SUPPLEMENTARY METHODS
Generation of mice humanized for hSERT
A 5.0-kb ‘‘long arm’’ and a 1.6-kb ‘‘short arm’’ were cloned by PCR with EXL
DNA polymerase (Stratagene, La Jolla, CA) using genomic DNA obtained from
129S6/SvEv mice as template. The long arm corresponded to sequences from
intron 1 and exon 2 of mouse SERT genomic DNA, whereas the short arm
comprised sequences from intron 2 of SERT genomic DNA. The cDNA of WT
and
mutant
SERT
(A505V/L506F/I507L/S574T/I575T),
including
the
polyadenylation sequences in a mammalian expression plasmid (pcDNA3) were
amplified. A unique restriction enzyme site (EcoR I) downstream of the initiation
start codon in exon 2 was used to insert the cDNA in order to express human
versions of WT and mutant SERT.
The long and short arms were then
subcloned into a targeting vector resulting in the insertion of a floxed herpes virus
thymidine kinase/neomicin (TK/NEO) selection cassette (Figure S1). The
targeting construct was transfected into 129S6/SvEv mouse ES cells. Clones
carrying humanized SERT alleles were selected using a standard diphtheria toxin
(DT)/G418 double selection protocol. Positive ES cell clones were subsequently
karyotyped, and the occurrence of homologous recombination was confirmed by
PCR.
Recombinant ES cell lines were transiently transfected with a CRE
recombinase expression construct to remove the TK/NEO selection cassette.
Chimeras generated from selected ES cells were intercrossed with C57BL6/J
mice; offspring (F1) that inherited the humanized SERT allele were identified by
PCR analysis.
SERT mRNA Analysis
The presence or absence of hSERT and mSERT mRNA transcripts were
determined using cDNA restriction enzyme analysis. RNA was extracted with
Trizol and RNeasy minikits according to the manufacturer’s protocol (Qiagen,
Valencia, CA). RNA was reverse transcribed into cDNA using the iScript cDNA
synthesis kit according to the manufacturer’s protocol (Bio-Rad, Hercules, CA).
Two sets of SERT-specific primers were used to amplify SERT in all genotypes
(Primer
set
1:
Forward:
TCCATTCCACGTCMCCTGCTG,
AAGCTCGTCATGCAGTTCACCAC,
TGGAGTATCATCTTGGGYTACTGC,
Primer
set
2:
Reverse:
Forward:
Reverse:
ACACAGCATTCAWGCGGATGTC). Primer set 1 results in a 454-bp amplicon,
and Primer set 2 generates a 175-bp amplicon. The PCR protocol consisted of
an initialization step at 95 °C for 5 minutes, followed by 40 cycles of 95 °C for 30
seconds, 55 °C for 45 seconds and 72 °C for 1 minute. A final elongation step at
72 °C for 5 minutes was also performed. The PCR products from each primer set
were divided into two aliquots, the first of which was examined using agarose gel
electrophoresis to confirm SERT expression. The second aliquot of PCR
products from Primer sets 1 and 2 were then digested with MfeI and AflII,
respectively. MfeI cleaves hSERT cDNA (but not mSERT cDNA, which has a
single nucleotide change in the MfeI consensus sequence) to form 281-bp and
173-bp fragments. In contrast, AflII cleaves mSERT, but not hSERT, cDNA due
to a single nucleotide change in the AflII consensus sequence in the latter. AflII
cleavage generates a 94-bp and an 81-bp fragment. SERT cDNA cleavage by
MfeI or AflII was analyzed by agarose gel electrophoresis using a 2% agarose
gel. Genotype of hSERT mice was determined using a standard RT-PCR
protocol.
SERT Autoradiography
Cryosections were collected on slides at the level of the dorsal hippocampus
(Allen Brain Atlas, Coronal Plate 72) and stored at 80 ˚C. Upon experiment, the
slides were defrosted in a desiccator. The sections were incubated at RT for 60
min with assay buffer (50 mM Tris, 150 mM NaCl, 5 mM KCl, pH=7.4) containing
50 pM [125I]-RTI-55 (Perkin Elmer, Waltham, MA), 50 nM GBR12935 (Tocris,
Brsitol, UK), and 1µM maprotiline (Tocris). GBR12935 was included to minimize
binding to the dopamine transporter. Maprotiline was included to block binding to
the noradrenaline transporter. Non-specific binding was defined by including 1
µM escitalopram. The sections were washed twice for 5 min in ice-cold assay
buffer and dipped in ice-cold ddH2O. Sections were exposed to Kodak Biomax
MR films (Sigma, St. Louis, MO) for 48h.
Brain Membrane Preparation
Whole mouse brains (excluding the cerebellum), or dissected frontal cortices and
hippocampi, were homogenized with an Ultra-turrax homogenizer for 10 sec at ¾
maximal speed in binding buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2
mM CaCl2, 1.2 mM MgCl2, pH = 7.4) at 0 ˚C. The homogenate was centrifuged
for 15 min at 30,000g and the membranes washed with 8 ml of the same buffer.
Finally, the membranes were suspended in binding buffer containing 10%
sucrose at a protein concentration of about 5 mg/ml and kept at -80 ˚C until use.
[3H]-Escitalopram Saturation Binding
BMax and Kd values for [3H]-escitalopram binding were determined as previously
described (Plenge et al. 1990) In brief, [3H]-escitalopram binding was carried out
in 24-well plates at room temperature in a total volume of 400 µL containing 50
µL brain membrane suspension, 300 µL binding buffer (25 mM HEPES, 120 mM
NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, pH 7.4)) and 50 µL [3H]escitalopram. The [3H]-escitalopram was added in 6 concentrations ranging from
0.2 nM to 12 nM final concentration. All determinations were performed in
triplicate. Non-specific binding was determined by adding 1 µM paroxetine. The
samples were incubated for 2 hours at room temperature to obtain binding
equilibrium and stopped by rapid application of the membranes on GF/B filters
using
a
Connectorate
Cell
Harvester
(Connectorate
AG,
Reppischhof,
Switzerland) mounted with a head for 24 well plates, followed by immediate
washing with 0,5 L ice-cold 0,2 M NaCl, and dried on a hot plate. To determine
the amount of bound [3H]-escitalopram, a MeltiLexTM scintillation sheet is melted
into the filter and counted on a Micro Beta2 counter (Perkin Elmer). BMax and KD
values were calculated from the saturation binding curves by non-linear
regression using GraphPad Prism vs. 5.0.
[3H]-Escitalopram Dissociation Binding
Dissociation rates were determined as previously described (Plenge et al. 2007).
In brief, [3H]-escitalopram (15-20 nM, 70 Ci/mmol) was added to the brain
membranes (see above) and incubated for 30 minutes at 0 ˚C in binding buffer.
Dissociation was initiated by diluting samples 20x in binding buffer containing 0.1
µM paroxetine and escitalopram or R-citalopram in the indicated concentrations.
(Paroxetine was added to avoid re-association of dissociated [3H]-escitalopram
and at this concentration has no allosteric effect on [3H]-escitalopram
dissociation). The dissociation was assessed between 5 and 70 minutes and
stopped by rapid filtration through GF/B filters using a Connectorate Cell
Harvester mounted with a head for 24 well plates. Non-specific binding was
determined with 1 µM paroxetine at 37 oC for 70 minutes. The temperature for the
dissociation rate was set at 18 ˚C. Dissociation rates were determined in triplicate
in at least three independent experiments.
Escitalopram Pharmacokinetics
Escitalopram 0.25, 0.5,1 and 2 mg/kg were administered (I.P.) to mSERTWT mice
and brains and plasma collected at 30, 60 and 90 min at stored at –80 ˚C.
Escitalopram levels in plasma and brain samples were determined using highperformance liquid chromatography (HPLC, Sparks Holland, Netherlands)
coupled with mass spectrometry (ThermoFinnigan TSQ Quantum Ultra, San
Jose, CA). Plasma samples were thawed at room temperature. An aliquot of 50
µL plasma was mixed with 150 µl of a dimethyl sulfoxide (DMSO)/acetonitrile
(ACN) (20:80) solution that contained a proprietary internal standard (50 ng/ml)
and centrifuged (2700 RPM; 15min at 10 °C). Frozen brains were weighed and
homogenized in in 4x (w/v) of buffer consisting of 50 % water, 30 % 2-propanol
and 20 % DMSO and centrifuged (2700 RPM; 15min at 10 °C). Ten µl of the
supernatant was injected unto the HPLC-MS (see below).
HPLC-MS:
was used for analytical separation. A 5 minute gradient with the following mobile
phases was used: 0.1 % formic acid in water (solvent A) and 0.01% formic acid
in ACN (solvent B). The gradient was kept at 95/5 (solvent A/solvent B) for 0-1.67
min, then held for 2.16 min at 5/95 and then returned to initial conditions of 95/5
for 1.16 min. The MS was equipped with a HESI probe and the source conditions
were as follows: Vaporizer temp = 400 °C; spray voltage 3000 V; sheath and aux
gas = 40psi; ion sweep gas = 20 psi; capillary temperature = 275 ˚C. Spectra
were acquired in positive SRM mode with the parent mass of 260540 being
325.17 (m/z) and 3 daughters (1) 109 (m/z) at 30 CE (Collision Energy), (2) 166
at 25 CE and (3) 262 at 20 CE with a tube lens of 125. Spectra were analyzed
using Xcalibur software (Thermo Scientific, Wyman, MA).
Table S1
Effect of unlabeled escitalopram on [3H]-escitalopram dissociation from hSERTWT and hSERTALI/VFL+SI/TT
Escitalopram (µM)
0
1.23
3.7
11.1
33.3
100
300
hSERTWT
Dissociation T1/2 (min)
12.7
16.0
21.9
40.6
89.4
179.5
ND
SEM
0.76
0.87
0.74
1.55
6.72
14.31
ND
hSERTALI/VFL+SI/TT
Dissociation T1/2 (min)
ND
23.7
26.0
25.1
26.6
23.7
22.2
Effect of unlabeled es- and R-citalopram on [3H]-escitalopram dissociation from hSERTWT
Escitalopram (µM)
0
3
3
3
3
3
3
3
R-citalopram (µM)
0
0
1.23
3.7
11.1
33.3
100
300
hSERTWT
Dissociation T1/2 (min)
14.9
26.1
27.3
32.3
35.6
44.3
63.8
90.7
SEM
0.19
0.35
3.32
3.26
2.67
1.72
4.91
2.34
SEM
ND
1.25
1.54
1.59
1.63
2.25
1.13
SUPPLEMENTARY REFERENCES
Plenge P, Gether U, Rasmussen SG (2007) Allosteric effects of R- and S-citalopram on
the human 5-HT transporter: evidence for distinct high- and low-affinity
binding sites. European journal of pharmacology 567: 1-9.
Plenge P, Mellerup ET, Laursen H (1990) Regional distribution of the serotonin
transport complex in human brain, identified with 3H-paroxetine, 3Hcitalopram and 3H-imipramine. Progress in neuro-psychopharmacology &
biological psychiatry 14: 61-72.