligand binding experiment (described under
"Experimental Procedures"). Consistent with an estimate of the TBZ
dissociation half-life of less than 1 min, based on the common on-rate
value for a small molecule ligand binding to a protein of
106 M1
s1, and a Kd of 12.5 nM (34), the ligand exchange rate is fast enough that,
during the 20-min ligand binding incubation period, excess
non-radioactive TBZ exchanges with bound [3H]TBZOH to
allow detection of specific [3H]TBZOH binding. Human
VMAT2 constructs tested included WT, +2.3.8, +1.4.5.6.7.9.10, and the
single cysteine replacements Cys2 Ser,
Cys3 Ala, and Cys8
Ala. As seen in Fig. 7 (A,
B, D, and E), MTSEA inhibition of
[3H]TBZOH binding by the four constructs that possessed
the primary target cysteine (Cys8) was
significantly and reproducibly protected against MTSEA inhibition by
the presence of [3H]TBZOH during the MTSEA reaction. The
amount of binding activity protected at 5 mM MTSEA was
between 65% and 80% of maximal (compared with 10% or less binding in
the unprotected condition), consistent with protection of the primary
MTSEA target (Cys8), but not the secondary MTSEA
target cysteine(s). This supports a model in which the primary MTSEA
target mediating [3H]TBZOH binding inhibition,
Cys8 (Cys439) in TM 11, is at the
TBZ binding site. In the two constructs that lacked
Cys8 (+1.4.5.6.7.9.10, and the single cysteine
replacement Cys8 > Ala, Fig. 7 (C
and F)) [3H]TBZOH binding was insensitive to
the primary effect of MTSEA, but was inhibited up to 20% by reaction
at the secondary MTSEA target. The 20% effect of MTSEA inhibition
mediated by the secondary cysteine target(s) was not protected by
[3H]TBZOH, consistent with conformational effects rather
than direct steric blocking at these sites. Although it is difficult to
absolutely rule out the possibility that protection at
Cys8 may be due to a conformational effect of
[3H]TBZOH binding, the observed effect of protection is
specific to Cys8 and not shared with the
secondary MTSEA target cysteines. Further, TBZ-protectable
Cys8 is positioned centrally with regard to
previously available data from photoaffinity labeling (particularly
compelling data for a direct effect), mutagenesis, and chimera analysis
(as reviewed below under "Discussion"), clearly defining a region
involved in TBZ binding.
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Fig. 7.
[3H]TBZOH occupancy at the
tetrabenazine binding site protects against MTSEA reaction at human
VMAT2 Cys8 (Cys439). The ability of 500 nM [3H]TBZOH to protect against modification
by 1-5 mM MTSEA was assessed (as described under
"Experimental Procedures") by comparison of [3H]TBZOH
binding when radioligand was added before (cross,
dashed lines) or after (solid
square, solid lines) treatment with
MTSEA. Panels A-F show the results of this assay
for different human VMAT2 cysteine replacement constructs. Statistical
significance of differences at a given MTSEA concentration between the
protected ([3H]TBZOH first, cross and
dashed line) and unprotected (MTSEA first,
solid square and solid
line) conditions was assessed as described under
"Experimental Procedures" and is indicated in the figures between
the protected and unprotected curves (*, p < 0.1; **,
p < 0.01; ***, p 0.001).
Error bars show the S.E. of multiple measurements
at the indicated concentration point. Panels A-C
and panels D-F were from experiments performed
on separate occasions. A, WT human VMAT2; B,
+Cys2.3.8 VMAT2; C, +Cys1.4.5.6.7.9.10 VMAT2; D,
Cys2 Ser VMAT2; E,
Cys3 Ala VMAT2; F,
Cys8 Ala VMAT2. The four constructs in which
Cys8 is present (panels A,
B, D, and E) all show
[3H]TBZOH protection of MTSEA inhibition, whereas the two
constructs in which Cys8 is absent
(panels C and F) have
[3H]TBZOH binding that is much less sensitive to
MTSEA.
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DISCUSSION |
In instances where x-ray crystallography or NMR studies have not
yet been feasible (especially the case for the majority of integral
membrane proteins), creative approaches to the study of structure,
function, and ligand binding sites have been applied that take
advantage of the unique chemical reactivity of native or engineered
cysteines. An important prerequisite for the application of these
methods is either a functional cysteine-less background, or a
background of cysteines that does not interfere with the particular
assay (i.e. derivatization, cross-linking, etc.) being applied. We sought to apply such methods to the study of human VMAT2,
which has 10 naturally occurring cysteines (Fig. 1A). We first examined the effect of replacement of human VMAT2 cysteines on
[3H]TBZOH binding. None of these cysteines individually
are critical for human VMAT2 expression or ligand binding (Fig.
1C). However, although a cysteine-less VMAT (all 10 cysteines replaced with serines) is expressed at levels comparable to
wild type (Fig. 2C), it neither binds
[3H]TBZOH (Fig. 2A) nor transports serotonin
(data not shown). Based on this observation, we sought to identify
subsets of native cysteines that would allow high affinity ligand
binding and monoamine transport and, at the same time, provide the
essential non-reactive cysteine background for cysteine-based
biochemical structure and function studies. The number and position of
the C. elegans VMAT cysteines suggested a cysteine
combination that might restore function to Cys-less human VMAT2.
Indeed, +2.3.8 human VMAT2 with a subset of only 3 (out of 10)
cysteines, at the corresponding positions of the three cysteines in
C. elegans VMAT (Fig. 3A), was found to have
wild-type binding affinity (Kd, Fig. 3B)
and serotonin transport (uptake normalized to the number of
[3H]TBZOH binding sites, Fig. 4 (A and
B)), with a reduction (by ~75%) in high affinity ligand
binding sites (Bmax, Fig. 3B), but not significantly in total expression (assessed by Western blot, Fig.
3C). This subset of cysteines (present in C. elegans VMAT, and in human, rat, mouse, and Bos taurus
VMAT2) is sufficient but not necessary to restore VMAT2 function.
Replacement in wild type human VMAT2 of this set of three cysteines
still resulted in a similarly functional transporter (near wild type
[3H]TBZOH binding Kd, decreased
Bmax, Fig. 3B). Compared with wild
type, this latter construct (lacking the three transmembrane cysteines
present in C. elegans VMAT, see Fig. 3A) was
found to have two very interesting properties, i.e.
significant enhancement of the rate of tetrabenazine-protectable
serotonin transport (normalized to the number of
[3H]TBZOH binding sites, Fig. 4) and pronounced
insensitivity of [3H]TBZOH binding to inhibition by the
thiol-modifying reagent MTSEA (methanethiosulfonate ethylamine, Fig.
5A). Cysteine derivatization experiments on a series of
human VMAT2 cysteine replacement mutants allowed identification of
Cys8 in TM 11 (Cys439) as the
primary target mediating ~80% of the MTSEA inhibition of
[3H]TBZOH binding. Permanently charged or more
hydrophilic sulfhydryl-reagents did not react at
Cys8 (Figs. 5 (C and D)
and 6 (C and D)). A secondary component of MTSEA
inhibition (responsible for ~20% of inhibition) is due to reaction
at one or more of the non-TM cysteines Cys1,
Cys5, Cys9, and
Cys10 (Fig. 5A). No significant
difference in MTSEA inhibition of [3H]TBZOH binding was
detected in the presence or absence of a proton gradient (Fig.
6B). The secondary target cysteine(s) appeared to be
accessible for reaction with the permanently charged
sulfhydryl-modifying reagent MTSET (Fig. 5C) and
-mercaptoethanol (Fig. 6, C and D), but were
not protected from MTSEA reaction by bound [3H]TBZOH
(Fig. 7). However, preincubation with 500 nM
[3H]TBZOH blocked modification at
Cys8 (Cys439 in TM 11) by a
10,000-fold molar excess of MTSEA. The most straightforward explanation
of this result is that Cys439, the primary target mediating
MTSEA inhibition of [3H]TBZOH binding, is positioned at
the tetrabenazine binding site.
Previously available evidence regarding the location of the TBZ binding
site of VMAT2 has been obtained from four lines of experimentation:
estimation of the hydrophobicity of the TBZ binding site (33),
photoaffinity labeling (21), site-directed mutagenesis (15), and
analysis of VMAT2/VMAT1 chimeras (16-19). The earliest of these
studies utilized VMAT present in bovine chromaffin granules, and a
series of five transporter substrates and seven TBZ derivatives of
differing physicochemical properties to examine the hydrophobicity of
the TBZ binding site on VMAT2. The apparent partition coefficient between octanol and buffer, which correlated with measurements of
membrane bilayer/water partitioning, was used as a measure of drug
hydrophobicity. Over a range of 5 orders of magnitude, ability to
displace [3H]TBZOH binding by the TBZ derivatives or
transport substrates correlated well with hydrophobicity as estimated
from octanol/water partition coefficients. The authors suggested that
this correlation should be interpreted as a demonstration that bound
TBZ (or derivatives) is in equilibrium with TBZ concentrated in the
lipid membrane, and that TBZ binds in a hydrophobic site, presumably
within the TM segments (33). The photolabeling, mutagenesis, and
chimera experiments utilized cloned VMAT2 and VMAT1 isoforms from rat. Photoaffinity labeling studies from our laboratory (21) using the TBZ
photolabel [125I]azidoiodophenylpropionyl tetrabenazine
([125I]TBZ-AIPP) demonstrated that the majority of
labeling (60%) occurred on a small peptide containing the cytoplasmic
loop between TM10 and TM11, and most of TM11. Significantly, there was
some [125I]TBZ-AIPP photoinsertion (40%) into an
N-terminal peptide that contained lysine 20, which is the predominant
(>90%) site of photoinsertion by the ketanserin photolabel
[125I]7-azido-8-iodoketanserin at the predicted TM 1 membrane/cytoplasm interface. [125I]TBZ-AIPP
photolabeling of two peptides distant in primary sequence suggests that
these portions of the transporter may be juxtaposed in the folded
protein structure. This is also suggested by identification of a
functional ion pair between charged residues in TM 2 and TM 11 (20).
VMAT1 isoforms have a significantly lower affinity for TBZ than VMAT2
isoforms. Mutagenesis studies of aspartate residues in transmembrane
segments of rat VMAT1 identified an aspartate in TM10 (also an
aspartate in VMAT2 isoforms), which, when mutated to glutamate,
resulted in gain of a measure of TBZ binding affinity (15). Analysis of
transporter chimeras between rat VMAT1 and VMAT2 allowed identification
first of domains that contributed to VMAT2 higher affinity TBZ binding
(TM 5-8 and TM 9-12; Ref. 16), and then of individual amino acids
(17, 18) in VMAT2, which, when replaced by the VMAT1 residue, led to an incremental, additive transition from the higher VMAT2 tetrabenazine affinity to the lower VMAT1 affinity. Similarly, analysis of human VMAT1/VMAT2 chimeras identified VMAT1 domains, which, when substituted into a VMAT2 background, led to a large reduction in TBZ affinity, manifest by a large increase in IC50 for transport
inhibition (19).
Cys439 in TM 11, discovered through this work to be the
primary, TBZ-protectable target mediating MTSEA inhibition of human
VMAT2 [3H]TBZOH binding, is positioned centrally with
regard to almost all previously available information on the
tetrabenazine binding site (Fig. 8).
Cys439 is positioned in a hydrophobic environment (as
predicted for the tetrabenazine binding site; Ref. 33), in the center
of four mutations previously found to affect tetrabenazine binding (15, 17, 18), on a peptide that is photolabeled by a tetrabenazine photolabel (21), and on a transporter domain involved in TBZ binding
identified from rat (16) and human (19) VMAT1/VMAT2 chimera studies.
The cysteine derivatization and protection experiments described in
this report support these earlier findings and significantly strengthen
them against argument of the inherent uncertainties in interpreting
site-directed mutagenesis data, or the possibility of photolabel
insertion at a site somewhat distant from pharmacophore binding.
Multiple independent experimental methods all identify the same
transporter domain, converging on a tetrabenazine binding site for
VMAT2, which directly involves TM 10-12, with important contributions
from the N-terminal portion of the molecule (TM 1), and the lumenal
loop between TM 7 and TM 8. Since human VMAT2 with a cysteine 439 to
alanine replacement is both functional and insensitive to MTSEA, this
now opens the possibility of future detailed mapping studies of this
and other VMAT2 ligand binding sites by derivatization and protection
experiments of "engineered" cysteines at different locations
throughout the transporter which may be distant in primary sequence,
but nearby in tertiary structure at the TBZ binding site.
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ABSTRACT
INTRODUCTION
