1-Methylpyridinium-4-(4-phenylmethanethiosulfonate) iodide, MTS-MPP+, a novel scanning cysteine accessibility method (SCAM) reagent for monoamine transporter studies

Article abstract of DOI:10.1016/j.bmc.2006.09.058

A novel substituted cysteine accessibility method (SCAM) reagent was developed for monoamine uptake transporters. The new reagent, MTS-MPP⁺, was a derivative of the neurotoxin and transporter substrate MPP⁺. MTS-MPP⁺ label

Full text of DOI:10.1016/j.bmc.2006.09.058

Bioorganic & Medicinal Chemistry 15 (2007) 305–311
1-Methylpyridinium-4-(4-phenylmethanethiosulfonate) iodide,
MTS-MPP+, a novel scanning cysteine accessibility method
(SCAM) reagent for monoamine transporter studies
Alejandra Gallardo-Godoy, Melissa I. Torres-Altoro, Kellie J. White,
Eric L. Barker and David E. Nichols*
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences,
Purdue University, West Lafayette, IN 47906, USA
Received 24 July 2006; revised 21 September 2006; accepted 26 September 2006
Available online 29 September 2006
Abstract—A novel substituted cysteine accessibility method (SCAM) reagent was developed for monoamine uptake transporters.
The new reagent, MTS-MPP+, was a derivative of the neurotoxin and transporter substrate MPP+. MTS-MPP+ labeled cysteine
residues introduced into the serotonin transporter protein. Although it did not prove to be a substrate, as is MPP+, it appears to
label cysteine residues lining the permeation pore of the transporter more readily than currently available nonspecific SCAM
reagents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
porter (SERT). We hypothesized that a substituent
in that position of MPP+ might similarly provide a
molecule that was a substrate at the SERT. Thus,
we synthesized 4-(1-methylpyridinium)phenylmethan-
ethiosulfonate iodide (MTS-MPP+) 6. This report de-
scribes the synthesis of this new reagent, and a
comparison with a conventional MTS reagent [2-(tri-
methylammonium)ethyl]methanethiosulfonate (MTSET)
that lacks specificity for any features of the
transporter.
Methanethiosulfonate cysteine scanning reagents have
proven very valuable in mapping out protein structure
by thiol-disulfide interchange.1–11 Unfortunately, most
methanethiosulfonate reagents are relatively nonspecif-
ic, and their reactivity depends on solution accessibility
of cysteine residues within the protein. For ongoing
studies of monoamine transporters in our laboratory,
we envisioned that a methanethiosulfonate reagent
derived from a substrate for the transporter might
prove useful. In particular, N-methylphenylpyridinium,
MPP+, is a substrate for all biogenic amine (norepi-
nephrine, dopamine, and serotonin) transporters.12–15
We speculated that if the reagent retained its substrate
characteristics, it might be useful to map the perme-
ation pathway through the transporter using the
substituted cysteine accessibility method.8,10 A good
deal of prior work in our laboratories with phenethyl-
amine type substrates at monoamine transporters had
established that chemical substitution at the 4-position
was particularly well tolerated by the serotonin trans-
2. Chemistry
The synthesis for the MTS-MPP+ reagent is shown in
Scheme 1. The initial biaryl compound was readily pro-
duced by a palladium-mediated Suzuki coupling reac-
tion. Synthesis of the methylmethanesulfonate
thioester then proved to be problematic due to facile
dimerization of the thiol intermediate 3. We found, how-
ever, that dimer (4) could easily be converted into thio-
ester 5.16 Thus, 3 was completely dimerized to 4, which
was then derivatized as the methanesulfonate 5, as
shown in Scheme 1.
Keywords: SCAM reagent; MPP+; Serotonin transporter; Mutagenesis.
*
Corresponding author. Tel.: +1 765 494 1461; fax: +765 494 1414;
e-mail: drdave@pharmacy.purdue.edu
The final step was quaternization of the pyridine nitro-
gen. When carried out in more polar solvents such as
nitromethane, both the pyridine nitrogen and the aryl
Present address: Vertex Pharmaceuticals, Inc., 11010 Torreyana
Road, San Diego, CA 92121, USA.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.058

306
A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 15 (2007) 305–311
SMe
SH
SMe
Br
SMe
Br
i. 73%
ii. 83%
iii. 81%
+
N
.
HCl
B(OH)2
N
N
1
2
3
O
O
H3C
H3C
S
S
S
S
S
O
O
iv. 90%
v. 67%
vi. 61%
N
N
N
I
2
CH3
4
5
6
Scheme 1. Reagents: (i) t-BuLi, (i-PrO)3B; (ii) KOH, (Bu)4NBr, (Ph3P)4Pd, DME, H2O; (iii) t-BuSNa, DMF; (iv) t-BuOOH, V(acac)2; (v) AgNO3,
CH3SO2Na; (vi) CH3I, THF.
sulfur were methylated, leading to decomposition of the
methanesulfonate ester and production of N-methyl-
quaternized 2. In THF, however, the quaternization of
the pyridine nitrogen proceeded more rapidly, affording
the N-methylpyridinium salt, which was insoluble in
THF and precipitated upon formation. This simple
strategy prevented formation of the undesired S-methyl
side product. It might also be noted that this route is
very attractive for the production of a radiolabeled ver-
sion of the reagent when [3H]CH3I is used for this last
quaternization step. Thus, alkylated transporters could
be identified readily simply by quantifying tritium irre-
versibly bound to the membrane fraction of cells
expressing transporter, potentially avoiding the addi-
tional effort required for functional characterization.
wild-type hSERT.17,18 MTS-MPP+ was significantly less
potent at inhibiting [3H]5-HT uptake than MPP+
(Fig. 1, Ki = 48 26 lM vs. 1300 100 lM, n = 2).
3.2. MTS-MPP+ is not a substrate at hSERT
MTS-MPP+ does inhibit [3H]5-HT uptake at C109A/
hSERT, albeit with a higher Ki value than MPP+. With-
out a radiolabel, however, the ability of MTS-MPP+ to
be transported by SERT cannot be directly assessed in
125
MPP+
MTS-MPP+
100
3. Results and discussion
75
50
25
0
3.1. MTS-MPP+ inhibition of [3H]5-HT uptake at human
serotonin transporter (hSERT) is less potent than MPP+
We anticipated that MTS-MPP+ 6, an MPP+ analog
with a methanethiosulfonate group attached to the
phenyl ring, might serve as a selective substrate for
SCAM studies. Because we wished to take advantage
of the methanethiosulfonate moiety on MTS-MPP+,
we first assessed whether MTS-MPP+ would interact
with hSERT. The ability of MTS-MPP+ to inhibit
[3H]5-HT uptake was compared to MPP+ at the
C109A/hSERT mutant. The C109A mutant was used
in all studies because C109 is the only endogenous cys-
teine in SERT sensitive to modification by methanethio-
sulfonates, and the 5-HT uptake activity and
pharmacology of C109A/hSERT is similar to that of
-6
-5
-4
-3
-2
Log [MTS reagent], M
Figure 1. MTS-MPP+ inhibition of [3H]5-HT uptake at SERT is less
potent than MPP+. Dose–response of MTS-MPP+ vs. MPP+ for [3H]5-
HT uptake inhibition at C109A/hSERT. C109A/hSERT cDNA was
transiently transfected into HEK cells and uptake inhibition performed
as described in Section 5. Ki values (means SD) were MPP+
48 26 lM vs. MTS-MPP+ 1300 100 lM. Data represent two
experiments performed in triplicate. R2 values for each curve fit were
MPP+ 0.916 and MTS-MPP+ 0.877.

A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 15 (2007) 305–311
307
standard uptake assays. Thus, the ability of MTS-MPP+
to function as a substrate was determined by evaluating
the MTS-MPP+-induced current in hSERT-expressing
oocytes. Surprisingly, unlike MPP+ (Fig. 2A), MTS-
MPP+ was incapable of inducing inward current in
C109A/hSERT, but instead, caused an outward current
consistent with blockade of the transporter-associated
‘leak’ current that is indicative of ligand binding without
translocation (Fig. 2B). Transporter-associated currents
for hSERT have been well characterized such that sub-
strates are known to induce inward currents.19 In addi-
tion to substrate-activated currents, hSERT also
exhibits a constitutive ‘leak’ current that can be blocked
by transporter antagonists. This block of the ‘leak’ cur-
rent is revealed as an apparent outward current upon
application of a transporter blocker.19,20 Although
MTS-MPP+ was not a potent blocker of 5-HT transport
(Fig. 1), the ability of MTS-MPP+ to block hSERT-as-
sociated ‘leak’ currents at lower micromolar concentra-
tions (i.e., 30 lM) suggests specific interactions with the
transporter are occurring at concentrations below the Ki
value for 5-HT uptake inhibition.
the G498C mutation with a potency comparable to that
of the nonselective reagent MTSET.
Based on our homology model of hSERT, W103 in
transmembrane helix I is predicted to be in the region
lining the permeation pathway. The W103C mutant also
has been shown to be sensitive to inactivation by
MTSET.17,22 Because W103 resides deeper in the protein
region surrounding the pore region than G498, however,
we predicted that MTS-MPP+, which presumably binds
to SERT at sites within the substrate permeation path-
way, would inactivate the W103C mutant with greater
potency than the nonselective reagent MTSET. Indeed,
MTS-MPP+ inactivated the W103C mutant with a 10-
fold greater potency than MTSET (Fig. 4B). These data
are consistent with the hypothesis that MTS-MPP+ re-
acts more rapidly with residues in or near the substrate
permeation pathway, in contrast to MTSET, which sim-
ply interacts with any freely accessible cysteine residue.
We speculated that both MTSET and MTS-MPP+
would be capable of reacting with residues such as
G498C and W103C that are located in the aqueous re-
gion of the permeation pathway. Even though MTS-
MPP+ proved not to be a substrate, we envisioned that
its structural similarity to MPP+ would allow it to be
more concentrated in the permeation pathway, and
hence would produce more rapid covalent labeling of
those residues deeper in the pore.
3.3. MTS-MPP+ significantly inhibits G498C and
W103C mutants compared to C109A/hSERT
A SERT homology model based on the crystal structure
of the Aquifex aeolicus leucine transporter21 allowed us
to identify locations for several residues that appear to
reside at the entrance to the substrate permeation path-
way (Fig. 3). Our laboratory has observed that the
hSERT G498C mutation was sensitive to inactivation
by MTSET (M. Torres-Altoro and E. Barker, unpub-
lished results). To explore whether MTS-MPP+ could
interact with residues at the entrance to the permeation
pore, G498C/hSERT was treated with increasing con-
centrations of either MTSET or MTS-MPP+
(Fig. 4A). MTS-MPP+ inactivated [3H]5-HT uptake at
By contrast, with its nonspecific interactions and rela-
tively small size, MTSET would not be concentrated
in the permeation pathway and would have free access
to cysteine residues in any region that was exposed to
the aqueous environment. These interactions of MTSET
could alter transporter function by disrupting conforma-
tions required for substrate translocation. We believe
the experiments with the G498C and W103C mutants
MPP+ (5 μM)
A
5-HT (10μM) MPP+ (3 μM)
MPP+ (5 μM)
fluoxetine (20 μM)
25 nA
20 s
30 μM MTS-MPP+
50 μM MTS-MPP+
B
10 μM 5-HT
25 nA
10 s
Figure 2. Unlike MPP+, MTS-MPP+ does not activate inward current at hSERT. MPP+ (A) or MTS-MPP+ (B) were applied to C109A/hSERT-
expressing oocytes as described in Section 5. Oocytes were clamped at ꢀ120 mV and perfused with drug solutions as indicated. MTS-MPP+ did not
induce SERT-associated inward currents but blocked C109A/hSERT leak current. Data are representative of six oocytes from two separate batches.

308
A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 15 (2007) 305–311
Figure 3. Approximate location of residues G498 and W103 in a homology model of the SERT. The view is looking down into the proposed entrance
to the substrate permeation pathway, approximately in the center of the protein, just to the left of G498, which is evident on the exposed surface of
the protein. W103, by contrast, is buried slightly within the protein and we hypothesize only becomes accessible when a ligand binds within the
permeation pathway.
give results consistent with this reasoning. G498C is pre-
dicted to reside at the surface of the pore and is thus
freely accessible to both MTSET and MTS-MPP+.
W103C is predicted to lie deeper within the permeation
pathway, and reacts more potently with MTS-MPP+
than MTSET, possibly even becoming more accessible
as a result of the functional processes involved in sub-
strate binding.
MTSET might allow more ready access to the W103C
mutant. Additional studies of the MTS-MPP+ reagent
at other biogenic amine transporters as well as in com-
parisons to other nonspecific MTS reagents are warrant-
ed. Regardless, it therefore seems possible that the
differential reactivity to MTS-MPP+ vs. a nonspecific
SCAM reagent such as MTSET may be useful in identi-
fying residues that line the extracellular face of the sub-
strate permeation pathway.
4. Conclusions
5. Experimental
5.1. General
We have synthesized a novel analog of MPP+ that con-
tains a reactive methanethiosulfonate group similar to
commercially available methanethiosulfonate com-
pounds. Synthesis of an MTS-containing MPP+ mole-
cule should allow for better understanding of specific
residues that interact with MPP+. The ability of this
compound, MTS-MPP+, to inhibit [3H]5-HT uptake at
C109A/hSERT vs. MPP+ was determined to character-
ize the interaction of this molecule with SERT. Unfortu-
nately, MTS-MPP+ was not as potent as MPP+ at
inhibiting [3H]5-HT uptake. Furthermore, an inability
to induce current at C109A/hSERT suggests that, unlike
MPP+, MTS-MPP+ does not function as a substrate. In
fact, MTS-MPP+ appeared to act as an antagonist, as
outward movement of current was evident in the pres-
ence of MTS-MPP+, suggesting the blockade of the
SERT ‘leak’ current.19
All reagents were commercially available and were used
without further purification unless otherwise indicated.
Anhydrous THF was obtained by distillation from ben-
zophenone-sodium under nitrogen immediately before
use. Melting points were determined using a Thomas–
1
Hoover apparatus and are uncorrected. H NMR spec-
tra were obtained with a Bruker AXR300 (300 MHz)
instrument. Chemical shifts are reported in d values
(ppm) relative to an internal reference of TMS. Chemi-
cal ionization mass spectra (CI-MS), using isobutene as
the carrier gas, were obtained with a Finnigan 4000
spectrometer. Electrospray ionization analyses were car-
ried out on a Finnigan MAT LCQ Classic (ThermoElec-
tron Corp, San Jose, CA) mass spectrometer. Elemental
analyses were performed by the Purdue University
Microanalysis Laboratory and are within 0.4% of the
calculated values. Thin-layer chromatography was per-
formed using J. T. Baker flex silica gel IB2-F, plastic-
backed sheets with fluorescent indicator, visualizing
with UV light at 254 nm and plates developed with ethyl
acetate, unless otherwise noted. When indicated, com-
A more potent interaction of MTS-MPP+ with a residue
that lines the actual permeation pathway was observed
when compared to the nonselective reagent MTSET.
Alternative explanations for our results could include
that the more lipophilic nature of MTS-MPP+ or its
more linear conformation compared to the spherical

A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 15 (2007) 305–311
309
(2.3 g, 11.9 mmol) were dissolved in 50 mL DME. To
this stirring mixture were added KOH (3.4 g, 60 mmol)
and (Bu)4NBr (0.5 g, 1.5 mmol) in 30 mL of water.
The mixture was stirred and degassed by sparging with
Ar for 20 min at rt. Tetrakis(triphenylphosphine)palla-
dium catalyst (0.5 g, 0.43 mmol) was added and the mix-
ture was heated at 80 ꢁC for 48 h. The reaction mixture
was then cooled to rt, the solvents were removed under
vacuo, the residue was taken up into CH2Cl2, washed
with water and brine, dried (anhydrous Na2SO4), fil-
tered, and the solvent evaporated to yield the product.
The crude base was then purified by column chromatog-
raphy over silica, using ethyl acetate/hexane (1:9) to
yield the product as white crystals 1.50 g (63%). 1H
NMR (300 MHz, CDCl3): d 8.63 (d, 2H, J = 6.07 Hz),
7.55 (d, 2H, J = 8.36 Hz), 7.47 (d, 2H, J = 6.09 Hz),
7.33 (d, 2H, J = 8.37 Hz), 2.51 (s, 3H, SCH3). Mp
112–113 ꢁC; CI-MS (m/z) 202 (M+ H). Anal.
(C12H11NS) C, H, N, S.
A
hSERT G498C
MTSET
MTS-MPP+
100
50
0
-6
-5
-4
-3
log [MTS reagent], M
B
hSERT W103C
100
50
0
5.1.3. 4-(4-Thiophenyl)pyridine (3). To an oven-dried
250 mL flask, connected to a condenser and flushed with
Ar, was added 1.0 g (5.0 mmol) of 2, followed by 1.12 g
(10.0 mmol) of commercial t-BuSNa powder. Dry DMF
(50 mL) was injected via syringe while stirring vigorous-
ly. The mixture was placed in an oil bath preheated to
160 ꢁC and stirred for 5 h. The reaction mixture was al-
lowed to cool to rt, maintaining the Ar atmosphere. The
reaction mixture was quickly poured over 100 g of
crushed ice and stirred until the ice had melted. The
pH was adjusted to 7 and the mixture was extracted with
3 · 70 mL CH2Cl2. This organic solution was washed
repeatedly with water to remove DMF, then dried, fil-
tered, and the solvent evaporated to afford the product
as dark white crystals 0.75 g (81.1%). 1H NMR
(300 MHz, CDCl3): d 8.65 (d, 2H, J = 6.18 Hz), 7.55–
7.30 (m, 6H), 3.55 (s, 1H, SH). Mp 171–172ꢁC, lit24
mp 171 ꢁC; CI-MS (m/z) 188 (M+ H).
MTSET
MTS-MPP+
-6
-5
-4
-3
log [MTS reagent], M
Figure 4. MTS-MPP+ inactivates [3H]5-HT uptake at the hSERT
G498C and W103C mutants. G498C/hSERT, or W103C/hSERT
transiently expressed in HEK-293 cells were treated with increasing
concentrations of either MTSET or MTS-MPP+ as described in
Section 5. (A) G498C/hSERT showed a decrease in [3H]5-HT uptake
following treatment with MTS-MPP+ and MTSET. The potencies of
MTS-MPP+ and MTSET were similar (log EC50 values: MTS-MPP+
ꢀ5.4 0.2 vs. MTSET ꢀ5.5 0.1). (B) W103C/hSERT was inactivat-
ed by MTS-MPP+ at lower concentrations than MTSET (log EC50
values: MTS-MPP+ ꢀ4.5 0.1 vs. MTSET ꢀ3.3 0.2). Data repre-
sent means SEM from three separate experiments performed in
duplicate. R2 values for each curve fit were: G498C, MTSET 0.999,
MTS-MPP+ 0.995; W103C, MTSET 0.996, MTS-MPP+ 0.999.
pounds were purified by column chromatography over
using silica gel 60, 230–400 mesh (J. T. Baker). All reac-
tions were carried out under an inert atmosphere of
argon unless otherwise indicated.
5.1.4. 4-(4-Pyridyl)phenyldisulfide (4). Following the
method of Raghavan et al.25 a solution of 3 (1.0 g,
5.3 mmol) in dry CH2Cl2 (10 mL) was cooled to
ꢀ15 ꢁC. Vanadyl acetylacetonate (0.5 mol%) and tert-
butylhydroperoxide (1.0 mL of ca. 5.5 M in decane;
5.30 mmol) were then added. The reaction mixture was
stirred overnight, with gradual warming to rt. The mix-
ture was then diluted with CH2Cl2 and washed with
water, 0.2 N NaOH, water, and brine. The organic layer
was dried, filtered, and the solvent evaporated to pro-
vide the product as yellow crystals; 1.0 g (50%). 1H
NMR (300 MHz, CDCl3): d 8.66 (dd, 4H, J = 1.64 Hz,
J0 = 6.18 Hz), 7.62 (m, 8H), 7.46 (dd, 4H, J = 1.64 Hz,
J0 = 6.19 Hz). CI-MS (m/z) 373 (M+ H). Mp 171–
172 ꢁC. Anal. (C22H16N2S2) C, H, N, S.
5.1.1. 4-Methylthiophenylboronic acid (1). A solution of
4-bromothioanisole (5.0 g, 24.6 mmol) in 100 mL of
dry THF was cooled to ꢀ78 ꢁC. A solution of 2.5 M
n-BuLi in hexane (34.0 mL, 85 mmol) was slowly added.
After 15 min stirring, triisopropoxy borate (5.64 g,
6.90 mL, 30 mmol) was added in one portion. The mix-
ture was stirred at ꢀ78 ꢁC for 4 h, then warmed to rt and
stirred overnight. The reaction was quenched by addi-
tion of 50 mL of water, the THF was removed by rotary
evaporation, and the product was extracted into Et2O.
The organic layer was washed with brine, dried, and
1
evaporated to yield 2.56 g of crude product (72%). H
NMR (300 MHz, CD3COCD3):
5.1.5. 4-Pyridyl-(4-phenylmethanethiosulfonate) (5). This
step was based on the procedure of Bentley et al.16 To
a solution of 4 (0.2 g, 0.53 mmol) in 50% acetone–H2O
(5 mL) was added a solution of silver nitrate (100 mg,
0.60 mmol) and sodium methanesulfinate (60 mg,
0.60 mmol) in 50% acetone–H2O (5 mL). The mixture
was heated at reflux for 1 h, during which the mixture
became bright yellow. The product mixture was filtered
d 7.85 (d, 2H,
J = 8.3 Hz), 7.25 (d, 2H, J = 7.50 Hz), 7.00 (s, 2H,
OH), 2.60 (s, 3H, SCH3). Mp 211–212 ꢁC; lit23 mp
206–207 ꢁC; CI-MS (m/z) 169 (M+ H).
5.1.2. 4-(4-Methylthiophenyl)pyridine (2). Boronic acid 1
(2.0 g, 11.9 mmol) and 4-bromopyridine hydrochloride

310
A. Gallardo-Godoy et al. / Bioorg. Med. Chem. 15 (2007) 305–311
and the filtrate was extracted with 3 · 50 mL of CH2Cl2.
The combined organic extracts were dried, filtered, and
KRH/glucose (total) and incubated for 10 min at
37 ꢁC. Next, 25 lL of [3H]5-HT (Amersham, Piscata-
way, NJ, ꢁ125 Ci/mmol; final assay concentration
20 nM) diluted in KRH/glucose buffer supplemented
with 100 lM pargyline and 100 lM ascorbic acid was
added to each well, followed by incubation for an addi-
tional 10 min at 37 ꢁC. Cells were then washed three
times with cold KRH/glucose buffer, solubilized with
scintillation cocktail (Microscint-20, Packard Instru-
ments), and shaken overnight before determining accu-
mulated tritium using a Packard TopCount NXT.
Data were analyzed using the nonlinear regression curve
fit for a sigmoidal dose–response curve (four parameter
logistic equation) from GraphPad Prism v3.0 (Graph-
Pad Software, San Diego, CA). Total specific accumula-
tion of tritium equaled ꢁ25,000 cpm which is <10% of
the total available tritium in each assay
(>400,000 cpm), thus, validating the use of this equa-
tion. The concentration of substrate (20 nM) used in
our uptake inhibition assays is far below the Km value
for 5-HT uptake (500–700 nM) making the conversion
from IC50 to Ki values negligible (<4%).
1
evaporated to yield yellow crystals; 100 mg (71.4%). H
NMR (300 MHz, CDCl3): d 8.72 (dd, 2H, J = 1.52 Hz,
J0 = 6.10 Hz), 8.84 (m, 2H), 7.74 (m, 2H), 7.51 (dd,
2H, J = 1.67 Hz, J0 = 6.15 Hz), 3.43 (s, 3H, SO2Me).
Mp 133–135 ꢁC; CI-MS (m/z) 373 (M+ H).
5.1.6. 1-Methylpyridinium-4-(4-phenylmethanethiosulfo-
nate) iodide (6). To a solution of 5 (50 mg, 0.19 mmol)
in dry THF (0.5 mL) was added dropwise a solution
of methyl iodide (26.7 mg, 0.011 mL, 0.19 mmol) in
0.5 mL of dry THF. The mixture was stirred for 24 h
at rt as the pyridinium salt precipitated. The product
was collected by filtration, washed with THF (5 mL),
1
and air-dried. IR 1315, 1132 cmꢀ1 (S–SO2). H NMR
(300 MHz, D2O): d 8.75 (d, 2 H, J = 5.69 Hz), 8.25 (d,
2H, J = 5.51 Hz), 7.97 (m, 4H), 4.32 (s, 3H, NMe),
3.37 (s, 3H, SO2Me). Mp 164–167 ꢁC; ESI-MS (m/z)
280 (M+). Anal. (C13H14INO2S2) C, H, N, I.
5.2. SERT expression and electrophysiology in oocytes
hSERT contains one native cysteine, C109, that is reac-
tive with MTS reagents, thus all studies were performed
with the hSERT C109A mutant.18 hSERT C109A/
pcDNA3.1+ was linearized and transcribed in vitro
using the AmpliCapTM T7 High Yield Message Maker
Kit (Eppicentre, Madison, WI). Following injection of
SERT cRNA, Xenopus laevis oocytes were maintained
at 18 ꢁC in Ca2+-Ringer’s solution. Three or four days
post-injection oocytes were subjected to two-electrode
voltage-clamp. Recording solutions consisted of rt
Ca2+-Ringer’s solution with concentrations of 5-HT, flu-
oxetine, MTS-MPP+ or MPP+ (Sigma), as indicated.
Transporter-associated currents were recorded by
clamping the oocyte membrane potential at ꢀ120 mV,
perfusing the oocytes with 5-HT or drug for 15–30 s,
and then washing with Ca2+-Ringer’s solution for at
least 40 s. Perfusion was controlled by gravity. For
MTS-MPP+ experiments, MTS-MPP+ was made fresh
in deionized water and then diluted in Ca2+-Ringer’s
for perfusion. The oocyte was clamped at ꢀ120 mV
for the duration of the experiment. Data were acquired
digitally using Clampex 8.1 (Axon Instruments) and
analyzed using Clampfit 8.1 (Axon Instruments) and
SigmaPlot 5.0 (SPSS Science, Chicago, IL). Current
averages and statistics were performed using Prism 3.0
(GraphPad Software, San Diego, CA). Water-injected
oocytes were assayed in parallel with SERT-injected oo-
cytes to determine nonspecific effects on currents.
For the MTS-MPP+ inactivation assay, C109A/
hSERT, W103C/hSERT, or G498C/hSERT cDNAs
were transfected into HEK cells as described above.
Forty-eight hours post-transfection, the cells were
washed once with PBS/CM buffer (137 mM NaCl,
2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4,
0.1 mM CaCl2, and 1.0 mM MgCl2). For the assay,
225 lL PBS/CM was added to each well plus 25 lL
of increasing concentrations of MTS reagent or
KRH/glucose (total and nonspecific wells) followed
by incubation at rt for 10 min. Following incubation
with MTS-MPP+, cells were washed twice with
PBS/CM. Next, 225 lL of 37 ꢁC KRH/glucose
(200 lL KRH/glucose + 25 lL 10 lM fluoxetine for
nonspecific wells) were added to each well, as well as
25 lL of [3H]5-HT (20 nM final). The cells were incu-
bated for 10 min at 37 ꢁC. Transport was terminated
by washing twice with cold KRH/glucose. Cells were
solubilized and data analyzed as described above.
Acknowledgments
This work was supported by NIDA Grant DA02189
(D.E.N.) and NIMH Grant MH60221 (E.L.B.).
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