Do the binding sites of substrates and tricyclic antidepressants overlap on the
human serotonin transporter?
Doctoral thesis at the Medical University of Vienna in partial fulfillment for the degree of
Doctor of Philosophy (PhD)
Submitted by
Subhodeep Sarker
Supervisor:
Univ. Prof. Dr. Michael Freissmuth, MD Professor and Chairman Institute of Pharmacology
Center for Physiology and Pharmacology Medical University of Vienna
Waehringer Strasse 13a A-1090 Vienna, Austria
Vienna, August 2010
‘With utmost reverence and humility within, I dedicate this sojourn to my Guru, Misha and dearest friend Sonja, who symbolize to me the quintessential epitome of human
values’
1
ACKNOWLEDGEMENTS
Albeit having inherited the oldest living tradition on Earth, with roots reaching
back into prehistory, one of its very few customs I follow blindfolded, without ever
questioning its very rational basis, is that of the ancient Gurukul, wherein one’s teacher is
held Supreme in all conceivable realms of conscious existence.
Without the unstinted faith of my Guru, Prof. Michael Freissmuth, I may never
have walked the conglomerate steps I did during the course of this thesis. He inspired me
every moment I breathed during this sojourn. I shall forever be grateful to him for
providing me with the necessary leverage I needed to come out of my shy, inhibited and
reclusive nature and for setting to me a benchmark for self-accomplishment. The doors to
his mind and magnanimous heart were perennially left open to me and he was never
imposing – a lesson, to which I will fall back upon for inspiration to leave with posterity
to ponder upon. Perhaps his greatest legacy that I will carry within is the knack of asking
questions - questions perennially reframed to testable hypotheses and amenably
addressed through scientific rigor. He leaves the indelible impression of a great man
within.
With equal sentiment, I am inexplicably indebted for life, to Dr. Sonja Sucic. Her
inimitable selflessness, contagious enthusiasm and honest passion for her life and work
inspired me to emulate her ethic and incorporate in my own. Her steadfast friendship and
contribution to every conceivable aspect of my life crystallized to be the definitive
inspiration towards fruitful execution of my work and in completion of this thesis. The
joy of discovery and experimentation was demonstrated to me by my parents and Guru
but it is what I cherished exploring most, with Dr. Sucic. I, especially am grateful to her
for having refurbished, with a pinch of satire, my very perception towards the ironical
vagaries of life. She commands the most profound influence on my philosophical
underpinnings in the way I approach life today.
2
Besides, I am grateful to Prof. Harald H. Sitte and his group who helped expand
my work to include a more holistic understanding of monoamine transporter inhibition. I
also express my gratitude to Prof. Gerhard F. Ecker and his graduate student Mag. René
Weissensteiner, for their insights from computational biology enabled me to generate
hypotheses towards designing experiments in an iterative approach. I wish to congratulate
all my colleagues in the laboratory for providing me with a productive atmosphere
necessary to work in an efficient manner. Especially, Mr. Ali El-Kasaby with whom I
enjoyed my humble origins in the laboratory and developed a formidable friendship to
last for a lifetime. I am thankful to Mrs. Ludmilla Hertting and late Mrs. Alice Zafaurek
for their kind support with administrative affairs and extend my gratitudes to Prof.
Werner Sieghart (Medical University of Vienna, Austria), Prof. Stefan Böhm (Medical
University of Vienna, Austria), Prof. Nicholas Singewald (Medical University of
Innsbruck, Austria), Prof. Cord-Michael Becker (University of Erlangen-Nürnberg,
Germany) and Prof. Menahem Segal (The Weizmann Institute of Science, Rehovot,
Israel), for their critique and encouragement served to maintain direction during my
research.
In addition, I humbly acknowledge the financial support received for my research
from the Austrian Science Fund (FWF) and the Medical University of Vienna in the Cell
Communication in Health and Disease (CCHD) Doctoral Program.
Finally, my endearing set of parents, is the very reason I ever had the opportunity
to walk in pursuit of my childhood fascination to discover the world I inherited in a
dedicated endeavor. Their steadfast friendship, love, affection, belief and towering
inspiration helped me to withstand rigors in between. I am grateful to life for letting me
know my parents closely!
Shubho
August 6, 2010
Vienna, Austria
3
ORIGINAL PUBLICATIONS
Sarker, S., Weissensteiner, R., Steiner, I., Sitte, HH., Ecker, GF., Freissmuth, M., Sucic,
S. (2010) The high-affinity binding site for tricyclic antidepressants resides in the outer
vestibule of the serotonin transporter (in revision).
MEETING ABSTRACTS
Sarker, S., Steiner, I. (2008) Do the binding sites of substrates and tricyclic
antidepressants overlap on the human serotonin transporter? BMC Pharmacol.8
(Suppl.):A9. 14th Scientific Symposium of the Austrian Pharmacological Society
(APHAR), Innsbruck, Austria, 21-22 November, 2008.
4
To: Michael Freissmuth <[email protected]>
From: [email protected]
ReplyTo: Richard Kim <[email protected]>, [email protected]
Subject: MOLPHARM/2010/067538-Revision Invitation
Cc: Richard Kim <[email protected]>
RE: MOLPHARM/2010/067538
Dear Dr. Freissmuth:
Your manuscript entitled "THE HIGH-AFFINITY BINDING SITE FOR TRICYCLIC
ANTIDEPRESSANTS RESIDES IN THE OUTER VESTIBULE OF THE SEROTONIN
TRANSPORTER" has been reviewed by two experts familiar with this field. Please visit
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6
Reviewer 1 Comments for the Author...
This manuscript presents the results of an investigation into the interaction of tricyclic
antidepressants (TCAs) with the serotonin transporter (SERT). Crystallographic analysis
of the bacterial LeuT homolog identified specific binding sites for TCAs, and some
authors have postulated that the same regions and types of interactions apply to SERTs.
However, while TCAs are competitive inhibitors of SERTs, they are low-affinity,
noncompetitive inhibitors of LeuT, suggesting a different type and site of interaction. In
this work Sarker et al combine molecular modeling with functional measurements
(including mutagenesis) to provide a working model for the TCA binding site of SERT.
The work is carefully done, and the results support the conclusions. However, there are
several aspects of the work that need expansion, as detailed below.
p. 7: “We also verified that the model was consistent with the results from previous
mutagenesis studies..” Please elaborate on what the criteria were and how the model was
consistent with published data.
p. 8: It appears from the examination of the models in the manuscript that while the main
chains were fixed during the docking process, the side chains were allowed to move. Is
this true? If so, please state so; if not, please explain why the side chains are in different
orientations in Fig 1 a-c.
p. 14, Fig. 1: The authors posit various interactions with the various dockings (i.e.,
“Further interactions of the tricyclic ring with the aromatic residues Y175, Y176, F35
was (sic) observed.” Please elaborate on how they distinguish between true interactions
(hydrogen bonds, ionic bonds, etc.) and two residues just being near. What criteria were
applied?
p. 14, Fig 1b: “The nitrogen atom shows an ionic interaction with D98.” I don’t see that
D98 is close enough to the nitrogen to make an ionic interaction.
7
p. 15, Fig 1: An important discriminator used in evaluating the three docking poses is the
role of Y95. In poses 2 and 3 the authors hypothesize that Y95 makes a hydrogen bond
with the nitrogen in imipramine. What criteria were applied to determine that such a
hydrogen bond might occur, especially for pose 3?
p. 16: The authors use the effect of the Y95F mutation to help them to discriminate
between docking poses, assuming that a hydrogen bond between the hydroxyl of Y95 and
the nitrogen in imipramine plays an important role. The mutation certainly weakens the
interaction, as shown in Fig. 2. If the mutation disrupts a hydrogen bond, then the KD
values should reflect the loss of 0.5-2 kcal/mol. Do they? If so, the authors should
mention this; if not, they need to explain this.
p. 16, Fig 3: The authors show that a number of tricyclics assume a similar orientation in
the proposed imipramine binding site as docking group 3. What about similarity to the
other docking poses? Where they observed at all?
p. 16: It is clear that the Y95F mutation allows discrimination between binding pose 1
and poses 2 and 3. However, none of the experimental data can discriminate between
poses 2 and 3. In order to be able to discriminate between poses 2 and 3, the authors
should identify a potential mutation that should affect binding in pose 2 but not 3 or vice
versa. The position of imipramine in the two poses should be different enough to identify
such a mutation that could be done and tested.
p. 23: A cartoon of the proposed architecture of SERT with the various binding sites
would be helpful in following the Discussion.
8
Reviewer 2 Comments for the Author...
Using sophisticated modeling procedures combined with binding measurements to
wildtype SERT transporter and a well selected mutant evidence has been presented
suggesting how tricyclic antidepressants bind to the vestibule of SERT close to the
substrate binding site. This may be of clinical importance. The drawn conclusions are
supported by the data. Some points should be clarified and some small changes are
suggested.
1)Page 15, three bottom lines: "This indicates that proteins were correctly folded..." This
statement appears to be too strong. Plasma membrane location suggests absence of gross
misfolding but it does not prove that the tertiary structure is completely intact.
2) Page 16, lines 1-3, Figs. 2b and c: "Differences in Vmax were likely to be accounted
for by differences in expression levels...."
Why is the 5-HT uptake by SERT(WT) in Fig. 2b lower than the 5-HT uptake by
SERT(Y95F) whereas the imipramine binding to SERT(WT) in Fig. 2c is higher than the
imipramine binding to SERT(Y95F)? Since stably transfected cell lines were used this
cannot be explained by different expression levels.
Minor points
Page 16 line 11: Please indicate the significance of the difference of the IC50 values!
Page 14 lines 18, 20 and Fig. 1: The positions of all mentioned mentioned amino acids
should be indicated in the figure. Y176, I168, V343 are missing.
Fig. 3: For clarity, I suggest to present the structural formulas of the docked compounds
also separately.
9
TABLE OF CONTENTS PAGE
LIST OF ABBREVIATIONS 12
LIST OF FIGURES 14
ZUSAMMENFASSUNG 15
ABSTRACT 17
1. INTRODUCTION
1.1 Neurotransmitter:sodium symporters (NSS; SLC 6 gene family) 19
1.2 Monoamine transporters 19
1.3 Pharmacology of monoamine transporters 20
1.4 Molecular biology of monoamine transporters 23
1.5. Structure-function studies of monoamine transporters 25
1.6. The alternating access model of transport 27
1.7. X-ray crystallography of NSS proteins 28
1.8. The cytoplasmic permeation pathway 29
1.9. Structural repeats in NSS proteins 30
1.10.Structural insights from new crystal structures 31
2. METHODS AND MATERIALS
2.1. Materials 34
2.2. Site-directed mutagenesis, cell culture and transfections 34
2.3. Confocal laser scanning microscopy 35
2.4. Radioligand binding assays 35
2.5. Uptake and release assays 36
2.6. Data analysis 36
3. RESULTS
3.1. The TCA dimethyl-aminopropyl side chain resides in the primary
substrate binding pocket 37
3.2. Carbamazepine and imipramine share a common binding pocket
in SERT 41
3.3. Substrates and inhibitors bind to SERT in a mutually non-exclusive
10
manner in the presence of carbamazepine 45
3.4. Amphetamines and carbamazepine cannot be bound to SERT
simultaneously 48
3.5. Ibogaine and carbamazepine bind to SERT in a mutually
non-exclusive manner 48
4. DISCUSSION 52
5. BIBLIOGRAPHY 58
6. APPENDICES 68
7. CURRICULUM VITAE 70
11
LIST OF ABBREVIATIONS
5-HT 5-hydroxtrytamine (serotonin)
b bovine (species)
CFT (WIN35, 428) 2β-carbomethoxy-3β-(4-fluorophenyl)tropane
COOH carboxyl terminal
d Drosophila (species)
DA dopamine
DAT dopamine transporter
DMEM Dulbecco’s modified Eagle’s Medium
EGFP enhanced green fluorescent protein
EL extracellular loops
GABA γ-aminobutyric acid
h human (species)
IL intracellular loop
m mouse (species)
MPP+ 1-methyl-4-phenylpyridinium ion
MTS methanethiosulphonate
MTSEA MTS-ethylammonium
MTSES MTS-ethyl sulphonate
MTSET MTS-ethyltrimethylammonium
NE noradrenaline
NET noradrenaline transporter
NH2 amino terminal
PBS phosphate buffered saline
12
13
r rat (species)
RTI-55 (β-CIT) 2β-carbomethoxy-3β-(4-iodophenyl)tropane
SCAM substituted cysteine accessibility method
S.E.M. standard error of the mean
SERT serotonin transporter
TCA tricyclic antidepressant
TM transmembrane domain
Kinetic parameters
Bmax maximal binding
EC50 concentration of a drug resulting in 50% maximal response
IC50 concentration of an inhibitor resulting in 50% maximal inhibition
KD dissociation constant at equilibrium
Ki inhibitory constant at equilibrium
Km Michaelis’ constant
Vmax maximal rate of uptake
Single-letter abbreviations for amino acids
A Alanine
C Cysteine
D Aspartic acid
E Glutamic acid
F Phenylalanine
G Glycine
H Histidine
I Isoleucine
K Lysine
L Leucine
M Methionine
N Asparagine
P Proline
Q Glutamine
R Arginine
S Serine
T Threonine
V Valine
W Trytophan
Y Tyrosine
LIST OF FIGURES Figure 1. SERT, DAT and NET are presynaptically localized at the synaptic cleft Figure 2. Putative topology of monoamine transporters Figure 3. Cellular expression of SERTWT and SERTY95F Figure 4. Functional characterization of SERTWT and SERTY95F Figure 5. Imipramine affinity for SERTWT and SERTY95F Figure 6. Carbamazepine is a low affinity SERT ligand that competes for binding of [3H]imipramine Figure 7. Carbamazepine shares the binding site of imipramine on SERT Figure 8. Carbamazepine precludes amphetamine action on SERT Figure 9. Mutually non-exclusive binding of 5-HT and of carbamazepine to SERT Figure 10. Paroxetine and carbamazepine bind to SERT in a mutually non-exclusive
manner. Figure 11. Amphetamines and carbamazepine bind to SERT in a mutually exclusive
manner Figure 12. Mutually non-exclusive binding of ibogaine to SERT in the presence of carbamazpine Figure 13. Mutually non-exclusive binding of ibogaine to DAT in the presence of carbamazepine
14
ZUSAMMENFASSUNG Nach neuronaler Stimulation werden Neurotransmitter in den synaptischen Spalt
freigesetzt. Dort aktivieren sie prä- und postsynaptische Rezeptoren, um das elektrische
Signal weiterzuleiten und über Rückkopplung ihre eigen Freisetzung zu steuern. Die
synatische Neurotransmission unterliegt einer strengen räumlichen und zeitlichen
Regulation durch membranstänfige Transporter aus der NSS/SLC6Familie (
"neurotransmitter:sodium symporters" = solute carier 6). Diese Tranporter sind an der
präsynaptischen Nervendigung exprimiert und vermitteln die rasche Rückaufnahme des
freigestezten Neurotransmitters aus dem synaptischen Spalt. Erste Einblicke in ihre drei-
dimensionale Struktur wurden durch die Kristallisation eines bakteriellen Orthologen
gewonnen, nämlich LeuTAa ein Leucintransporter des thermophilen Bakteriums Aquifex
aeolicus. Die erste Kristallstruktur zeigt eine Konformation, die amehseten dem
Übergangszustand ("occluded state")zwischen der auswärts und der einwärts gerichteten
Konformation von LeuTAa entspricht. Von großem Interesse waren die in der Folge
gelösten Strukuren, die LeuTAa in einem Komplex mit den trizyclischen Antidepressiva
(TCA's) Clomipramin, Desipramin, Imipramin und Amitriptylin zeigten. Diese
ermöglichten es, Modellvorstellungen zu entwickeln, wie die menschlichen NSS-
Vertreter, inbesondere der Serotonintransporter (SERT), durch Inhibitoren gehemmt
werden. Allerdings ist die Hemmung von LeuTAa durch TCA's nicht-kompetitiv, während
TCA's den Serotonintransporters (SERT) kompetitiv hemmen. Dieser Widerspruch stellt
die Gültigket von LeutAa als Modell für SERT in Frage. Die vorliegenden Experimente
gehen von der Arbeitshypothese aus, dass diese Diskrepanz unter der Annahme erklärt
werden kann, dass TCA's im äußeren Vestibül binden und dass ihre Seitenkette in die
Substratbindungsstelle reicht. Weil Serotonin gößer als Leucin ist, verhindert dies die
gleichzeitige Bindung von Serotonin und TCA's. Diese Hypothese wurde durch zwei
Ansätze geprüft: (i) Verdränungsexperimente mit Carbamazepin (einem Analogon vn
Imipramin, das nur eine kurze aliphatische Seitenkette hat und das die Bindung von
[3H]Imipramin an SERT kompetitiv hemmt), (ii) gerichteter Mutagenese, mit der ich
eine diagnostische SERTY95F Mutation erzeugte. Die Entfernung der Hydroxylgruppe
setzte erwartungsgemäß die Affinität von [3H]Imipramin herab, hatte aber keinen
Einfluss auf Substratbindung und Translokation. Mit Dixon Plots ließ sich nachweisen,
15
dass Carbamazepin gleichzeitig mit Serotonin, Paroxetine oder Ibogain an den
Transporter gebunden sein konnten. Hingegen war die gelichzeitige Bindung von
Amphetaminen, nämlich para-Chloroamphetamin (PCA) oder Methylen-Dioxi-
Methamphetamine (MDMA = ‘ecstasy’), und von Carbamazepine nicht möglich. Diese
Beobachtungen sind daher mit dem folgendeb Modell vereinbar: (i) Der trizyclische Ring
von TCA's liebt im äußeren Verstbül von SERT und die Dimethylaminopropyl-
Seitenkette bestezt die Substratbindungsstelle. (ii) Die Bindung von Amphetaminen führt
zu einer Konformationsänderung im inneren und äußeren Vestibül, die ihre gleichzeitige
Besetzung durch den trizyclischen Ring verhindert. (iii) Die simultane Bindung von
Ibogain (das an die einwärts-gerichtete Konformation bindet) und von Carbamazepin an
SERT ist mit einer zweiten niederaffinen Bindungsstelle im inneren Vestibül vereinbar.
Dies stimmt mit der Pseudosymmetrie von NSS Proteinen und mit Hinweisen für eine
zweite niederaffine Bindungsstelle von Imipramin.
16
ABSTRACT
Upon neuronal stimulation, neurotransmitters are released into the synaptic cleft where
they activate both pre- and post-synaptic receptors for propagation of the electrical nerve
impulse. Neurotransmission across a synapse is under tight spatio-temporal regulation by
high-affinity membrane transport proteins of the neurotransmitter:sodium symporter
(NSS)-family. NSS proteins are localized on the pre-synaptic nerve terminals. They
mediate rapid retrieval of the released neurotransmitters from the synaptic cleft. The first
insight into the three-dimensional structure of this class of proteins was obtained by
crystallization of the prokaryotic NSS member, LeuTAa, isolated from the thermophilic
bacterium, Aquifex aeolicus. The first structure revealed a conformation likely
representing an intermediate (i.e., the occluded state) between the ‘outward-’ and
‘inward-’ facing conformations of LeuTAa. Subsequent crystal structures of LeuTAa
bound to tricyclic antidepressants (TCAs) viz., clomipramine, desipramine, imipramine
and amitriptyline afforded mehanistic insights into the inhibition of NSS proteins. A
major limitaion was noted: the inhibition of LeuTAa by TCAs is non-competitive, but
TCAs are competitive inhibitors of the human serotonin transporter (SERT). Hence there
is an obvious discrepancy, which questions the validity of the LeuTAa-based model. My
thesis is based on the working hypothesis that the discrepancy can be resolved by
assuming that TCA's bind in the outer vestibule and that their side chain reaches into the
substrate binding site. Because serotonin is larger, this precludes simultaneous binding of
serotonin and TCA's. I adressed this discrepancy via two approaches: (i) competition
binding experiments with carbamazepine (i.e., an imipramine analog with a short
aliphatic side chain, which was verifed to compete with [3H]imipramine binding to
SERT), (ii) site-directed mutagenesis where I generated a diagnostic SERTY95F mutation
which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding.
Dixon plots revealed that carbamazepine bound simultaneously to SERT in the presence
of serotonin, paroxetine or ibogaine. In contrast, the binding of amphetamines, viz. para-
chloroamphetamine (PCA) or methylene-dioxy-methamphetamine (MDMA or, ‘ecstasy’)
and of carbamazepine was mutually exclusive. My observations are consistent with the
following model: (i) the tricyclic ring of TCAs resides in the outer vestibule of SERT and
the dimethyl-aminopropyl side chain occupies the substrate binding pocket; (ii) binding
17
of amphetamines to SERT creates a structural change in the inner and outer vestibules
which precludes simultaneous occupancy of the tricyclic ring in the vestibules, (iii)
simultaneous binding of ibogaine (which binds to the ‘inward-facing’ conformation) and
of carbamazepine to SERT is indicative of a second low-affinity binding site in the inner
vestibule, consistent with the pseudo-symmetric fold of NSS proteins.
18
1. INTRODUCTION
1.1. Neurotransmitter:sodium symporters (NSS; SLC6 gene family)
Polytopic membrane proteins from the family of neurotransmitter:sodium
symporters (NSS; solute carrier 6, SLC6 gene family) inactivate neurotransmission
across a synaptic cleft by reuptake of released neurotransmitters into pre-synaptic nerve
terminals and/or surrounding glial cells. As secondary active transporters, they
accomplish this inward movement of their cognate substrates against their own
concentration gradient by coupling it to the inward movement of Na+ ions along an
electrochemical ionic gradient, in a co-transport/symport mechanism. The
electrochemical coupling of substrate transport to charge carriers (cations) results in a net
flow of charge during each transport cycle. Upon reuptake, the dissipated ionic gradient is
restored by the ATP-dependent Na+/K+-ATPase. The restoration of this ionic gradient,
predisposes the transporter in anticipation for the next transport cycle thus, assuming
spatio-temporal regulation over neurotransmission. In this regard, it was previously
suggested that coordinated conformational changes of the transporter accompany such
vectorial transport of solutes across a lipid bilayer in a single file mode (Jardetzky, 1966).
The SLC6 gene family is a diverse set of integral membrane transport proteins.
The SLC6 gene family includes among many others, the transporters for the inhibitory
neurotransmitters - γ-aminobutyric acid (GABA) and glycine, the monoaminergic
neurotransmitters - serotonin (5-HT), dopamine (DA) and noradrenaline (NE), and
osmolytes – betaine, creatine and taurine. These transporters regulate intercellular
communication between neurons and are the sites of action of various drugs of abuse. A
number of pharmacological agents have thus been identified that act specifically on these
proteins.
1.2. Monoamine transporters
The serotonin transporter (SERT) is a prototypical monoamine transporter in the
SLC6 gene family. SERT is comprised of 630 amino acids and it is a high affinity
transporter of its cognate ligand, serotonin (or 5-hydroxytryptamine, 5-HT). In addition,
SERT also transports 5,7-dihydroxytryptamine, fenfluramine (an anti-obesity drug), 3,4-
methylene-dioxy-methamphetamine (MDMA or ‘ecstacy’) and para-chloroamphetamine
19
(PCA) (Ramamoorthy et al., 1993). The transport cycle of SERT involves the coupling
of Na+ and Cl- gradients (in a co-transport/symport mechanism) and the antiport of K+.
This ascertains the electroneutral nature of 5-HT transport mediated by SERT (Rudnick
and Clark, 1993). SERT is implicated for its physiologic role in regulating mood,
aggression and cognition, among others. Abberant SERT physiology in terms of its
function, expression and gene polymorphisms have been implicated in various disorders
including, depression, anxiety and schizophrenia (Lesch and Mossner, 1998).
The dopamine transporter (DAT) is a monoamine transporter in the SLC6 gene
family. DAT is comprised of 620 amino acids and it is a high affinity transporter of its
cognate ligand, dopamine (DA). DAT is also an efficient transporter of noradrenaline
(NE) (Carboni et al., 1990; Giros et al., 1992; Giros et al., 1994; Moron et al., 2002). The
other substrates of DAT include sympathomimetic amines, amphetamines and the
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The nature of both
inward and outward dopamine transport by DAT is electrogenic.
The noradrenaline transporter (NET) is a monoamine transporter in the SLC6
gene family. NET is comprised of 617 amino acids and transports both noradrenaline
and dopamine (Pacholczyk et al., 1991). The other substrates of NET include
sympathomimetic amines and amphetamines. NET function is implicated in the
regulation of several central and sympathetic nervous system functions viz., learning and
memory, mood, attention, stress and blood flow.
1.3. Pharmacology of monoamine transporters
The monoamine transporters are established targets for various pharmacological
agents. These include psychostimulant drugs of abuse (e.g. cocaine, amphetamines),
neurotoxins (e.g. MPP+) and therapeutics like antidepressants (e.g. tricyclics, SSRIs).
Among the psychostimulant drugs of abuse, cocaine and its chemical congeners are
competitive inhibitors of monoamine transporters, albeit with better selectivity for DAT
(Beuming et al., 2008; Ritz et al., 1987). In contrast, amphetamines and its congeners are
substrates of monoamine transporters (Jones et al., 1998b; Rudnick and Wall, 1992;
Sulzer et al., 1995). Amphetamines are transported by monoamine transporters. They act
as weak bases causing a vesicular redistribution of monoamines. This is coupled to a
20
concomitant reversal in the transport direction which eventually leads to an efflux or
release of monoamines into the synaptic cleft (Jones et al., 1998b; Sulzer et al., 1995). In
contrast, 1-methyl-4-phenyl pyridinium ion (MPP+) which is the principal metabolite of
1-methyl-4-phenyl-1,2,3,6-tetrahydropridine (MPTP) is efficiently transported by DAT-
expressing dopaminergic neurons (Gainetdinov et al., 1997; Javitch and Snyder, 1984). In
addition, tricylic antidepressants (TCAs) viz., imipramine, desipramine, clomipramine
and amitriptyline competitively inhibit monoamine transporters. In particular, the
structural basis of the competitive inhibition of SERT by TCAs has garnered interest in
lieu of recent crystal structure determinations of the leucine transporter, LeuTAa – a
bacterial homologue from Aquifex aeolicus (Yamashita et al., 2005; Singh et al., 2007;
Singh et al., 2008; Zhou et al., 2007).
21
Figure 1. SERT, DAT and NET are presynaptically localized at the synaptic cleft
22
1.4. Molecular biology of monoamine transporters
The advent of molecular cloning to the field of transporter biology in the early
1990s was heralded by the cloning of NET and the GABA (γ-aminobutyric acid)
transporters (GAT) (Guastella et al., 1990; Pacholczyk et al., 1991). The human NET was
the first monoamine transporter to be cloned and characterized (Pacholczyk et al., 1991).
The comparison of the amino acid sequences of NET and GAT revealed a 46% sequence
similarity. This increased to 68% when accounted for conserved amino acid substitutions.
The later cloning of DAT and SERT (Kilty et al., 1991; Shimada et al., 1991) provided
the paradigm towards structural, functional and genetic studies of monoamine transporter
function. This led to the characterization of monoamine transporters as a distinct subset
of integral membrane transport proteins in the SLC6 gene family. The topological model
of monoamine transporters predicted from the hydrophobicity plots of their amino acid
sequences suggested 12 putative transmembrane domains (TMs) with cytoplasmic amino
(NH2-) and carboxyl (COOH-) termini tails as shown below. The model depicted the
presence of an extracellular loop between transmembrane domains 3 (TM3) and 4 (TM4)
with potential N-glycosylation sites (Figure 2) (Zahniser and Doolen, 2001). All
experimental data thus far have corroborated in favor of this topological model for the
monoamine transporters (Androutsellis-Theotokis and Rudnick, 2002; Bruss et al., 1995;
Chen et al., 1998).
23
Figure 2. Putative topology of monoamine transporters
NH2
COOH
TM TM TM TM TM 5
TM 6
TM 1 2 3 4 8
TM 7
TM 9
TM 10
TM 11
TM 12
24
1.5. Structure-function studies of monoamine transporters
The application of a chimeric approach helped delineate the transporter domains
involved in interactions with substrates and inhibitors. Using chimeras constructed out of
NET and DAT, Buck and Amara showed that the region encompassing TMs 1-3 were
involved in interactions with inhibitors (Buck and Amara, 1995). In contrast however,
Giros and colleagues using a similar approach argued in favor of the region between TMs
5-8 in recognizing inhibitors (Giros et al., 1994). The apparent discrepancy in the
observations can be attributed to the presence of different junction sites in the chimeras
used and species-specific differences in the human DAT (hDAT) and rat DAT (rDAT)
proteins (Buck and Amara, 1994; Buck and Amara, 1995; Giros et al., 1994). The
affinities of substrates for NET and DAT were attributed to the region from the amino
termini through to TM5 and the region between TM9 to the carboxyl termini (Buck and
Amara, 1994; Buck and Amara, 1995; Giros et al., 1994). TM3 of DAT was found to be
important for dopamine uptake as revealed in a study using hDAT and bovine DAT
(bDAT) chimeras (Lee et al., 1998). With relation to SERT, chimeras constructed out of
hSERT and rSERT found the importance of TM1 in substrate recognition (Barker et al.,
1998; Barker et al., 1994). The other domains involved in substrate translocation were
determined to be TM4, TM8, IL1 and the region between TMs 1-5 (Buck and Amara,
1995; Giros et al., 1994). The extracellular loops (ELs) were also ascertained to be
important with respect to the conformational changes associated with substrate
translocation (Smicun et al., 1999).
Site-directed mutagenesis enabled the characterization of individual amino acid
residues in transporter function. TM1 has been shown to be of particular importance in
maintaining transporter activity. Mutation of a highly conserved aspartic acid residue in
TM1 of SERT, NET and DAT reduced uptake of substrates and decreased inhibitor
binding affinity (Barker et al., 1999; Kitayama et al., 1992). Further studies investigated
the potential role of aromatic residues in recognizing substrates. Mutation of a
phenylalanine in TM3 of DAT reduced the affinities of substrates as well as inhibitors
(Chen et al., 2001; Lin et al., 1999; Lin et al., 2000) while aromatic residues
encompassing the regions between TMs 7–12 were found to be important in recognition
of substrates (Buck and Amara, 1994; Kamdar et al., 2001; Mitsuhata et al., 1998). In the
25
case of NET, previous studies have exhaustively studied the importance of TM2 and the
first intracellular loop (IL1) to ascertain the substrate interactions in NET. Sucic and
colleagues identified an absolutely conserved residue viz., E113 and a GQXXRXG motif
(residues 117 – 123) to be important determinants of NET expression and function.
Mutation of E113 to alanine (E113A) and aspartic acid (E113D) resulted in marked reduction
in cell surface expression, while a mutation to glutamine (E113Q) led to improvement in
surface expression and function (Sucic and Bryan-Lluka, 2002; Sucic et al., 2002).
Similarly, Korkhov and colleagues, studied the above mentioned residue which is also
conserved in SERT viz., E136. Interestingly, while E136A and E136Q failed to support
substrate influx into cells, E136D did so at a reduced rate (Korkhov et al., 2006). Binding
experiments with the cocaine analogue, 2β-[3H]carbomethoxy-3β-(4-iodophenyl)tropane
(β-[3H]CIT) supported the conjecture that the mutant transporters preferentially adopted
the inward-facing conformation as β-[3H]CIT interacted with SERT in a manner
consistent with its binding to the outward-facing conformation. Thus, it may be
concluded from the above two studies that in the absence of the glutamate in TM2 of
SERT and NET, the conformational equilibrium of the transporter is shifted to the
inward-facing conformation (Korkhov et al., 2006; Sucic and Bryan-Lluka, 2005; Sucic
et al., 2002). In contrast, both GAT-1 and DAT, in place of the conserved glutamate in
SERT and NET, possess a leucine heptad repeat in TM2 that is apparently involved in
stabilizing the oligomeric interface of the transporters (Scholze et al., 2002a; Torres et al.,
2003).
The application of the systematic cysteine accessibility method (SCAM)
facilitated the investigation of the translocation pathway in monoamine transporters. In
SCAM, amino acid residues are studied by substitution into cysteine and investigated for
surface accessibility by measuring their sensitivity to thio-specific modifying reagents.
Depending on the reactivity of the substituted cysteines, residues in IL3 were found to be
important in substrate recognition and in mediating conformational changes associated
with substrate translocation (Chen et al., 2000; Ferrer and Javitch, 1998; Loland et al.,
2002). In addition, DAT was found to possess an endogenous zinc (Zn+2) binding site
which apparently stabilized the outward-facing conformation of the transporter
(Norregaard et al., 1998). Mutation of an aromatic residue in IL3 of DAT abolished
26
transporter activity (Loland et al., 2002) while it improved in the presence of
Zn+2(Norregaard et al., 1998). This highlighted the importance of IL3 in DAT function.
Using a similar approach, Chen and colleagues identified two isoleucine residues
in TM3 of SERT which were found to be involved in high affinity recognition of
substrates and inhibitors (Chen and Rudnick, 2000; Chen et al., 1997). One of these
residues, I172 along with the conserved Y95 in TM1 of SERT have been suggested to
directly participate in 5-HT binding (Chen and Rudnick, 2000; Chen et al., 1997; Henry
et al., 2006). These observations concluded that TM1 and TM3 play an important role in
substrate and inhibitor binding and presumably in forming the translocation pathway of
SERT. These studies helped ascertain structural determinants towards understanding the
mechanism of monoamine transporter function.
1.6. The alternating access model of transport
The alternate access model provided the underlying framework for the mechanistic
basis of substrate transport across membranes (Jardetzky, 1966). Accordingly, substrates
bind to a central binding site in the protein moiety that could be alternately exposed to
either side of the membrane by a conformational change in the protein. In secondary
active transport, as is the case with SERT for example, 5-HT, Na+ and Cl- bind to SERT
in the outward-facing conformation. 5-HT, Na+ and Cl- are then released to the
intracellular milieu. Upon dissociation of the co-transported solutes, K+ is transported in
the opposite direction (antiport) in a counter-transport/exchange mechanism. This is
followed by reorientation of the transporter to its original conformation in anticipation for
the next transport cycle.
An intermediate state that binds the substrate is presumably involved in the
conformational changes that accompany the transition of a transporter from the outward-
to the inward-facing conformation. In secondary active transport, if the substrate binding
site is simultaneously accessible from both sides of the membrane, as in a putative
channel mode, such an intermediate state would result in an uncoupled flux of the bound
substrate and in the quick dissipation of electrochemical ionic gradients. Conceptually, it
can be visualized that substrate translocation by alternating access involves an occluded
intermediate state in which the substrate binding site is inaccessible from either side of
27
the membrane. This conjecture has been verified in recent crystal structure
determinations of the leucine transporter, LeuTAa – a bacterial homologue of mammalian
monoamine transporters from Aquifex aeolicus (Yamashita et al., 2005; Singh et al.,
2007; Singh et al., 2008; Zhou et al., 2007). It may be envisaged however, that the
substrate binding site lies in the middle of the membrane in order to account for an
intrinsic symmetry in the accessibility of the binding site from either side of the
membrane. This has been corroborated from crystal structure determinations of
transporters with bound ligands (Abramson et al., 2003; Huang et al., 2003; Yamashita et
al., 2005; Yernool et al., 2004). These observations suggested the importance of the
intrinsic symmetry of the substrate translocation process as regards the binding and
dissociation of substrates on either side of the membrane.
1.7. X-ray crystallography of NSS proteins
In 2005, Gouaux and colleagues provided the first atomic glimpse into the
molecular architecture of a member of the SLC6 gene family of solute carriers viz., with
the high-resolution X-ray diffraction structure of LeuTAa, a transporter from the
thermophilic bacterium, Aquifex aeolicus (Yamashita et al., 2005). The cognate ligand of
LeuTAa is the amino acid leucine Leu, which binds tightly to LeuTAa (Singh et al., 2008;
Yamashita et al., 2005). A comparison of the amino acid sequences of LeuTAa and the
mammalian monoamine transporters revealed a sequence homology of 20-25%.
However, the regions of highest homology cluster around the substrate binding site where
a Leu molecule and the two co-transported Na+ ions bind. The homology between the
amino acid sequences in the substrate binding pocket reaches upto 60% where six out of
ten residues lining the pocket are identical. In the LeuTAa structure with Leu bound in the
substrate binding pocket (Yamashita et al., 2005), an aqueous pathway is accessible from
the extracellular medium leading to the binding site for Leu and the two Na+ ions.
However, the permeation pathway leading from the substrate binding site to the
cytoplasm is inaccessible. This is indicative of the fact that the bound Leu cannot
dissociate to the intracellular milieu and that the bound state stabilizes an apparent
outward-facing conformation in an occluded state. This is also substantiated by ~25Å of
packed protein structure which prevents the dissociation of bound Leu to the cytoplasm.
28
The occluded structure of LeuTAa bound to Leu conformed well with the predictions
derived from the alternating access model of transmembrane solute transport.
The subsequent crystal structure determinations of LeuTAa showed that it could
bind to other amino acid substrates and inhibitors including TCAs (Singh et al., 2007;
Singh et al., 2008; Zhou et al., 2007). As regards substrates, LeuTAa catalyzed the uptake
of amino acids Ala, Gly, Met and Tyr (Singh et al., 2008). Compared to Leu, Ala elicited
a 5-fold higher turnover rate and 27% higher catalytic efficiency. In contrast, the LeuTAa-
Trp structure was different in comparison to the co-crystal structures with Ala, Gly, Met
and Tyr (Singh et al., 2008). Trp is not a substrate of LeuTAa. Trp is a classic competitive
inhibitor of Leu and Ala transport because it competes with Leu and Ala for binding to
LeuTAa and is itself not transported. In the LeuTAa-Trp structure, Trp was found to bind in
the same substrate binding pocket as Leu and Ala. However, the greater bulk of Trp
resulted in an open-to-out conformation which provided an accessible pathway leading
from the binding site to the extracellular medium. As regards the TCAs, the co-crystal
structures of TCAs bound to LeuTAa showed that TCAs non-competitively inhibit LeuTAa
(Singh et al., 2007; Zhou et al., 2007). This suggests that, although inhibition by Trp can
be overcome by addition of higher substrate concentrations (Singh et al., 2008), no
amount of substrate can overcome LeuTAa inhibition by a TCA like imipramine,
clomipramine or desipramine (Singh et al., 2007; Zhou et al., 2007). In the LeuTAa-TCA
co-crystal structures, Leu occupied the designated substrate binding site, while the TCA
was bound in the extracellular vestibule 11Å above the substrate binding site stabilizing
the extracellular gate in a closed conformation. By avoiding closure of the extracellular
pathway, TCAs could block transport if opening of the cytoplasmic pathway is coupled to
closure of access to the extracellular milieu. As the TCA is bound to a site distinct from
that for Leu, increasing substrate concentrations cannot overcome inhibition. The TCA
inhibition kinetics for LeuTAa is thus classically non-competitive.
1.8. The cytoplasmic permeation pathway
As mentioned above, a comparison of the amino acid sequences of LeuTAa and the
mammalian monoamine transporters revealed a sequence homology of only 20-25%.
However, SCAM analysis helped to determine the amino acid residues that presumably
29
line the cytoplasmic permeation pathway of SERT (Forrest et al., 2008). The access from
the substrate binding site to the cytoplasm was accounted by a permeation pathway
comprised of one face each of the TMs 1, 5, 6 and 8 (Forrest et al., 2008). More recently,
a similar reactivity profile of inserted cysteines to thiol-modifying reagents was observed
in TM8 of GAT-1 (Ben-Yona and Kanner, 2009). The model proposed for the
cytoplasmic permeation pathway conformed well with the accessibility of amino acid
residues in response to ligands and substrates. For example, addition of 5-HT increased
the reactivity of inserted cysteines, and this showed an ionic dependence on Na+ and Cl-
(Zhang and Rudnick, 2006). The validity of the model can be ascertained from the fact
that amino acid residues lining the cytoplasmic pathway of SERT were less accessible in
the presence of cocaine, which is known to bind to the transporter in the outward-facing
conformation. This leaves the extracellular pathway open and the cytoplasmic pathway
closed (Zhang and Rudnick, 2006). In contrast, the presence of ibogaine which stabilizes
SERT in a cytoplasmic-facing conformation led to the closure of the extracellular
pathway (Jacobs et al., 2007; Zhang and Rudnick, 2006).
1.9. Structural repeats in NSS proteins
LeuTAa has a topology highlighted by an inverted structural repeat as revealed from
its high resolution X-ray diffraction structure (Yamashita et al., 2005). This structural
tenet provided a key to understanding the opening and closure of the cytoplasmic
permeation pathway of SERT (Forrest et al., 2008). Within the 12 TMs that define the
LeuTAa architecture, TMs 1-5 are similarly arranged to TMs 6-10 as repeats A and B,
respectively. The topological orientation of repeat A is inverted with respect to that of
repeat B. Interestingly, this internal pseudosymmetry shows marked structural
conservation despite the lack of significant sequence homology between the two repeats.
Both repeats have an odd number of transmembrane helical spans. In addition the LeuTAa
structure elicited the presence of two broken transmembrane helices viz., TM1 and TM6
(Yamashita et al., 2005). The broken segments of helices TM1 and TM6 coordinate the
two Na+ ions in the substrate binding pocket. More specifically, TM1 and TM6 are found
within a four-helix bundle containing TM2 and TM7 (Forrest et al., 2008). The long,
tilted helices TM3 and TM8 lie alongside each other. This helical pair of TMs 3/8 is
30
scaffolded within two V-shaped helical pairs formed by TMs 4-5 and TMs 9-10. While
the loop between the helical pair TMs 4/5 resides on the cytoplasmic side, the loop of the
helical pair, TMs 9/10 lies on the extracellular side. In addition, the amino acid residues
in TMs 1, 5, 6 and 8 from the cytoplasmic pathway of SERT (Forrest et al., 2008) are
complemented by amino acid residues in the other repeat which form the extracellular
vestibule of LeuTAa in TMs 6, 10, 1 and 3, respectively (Singh et al., 2008; Yamashita et
al., 2005). Although most NSS family members possess more than 10 TMs it could still
be envisaged that the 10-TM core of LeuTAa containing these repeats comprises the basic
translocation machinery (Forrest et al., 2008; Quick et al., 2006). A comparison of the
relative orientations of the first two TM segments in each repeat viz., TMs 1-2 and TMs
6-7 suggests a unique structural arrangement of the broken segements of TM1 and TM6.
This is indicative of the fact that the two broken helices in their relative orientation with
respect to the protein architecture accounts for the main difference between the two
repeats. This is also insightful with regard to the mechanism of substrate translocation in
accordance with the alternate access model. The orientation of the two repeats results in
the formation of a four-helix bundle in LeuTAa viz., 1-2-6-7, as mentioned above. The rest
of the protein scaffolds the 1-2-6-7 bundle and prevents it from access to lipids in the
membrane bilayer. It was therefore suggested, that the difference in the orientation of the
1-2-6-7 helical bundle with respect to the scaffolds leads to the formation of an
extracellular pathway. Thus, it may be envisaged that the inverted repeat topology and
their structural orientation in the membrane results in the formation of the outward- and
inward-facing conformations of the transporter. This is explained by a ‘rocking-bundle
mechanism’ (Forrest et al., 2008). Accordingly, the ‘rocking’ of the 1-2-6-7 helical
bundle between two alternate conformations afforded the opening and closure of the
extracellular and cytoplasmic permeation pathways in a manner, consistent with the
alternating access model.
1.10. Structural insights from new crystal structures
X-ray crystallography of several other integral membrane transport proteins
characterized in divergent gene families, revealed that the 5-TM inverted-repeat fold is a
common feature in the molecular evolution of these proteins. In 2008, Faham and
31
colleagues reported a 2.7Å-resolution X-ray diffraction structure of a bacterial
homologue of the sodium/galactose transporter, vSGLT from Vibrio parahaemolyticus –
a member of the solute sodium symporter (SSS) family (Faham et al., 2008). vSGLT has
14 TMs and a low amino acid sequence identity with NSS family members. The vSGLT
structure represents an apparent inward-facing conformation. However, from a
comparison of the hydropathy plots (Lolkema and Slotboom, 1998a; Lolkema and
Slotboom, 1998b) and the biochemical data from the related sodium-iodide symporter
(De la Vieja et al., 2007), vSGLT was attributed a certain degree of structural similarity
to NSS family members. Furthermore, this was corroborated by the presence of an
inverted-repeat topology in vSGLT as observed in LeuTAa. Interestingly, the vSGLT
cytoplasmic pathway compares well with that of LeuTAa. TMs 2, 6, 7 and 9 of vSGLT
correspond to TMs 1, 5, 6 and 8 of LeuTAa. This predicts that vSGLT makes use of a
similar ‘rocking bundle mechanism’ to catalyze movements associated with substrate
translocation. LeuTAa and vSGLT represent the outward- and inward-facing
conformations, respectively in accordance with the alternating access model. In most
respects, it may be argued that the vSGLT structure proved insightful in verifying
predictions about the symmetric nature of the extracellular and intracellular permeation
pathways in these transporters and that the substrate binding site is formed by the relative
orientations of the pseudo-symmetric structural repeats (Forrest et al., 2008; Yamashita et
al., 2005). Subsequently, two structures of a sodium:benzyl-hydantoin symporter Mhp1
from Microbacterium liquefaciens - a member of the nucleobase:cation symporter (NCS)
were solved at 2.9Å and 4.0Å resolutions (Weyand et al., 2008). The Mhp1 structures
revealed an apparent extracellular-facing conformation akin to that of LeuTAa. Mhp1 has
12 TMs with LeuTAa-like with structural repeats viz., in TMs 1-5 and TMs 6-10. TMs 11
and 12 are oriented in a peripheral manner and pack differently in comparison to LeuTAa.
An X-ray crystal structure at 3.35Å resolution was also reported for the sodium:betaine-
glycine transporter, BetP from Corynebacterium glutamicum (Ressl et al., 2009). BetP
has a LeuTAa-like membrane topology fold with the structural repeats in TMs 3-7 and
TMs 8-12. The BetP structure is apparently in an occluded, substrate-bound state. There
is no accessible pathway on the perisplasmic side. However, a narrow cytoplasmic
permeation pathway is formed by helices that correspond to amino acid residues lining
32
the pathway in vSGLT and to those in the cytoplasm-facing model of LeuTAa.
Interestingly, in contrast to the structures of LeuTAa, vSGLT and Mhp1, the two structural
repeats of BetP structure are more similar to each other. This is indicative of a more
intermediate conformation in BetP. In addition the recent crystal structures of proteins
from the amino acid/polyamine/organocation or APC (or SLC7 gene) superfamily, were
published (Fang et al., 2009; Gao et al., 2009; Shaffer et al., 2009). Two APC family
members viz., AdiC and ApcT consistent with bioinformatics analysis (Lolkema and
Slotboom, 2008) elicited a LeuTAa-like topology. Thus, in keeping with the alternating
access model, the presence of an inverted-repeat topology affords an efficient
mechanistic explanation towards understanding the dynamics of the substrate
translocation process.
33
2. METHODS AND MATERIALS
2.1. Materials
Dulbecco’s modified Eagle’s medium (DMEM) and trypsin were purchased from
PAA Laboratories GmbH (Pasching, Austria). Fetal calf serum was purchased from
Invitrogen. [3H]WIN35,428 (-(-)-2-β-[3H]carbomethoxy-3-β-(4-fluorophenyl)tropane;
85.9 and 76Ci/mmol), [3H]imipramine (47.5Ci/mmol), and [3H]5-HT ([3H]5-
hydroxytryptamine; serotonin; 28.1Ci/mmol) were purchased from PerkinElmer, Boston,
MA. [3H]MPP+ (85Ci/mmol) was supplied by American Radiolabeled Chemicals (St
Louis, MO, USA). Serotonin (5-HT), s-(+)-3,4-methylenedioxy-methamphetamine
(MDMA), paroxetine, carbamazepine and para-chloroamphetamine (PCA) were
purchased from Sigma. 1-methyl-4-phenylpyridinium ion (MPP+) was purchased from
Research Biochemicals International, Natick, MA. Ibogaine was kindly donated by
Sacrament of Transition, Maribor, Slovenia.
2.2. Site-directed mutagenesis, cell culture and transfections
The SERTY95F mutation was generated in the pEYFP-C1-hSERT background
(Schmid et al., 2001), using the QuikChange® Lightning site-directed mutagenesis kit
(Stratagene, Carlsbad, CA, USA), and confirmed with automatic sequencing (AGOWA
Genomics, Berlin, Germany). The primer sequence (5’ to 3’ sense strands), with the
mutated nucleotides indicated in bold font, was:
CCTTCTCTCAGTGATTGGCTTTGCTGTGGACC. The generation of HEK293 T-REx
cells expressing the hSERT under the control of a tetracycline inducible promoter of the
HEK293 cells stably expressing DAT is described previously (Hilber et al., 2005;
Scholze et al., 2002b). For transient expression, HEK293 cells were transfected with wild
type or mutant SERT using CaPO4 co-precipitation. For generation of a stable cell line of
SERTY95F, HEK293 cells were transfected using the CaPO4 co-precipitation method and
geneticin (G418, Invitrogen) was added for clone selection.
34
2.3. Confocal laser scanning microscopy
Confocal microscopy was carried out with a Zeiss Axiovert 200-LSM 510
microscope equipped with argon and helium/neon lasers (30 and 1 mW, respectively) and
a 40x oil immersion objective (Zeiss Plan-Neofluar). Stably transfected cells (1x105
cells/1.6 cm glass coverslip) were placed in a chamber containing Krebs-HEPES buffer.
Yellow fluorescent protein (YFP)-tagged proteins were detected with a band pass filter
(475-525 nm) using the 458 nm laser line. Images, obtained as z-stacks (slice thickness of
~1µm), were analysed by the LSM Image Browser to study the surface expression of the
wild type and mutant SERTs.
2.4. Radioligand binding assays
Membranes were prepared from HEK293 cells stably expressing wild type or
mutant SERT or DAT as in Korkhov et al. (Korkhov et al., 2006) expression levels in
individual cell clones varied between 4 and 40 pmol/mg; buffers used for the preparation
of DAT-expressing membranes contained 10 µM ZnCl2 and were devoid of EDTA
(because Zn2+ promotes the outward-facing conformation that is required for high-affinity
binding of inhibitors (Scholze et al., 2002b). [3H]Imipramine equilibrium binding to
SERT was performed in duplicate incubations in an assay volume of 0.2 to 0.5 ml
(adjusted appropriately to avoid radioligand depletion). In competition binding
experiments, membrane preparations (8-25 µg/assay) were incubated with the radioligand
(~2 nM [3H]imipramine), the indicated concentrations of carbamazepine and increasing
concentrations of a second competing inhibitor in buffer (20 mM Tris-HCl, 1 mM EDTA,
2 mM MgCl2, 3 mM KCl, 120 mM NaCl, pH adjusted to 7.4). Non-specific binding was
determined in the presence of 3 µM paroxetine. Saturation experiments were done with
serial dilutions of [3H]imipramine ranging from ~0.1 to 50 nM. Equilibrium binding of
[3H]WIN35,428 to DAT was performed in a similar manner but in an assay volume of 0.1
mL containing ~10 nM [3H]WIN35,428 (for competition binding experiments), the
indicated concentrations of carbamazepine and ibogaine and buffer (20 mM Tris-HCl, 1
mM MgCl2, 3 mM KCl, 120 mM NaCl, 10 µM ZnCl2, pH adjusted to 7.4). In saturation
experiments, the concentrations of [3H]WIN35,428 ranged from ~1 to 100 nM. Non-
specific binding was determined in parallel in the presence of 3 µM methylphenidate.
35
Binding was allowed to proceed for 15-60 min at 20 °C and terminated by rapid filtration
onto GF/A glass microfiber filters (Whatman® International Ltd, Maidstone, UK),
presoaked in 0.5% polyethyleneimine (Sigma). The radioactivity trapped on the filter was
determined by liquid scintillation counting.
2.5. Uptake and release assays
HEK293 cells stably expressing wild type or mutant SERT were seeded onto 48-
well plates (0.5 x 105 cells/well), 24 h prior to the experiment. For saturation
experiments, the specific activity of [3H]5-HT was varied between 30 cpm/fmol (0.2 µM)
to 200 cpm/pmol (30 µM) by addition of unlabeled 5-HT. Assay conditions were as
outlined in (Korkhov et al., 2006). For release studies, HEK293 cells stably expressing
wild type SERT were grown on coverslips in 96-well plates (4x104 cells per well),
preloaded with 0.4 µM [3H]serotonin (specific activity ~30 cpm/pmol) or 0.1 µM [3H]1-
methyl-4-phenylpyridinium (MPP+, ~90 cpm/pmol), for 20 min at 37oC, in a final volume
of 0.1 mL/well. The coverslips were transferred into superfusion chambers and excess
radioactivity was washed out with Krebs-HEPES buffer at 25oC, for 45 min with
perfusion rates of 0.7 ml/min. Upon attainment of stable efflux, 2 min fractions were
collected for liquid scintillation counting.
2.6. Data analyses
Data from binding and uptake experiments were subjected to non-linear, least
squares curve fitting to equations for a rectangular hyperbola using a Marquardt-
Levenberg algorithm. The fit was not improved by employing a logistic equation (Hill
equation). Data transformed into Dixon plots were fitted by linear regression.
36
3. RESULTS
3.1. The TCA dimethyl-aminopropyl side chain resides in the primary substrate
binding pocket
Previously, it has been shown that Y95 in the serotonin transporter coordinates
high affinity recognition of antidepressants (Henry et al., 2006); accordingly I mutated
Y95 to phenylalanine. I hypothesized the candidate hydrogen bond donor in the hydroxyl
group in Y95 to mediate this high affinity interaction with the TCA side chain. When
expressed in HEK 293 cells, both SERTWT and SERTY95F were found predominantly at
the cell surface (Figure 3). This indicated that the proteins were correctly folded, because
incorrectly folded monoamine transporters are retained within the cell (Korkhov et al.,
2008). Similarly, the affinity of SERTWT and SERTY95F for substrate uptake was
comparable (Figure 4; KM, WT =1.2 ±0.8 µM and KM, Y95F=1.6±0.8 µM). Differences in
Vmax of uptake were likely to be accounted for by differences in expression levels (not
shown, see also Figure 4). As expected, removal of the hydroxyl group of Y95 resulted in
a pronounced drop in the affinity of imipramine. This was seen regardless of whether the
affinity of imipramine for SERTY95F was determined directly in a binding assay using a
membrane preparation (Figure 4, triangles) or via inhibition of substrate uptake (Figure
5, triangles). The affinity of SERTY95F for imipramine was lower than that of SERTWT;
KD values were 6.4 ± 0.4 nM and 46.7 ± 4.9 (means ± SD, n= 4) for SERTWT and
SERTY95F, respectively. Imipramine-induced inhibition of serotonin uptake was examined
as follows: IC50 values were 16.3 ± 1.4 nM and 90.9 ± 20.9 nM (means ± SD, n= 4) for
SERTWT and SERTY95F, respectively. These results argue for a role of hydrogen bonding
between the hydroxyl group of Y95 to the nitrogen in the dimethyl-aminopropyl side
chain of imipramine. To validate these results further, I sought for a complementary
approach using the tricyclic compounds carbamazepine and di-hydrocarbamazepine: di-
hydrocarbamazepine has the same ring system as imipramine but the dimethyl-
aminopropyl side chain is replaced by a carboxamido group. In carbamazepine, the
flexibility of the central epine ring is restricted by a double bond.
37
SERTY95F SERT
WT
Figure 3. Cellular expression of SERTWT and SERTY95F
HEK293 cells (1*105 cells/16 mm glass coverslip) stably expressing
YFP-tagged transporters indicate that the expression pattern of
SERTY95F is similar to that shown by SERTWT
38
0 3 6 9 12 150
20
40
60
80
100 SERTWT
SERTY95F
5-HT (µM)
Spec
ific
[3 H]5
-HT
upta
ke(p
mol
/106 ce
lls/m
in)
0 10 20 30 400
1
2
3 SERTWT
SERTY95F
Imipramine (nM)
[3 H]Im
ipra
min
e bo
und
(pm
ol/m
g)
Figure 4. Functional characterization of SERTWT and SERTY95F
HEK293 cells stably expressing SERTWT or SERTY95F were seeded onto 48-
well plates (0.5*105 cells/well), 24h prior to experiments. Cells were
incubated with [3H]5-HT, with or without 10 µM paroxetine to determine
non-specific uptake. The assay was conducted in duplicates and reaction
stopped after 1 min with ice-cold Krebs-HEPES buffer. Data, plotted
according to the hyperbolic model, are shown as means of a representative
experiment carried out in duplicates.
39
0.0
2.5
5.0
7.5
10.0
0.1 1 10 100 1000
SERTWT
SERTY95F
Imipramine (nM)
Spec
ific
[3 H]5
-HT
upta
ke (p
mol
/106 ce
lls/m
in)
Figure 5. Imipramine affinity for SERTWT and SERTY95F
Inhibition of [3H]5-HT uptake by imipramine was determined in HEK293 stably
expressing SERTWT or SERTY95F. The cells were incubated with 0.1 nM [3H]5-HT
and increasing concentrations of imipramine, in the presence or absence of 10 µM
paroxetine to determine non-specific uptake
40
3.2. Carbamazepine and imipramine share a common binding pocket in SERT
I verified this prediction using two approaches: (i) saturation experiments with
[3H]imipramine were carried out in the presence of increasing concentrations of
imipramine (Figure 6). Carbamazepine caused a rightward shift in the saturation curve of
[3H]imipramine in a manner that was consistent with competitive inhibition. This is most
readily evident from the Schild plot depicted in Figure 6: the slope of the Schild
regression (1.13) was close to unity and the intercept with the x-axis gave a pKB of 2.03,
i.e. an affinity estimate of 93 µM for carbamazepine. (ii) Alternatively, carbamazepine
was allowed to compete for [3H]imipramine binding at two different concentrations of the
radioligand (i.e., 0.8 and 9.9 nM). The shift in the apparent IC50values (119±15 µM vs.
563±61 µM) at 0.8 and 9.9 nM [3H]imipramine, respectively; n=3) was consistent with
that predicted from the Cheng-Prusoff equation (KI= IC50/[1+L/KD]) (Cheng and Prusoff,
1973). The resulting affinity estimates KI were in the range of 85 to 95 µM and thus
consistent with the affinity estimate obtained from the Schild regression (Figure 7).
Carbamazepine was reported to cause serotonin release from brain slices in a
manner independent of an action on SERT (Dailey et al., 1997). I confirmed that
carbamazepine per se failed to release [3H]serotonin from SERT-expressing preloaded
HEK293 cells (Figure 8). However carbamazepine blunted serotonin-release induced by
para-chloroamphetamine (Figure 8). This observation is consistent with the ability of
carbamazepine to occupy the imipramine binding site of SERT and to thereby antagonize
the releasing action of amphetamines.
41
[³H]Imipramine (nM)0 2 4 6 8 10 12
[³H]im
ipra
min
e bo
und
(pm
ol/m
g)
0
10
20
30control carbamazepine 50 µM carbamazepine 100 µM carbamazepine 250 µM
C arbam azep ine (µM )30 100 300
log
(CR
- 1)
-0 ,5
0 ,0
0 ,5
1 ,0
Figure 6. Carbamazepine is a low affinity SERT ligand that competes for
binding of [3H]imipramine. Saturation of [3H]imipramine binding to SERT in
the presence of increasing concentrations of carbamazepine: mean ± SEM
(n=3). Schild plot from the data presented in panel reveals the following
parameters: slope = 1.13, r = 0.976 with an x-intercept = pKB = 2.03 for
carbamazepine 42
0
5
10
15
20
1 10 100 1000
[3H]Imipramine 9.9 nM[3H]Imipramine 0.8 nM
Carbamazepine (µM)
[3 H]Im
ipra
min
e bo
und
(pm
ol/m
g)
Figure 7. Carbamazepine shares the binding site of imipramine on
SERT. Carbamazepine displaces [3H]imipramine with a KI predicted
by the Cheng-Prusoff approximation for competitive inhibition
43
0 2 4 6 8 10 12 14 16 18 20 22 24
-4
0
4
8
12
16 carbamazepinecarbamazepine ± paroxetinePCAcarbamazepine ± PCA
carbamazepine ± paroxetine
carbamazepine ± PCA
Time (min)
% [³
H] M
PP+
rele
ase
per 2
min
Figure 8. Carbamazepine precludes amphetamine action on SERT
For release assays, culture medium was removed from stably
transfected HEK293 cells (40*104 cells/well grown in 96-well plates on
coverslips). The cells were preloaded with 0.4 µM [3H]serotonin or
with 0.1 µM [3H]MPP+ for 20 min at 37 oC in a final volume of 100 µl
Krebs-HEPES buffer/well. The coverslips were placed into superfusing
chambers and excess radioactivity washed out with Krebs-HEPES
buffer at 25 oC, for 45 min at a perfusion rate of 0.7 ml/min. Upon
achieving stable efflux, 2-min fractions were collected and data
analysed
44
3.3. Substrates and inhibitors bind to SERT in a mutually non-exclusive manner in
the presence of carbamazepine
I surmised that carbamazepine allows for simultaneous occupancy of the substrate
binding site, because it lacks the aliphatic dimethyl-aminopropyl side chain of
imipramine. Thus, serotonin (5-HT) was allowed to compete for [3H]imipramine in the
absence and presence of carbamazepine to substantiate the binding modes of
carbamazepine and 5-HT (Figure 9). Dixon plots (where the reciprocal of bound
radioactivity is plotted as a function of one inhibitor at a fixed concentration of the
second inhibitor) allow to test if two inhibitors can occupy the same binding site
simultaneously or whether their binding is mutually exclusive (Segel, 1975). When the
data summarized in Figure 9 were replotted in a Dixon plot, they fell onto lines that
pivoted around an intersection point close to the x-axis (Figure 9). This was also seen in
membranes prepared from cells that expressed SERT at 10-fold higher density (inset to
Figure 9). If two inhibitors compete for the same binding site, their binding is mutually
exclusive and this results in parallel lines in the Dixon plot. Conversely, if both inhibitors
can be bound simultaneously, Dixon plots are expected to yield intersecting lines (Segel,
1975). Thus the data unequivocally show that binding of serotonin and carbamazepine is
not mutually exclusive.
45
5-HT (µM)0,0 0,1 1,0 10,0 100,0
[3 H]im
ipra
min
e bo
und
(pm
ol/m
g)0,0
0,5
1,0
1,5
2,0
controlcarbamazepine 0.3 mMcarbamazepine 1 mM
5-HT (µM)
46
0 20 40 60 80 100
1/[3 H
]imip
ram
ine
boun
d (p
mol
-1.m
g)
0
5
10
15
control carbamazepine 0.3 mM carbamazepine 1 mM
0 40 80
0,0
0,5
1,0
1,5
2,0control carbamazepine 0.5 mM
Figure 9. Mutually non-exclusive binding to 5-HT and of carabamezpine to
SERT
Competition of [3H]imipramine binding to SERT at equilibrium was performed in
duplicate incubations in an assay volume of 0.2 to 0.5 ml. Membrane preparations
(8-25 µg/assay) from HEK293 cells stably expressing wild type SERT were
incubated with the radioligand (~2 nM [3H]imipramine), the indicated
concentrations of carbamazepine and increasing concentrations of 5-HT in buffer
(20 mM Tris-HCl, 1 mM EDTA, 2 mM MgCl2, 3 mM KCl, 120 mM NaCl, pH
adjusted to 7.4). Non-specific binding was determined in the presence of 3 µM
paroxetine. Data is transformed into a Dixon plot for 5-HT by expressing the
reciprocal of [3H]imipramine bound (pmol.mg-1) as a function of 5-HT at a fixed
concentration of carbamazepine. Data are shown as means ± SD of three
independent experiments in duplicate
SERT is also inhibited by a class of compounds that are collectively referred to as
selective serotonin reuptake inhibitors (SSRI's). These are structurally unrelated to TCAs.
Several recent modeling studies argued for binding of SSRI's to the substrate binding site
(Andersen et al., 2010; Tavoulari et al., 2009). If these assignments were correct, all my
experiments would be consistent with a model, where simultaneous binding of
carbamazepine and paroxetine ought to be possible. This conjecture was tested by
measuring paroxetine-induced inhibition of [3H]imipramine binding in the absence and
presence of carbamazepine. A Dixon plot of the inhibition curves again yielded a family
of intersecting lines (Figure 10) showing that carbamazepine and paroxetine occupy
mutually non-exclusive binding sites in the transporter.
Figure 10. Paroxetine and carbamazepine bind to SERT in a mutually non-exclusive manner
Competition of [3H]imipramine binding to SERT at equilibrium was performed in duplicate
incubations in an assay volume of 0.2 to 0.5 ml. Membrane preparations (8-25 µg/assay) from
HEK293 cells stably expressing wild type SERT were incubated with the radioligand (~2 nM
[3H]imipramine), the indicated concentrations of carbamazepine and increasing
concentrations of paroxetine in buffer (20 mM Tris-HCl, 1 mM EDTA, 2 mM MgCl2, 3 mM
KCl, 120 mM NaCl, pH adjusted to 7.4). Non-specific binding was determined in the
presence of 3 µM paroxetine. The binding data from the competition experiments is
transformed into a Dixon plot.
paroxetine (µM)
1/[3 H
]imip
ram
ine
boun
d (p
mol
-1.m
g)
0
2
4
6
8
10
12 control carbamazepine 0.3 mM carbamazepine 1 mM
0 1 2 3 4
47
3.4. Amphetamines and carbamazepine cannot be bound simultaneously
Amphetamines are inwardly transported substrates of monoamine transporters
that trigger serotonin efflux. While the details remain enigmatic (Sitte and Freissmuth,
2010), it is clear that the conformation of SERT in its amphetamine bound state must
differ from that of the serotonin-liganded conformation. I therefore asked if
carbamazepine and methylene-dioxy-N-methylamphetamine (MDMA, "ecstasy") or
para-chloroamphetamine were bound simultaneously: in the presence of carbamazepine,
I observed a parallel shift in the Dixon plots for both, MDMA (Figure 11) and para-
chloroamphetamine (Figure 11). This is diagnostic of mutually exclusive binding of
carbamazepine and amphetamines.
3.5. Ibogaine and carbamazepine bind to SERT in a mutually non-exclusive manner
I also examined ibogaine another ligand of SERT, which has previously been
shown to stabilize SERT in the inward-facing conformation (Jacobs et al., 2007). Because
binding of imipramine and carbamazepine is to the outward-facing conformation,
ibogaine and carbamazepine are not predicted to be bound simultaneously. Surprisingly,
this was not the case (Figure 12). In the presence of ibogaine, I observed that the
inhibition curves generated in the absence and presence of carbamazepine intersected
when replotted as Dixon plots (Figures 12, 13). This was seen regardless of whether I
examined the interaction of the two compounds in SERT (Figure 12) or in DAT (Figure
13). This finding can be rationalized by considering the pseudo two-fold axis symmetry
predicted to exist for NSS transport proteins (Faham et al., 2008; Ressl et al., 2009; Singh
et al., 2008; Yamashita et al., 2005). A second binding site may exist in the inner
vestibule that can accomodate the tricyclic ring of carbamazepine if ibogaine is bound to
the substrate binding site in the inward-facing conformation. It is worth noting that a
second (low affinity) binding site has previously been described for imipramine in SERT
(Schloss and Betz, 1995; Sur et al., 1998).
48
-10 0 10 20 30-0.5
0.5
1.5
2.5carbamazepine 0.5 mMcontrol
MDMA (µM)
1/[3 H
]imip
ram
ine
boun
d (p
mol
-1.m
g)
-10 0 10 20 30
0.0
0.6
1.2
carbamazepine 0.5 mMcontrol
p-Chloroamphetamine (µM)
1/[3 H
]imip
ram
ine
boun
d (p
mol
-1.m
g)
Figure 11. Amphetamines and carbamazepine bind to SERT in a mutually
exclusive manner. Competition of [3H]imipramine binding to SERT at equilibrium
was performed in duplicate incubations in an assay volume of 0.2 to 0.5 ml. The
binding data from the competition experiments is transformed into a Dixon plot for
MDMA and PCA by expressing the reciprocal of [3H]imipramine bound (pmol.mg-1)
as a function of MDMA and PCA at a fixed concentration of carbamazepine,
respectively. Data are shown as means ± SD of three independent experiments in
duplicate.
49
50
SERT
0
5
10
15
20
25
carbamazepine 0.5 mMcontrol
0.1 1 10Ibogaine (µM)
[3 H]Im
ipra
min
e bo
und
(pm
ol/m
g)
0 10 20 30 40-0.25
0.25
0.75
1.25
1.75
carbamazepine 0.5 mMcontrol
Ibogaine (µM)
1/[3 H
]Imip
ram
ine
boun
d (p
mol
-1.m
g)
Figure 12. Mutually non-exclusive binding of ibogaine to SERT in the presence of carbamazepine Competition of [3H]imipramine binding to SERT at equilibrium was performed in duplicate incubations in an assay
volume of 0.5 ml. Membrane preparations (8-25 µg/assay) from HEK293 cells stably expressing wild type SERT
were incubated with the radioligand (~2 nM [3H]imipramine), the indicated concentrations of carbamazepine and
increasing concentrations of ibogaine in buffer (20 mM Tris-HCl, 1 mM EDTA, 2 mM MgCl2, 3 mM KCl, 120 mM
NaCl, pH adjusted to 7.4). Non-specific binding was determined in the presence of 3 µM paroxetine. The binding
data from the competition experiments is transformed into a Dixon plot for ibogaine by expressing the reciprocal of
[3H]imipramine bound (pmol.mg-1) as a function of ibogaine at a fixed concentration of carbamazepine, respectively.
DAT
0.0
0.2
0.4
0.6
0.8
carbamazepine 0.25 mMcontrol
0.1 1 10Ibogaine (µM)
[3 H]W
IN 3
5,42
8 bo
und
(pm
ol/m
g)
0 10 20
0
5
10
15 carbamazepine 0.25 mMcontrol
Ibogaine (µM)
1/[3 H
]WIN
35,
428
boun
d(p
mol
-1.m
g)
Figure 13. Mutually non-exclusive binding of ibogaine to DAT in the presence of carbamazepine Competition of [3H]WIN 35,428 binding to DAT at equilibrium was performed in duplicate incubations in an assay
volume of 0.2ml. Membrane preparations (8-25 µg/assay) from HEK293 cells stably expressing wild type DAT
were incubated with the radioligand (~10nM WIN 35,428), the indicated concentrations of carbamazepine and
increasing concentrations of ibogaine in buffer (20 mM Tris-HCl, 1 mM EDTA, 2 mM MgCl2, 3 mM KCl, 120 mM
NaCl, pH adjusted to 7.4). Buffers used for the preparation of DAT-expressing membranes contained 10 µM ZnCl2
and were devoid of EDTA. Non-specific binding was determined in the presence of 3µM methylphenidate. The
binding data from the competition experiments is transformed into a Dixon plot for ibogaine by expressing the
reciprocal of [3H]WIN 35,428 bound (pmol.mg-1) as a function of ibogaine at a fixed concentration of
carbamazepine, respectively.
51
4. DISCUSSION
The crystal structure of LeuTAa, a bacterial homologue to mammalian NSS family
members from the thermophilic bacterium Aquifex aeolicus, revealed a high resolution
occluded structure devoid of any water molecule (Yamashita et al., 2005). Herein, one
Leu molecule was shown to be in coordination with two Na+ ions viz., Na1 and Na2;
which together with Na1 tread the path to the cell interior. An ion pair comprising of
residues, R30 and D404 is suggested to act as a barrier at the atomic level between the
bound Leu molecule in the occluded state and the aqueous pathway connecting the
primary binding site to the extracellular and intracellular bulk solutions. Interestingly, the
occluded state despite the charged ion pair mentioned above, revealed extracellular
access to the Leu binding site against cytoplasmic access owing to ~25Å of ordered
protein structure preventing the latter. An interesting facet of this structure however, are
the internal repeats, viz., TMs 1-5 and TMs 6-10 (LeuTAa does not possess the extended
TM11 and TM12 as predicted for SERT, DAT or NET) related to each other by a pseudo
two-fold axis symmetry of 176.5° to the membrane plane. With subsequent crystal
structures of bacterial transporters from unrelated gene families (Faham et al., 2008;
Ressl et al., 2009; Singh et al., 2008; Singh et al., 2007; Yamashita et al., 2005; Zhou et
al., 2007) it is now increasingly clear that the pseudo-symmetric fold of internal repeats is
a common structural tenet evolved over time and conserved across the spectrum of
membrane transport proteins for mechanism of function. For instance, in a recent study
(Yousef and Guan, 2009), it was observed that although LeuTAa from Aquifex aeolicus
and the melibiose permease, MelB from E. coli (belonging to different gene families)
bore poor sequence similarity and homology, secondary structure weighted alignment
elicited a perfect match between the predicted secondary structures of LeuTAa and MelB.
Inspite of this quantum leap in understanding the function of neurotransmitter
transporters through use of their atomic structures, there are fundamental discrepancies
which do not conform to established pharmacology. Studies, thereafter, revealed that
LeuTAa, in addition binds to tricyclic antidepressants (TCA), clomipramine, imipramine,
desipramine and amitriptyline just outside this charged ion pair R30 and D404 in a
secondary binding site (Singh et al., 2007; Zhou et al., 2007), located in the extracellular
vestibule of the transporter (Shi et al., 2008). The LeuTAa co-crystal structures with TCAs
52
depicted the binding pocket harboring Leu, Na1 and Na2 similar in comparison to the
occluded structure, but for the guanidium group of R30 which is flipped over restraining
interaction with the hitherto existent extracellular aqueous pathway as observed in the
occluded structure. Desipramine binding however was inhibitory in function to LeuTAa,
but the mechanism stood unclear in lieu of contradictory conclusions derived from the co-
crystal structures. TCA binding took place in a site 11Å above the central substrate
binding site, thus retarding dissociation of Leu to bulk solution via the extracellular
pathway. For instance, clomipramine at a saturating concentration impacted [3H]Leu
dissociation from LeuTAa by upto 700-fold in a manner just mentioned (Singh et al.,
2007). In this regard, it was recently shown that two Leu molecules can simultaneously
occupy the primary and secondary binding sites on LeuTAa (Shi et al., 2008), and binding
of a second Leu molecule in the secondary binding site allosterically regulates the
downward translocation of Leu (in coordination with Na1) from the primary binding site.
Thus, given the ambiguity surrounding the non-competitive nature of inhibition of
LeuTAa as opposed to known competitive inhibition of monoamine transporters by TCAs,
it is of crucial importance to explore the mechanism of TCA binding to the secondary site
of monoamine transporters in the absence of a representative monoamine transporter
crystal structure bound to a TCA.
In addition, TCAs were instrumental in the discovery of neurotransmitter reuptake
and – in conjunction with other drugs and chemicals - in the dissection of the underlying
mechanisms (Hertting and Axelrod, 1961). The serotonin transporter has remained a
prime target for antidepressant drugs since this seminal discovery. It has remained
enigmatic how these drugs bind, because the transporter can accommodate a bewildering
variety of structures. Based on the recent work with the bacterial homologue LeuTAa, two
binding sites have been proposed: (i) a vestibular binding site (Singh et al., 2007; Zhou et
al., 2007) that also functions as a secondary substrate binding site (Shi et al., 2008), (ii)
the substrate binding site per se (Andersen et al., 2010; Tavoulari et al., 2009) the latter
assignment is also supported by a combination of computer-assisted modeling and site
directed mutagenesis studies which suggest that cocaine analogs occupy the substrate
binding pocket in DAT (Beuming et al., 2008).
53
The importance of the vestibular binding site has been a matter of debate. It can,
for instance, be argued that the low affinity of TCAs for LeuTAa render these models
questionable. In fact at the concentrations range in which TCAs bind to LeuTAa they also
bind to many other targets including mammalian Na+- and K+-channels and numerous G
protein-coupled receptors. At the current stage, it would seem doubtful that any insight
can be gained from the vestibular binding of desipramine to LeuTAa that would be
relevant to understand the function of the pore region of the Na+-channel. Here, I test this
by employing a chemical biology approach viz., the use of the tricyclic compound
carbamazepine. Carbamazepine can be viewed as a truncated version of imipramine
rendered more rigid by the presence of a double bond in the central epine ring. The
underlying rationale is the assumption that carbamazepine and imipramine compete for
the same binding site, i.e. the site to which the tricyclic ring system would dock. My
experiments provided formal proof for this assumption: in two independent assays,
carbamazepine fulfilled the criteria of a (low affinity) competitive inhibitor of
[3H]imipramine binding. In addition, it blunted amphetamine-induced reverse transport.
This explains both the mutually exclusive binding of imipramine and serotonin and the
non-exclusive binding of carbamazepine and serotonin as ascertained from the Dixon
plots. In addition, the drop in [3H]imipramine affinity that resulted from the Y95F
mutation is explained by the loss of a hydrogen bond between the hydroxyl group of the
tyrosine side chain at residue Y95 and the tertiary amine in the dimethyl-aminopropyl
group of imipramine. The importance of Y95 of SERT for the binding of tricyclic drugs
was originally appreciated based on a substitution by cysteine (Henry et al., 2006),
removing the phenyl ring and introducing a side chain can alter the flexibility of the
helices by hydrogen bonding to main chain amide bonds one helical turn removed. The
replacement of tyrosine with phenylalanine however, is more subtle and sufficed to
greatly reduce the affinity of imipramine (but not of serotonin). Thus, taken together my
observations are consistent with the presence of two overlapping binding sites in SERT:
the tricyclic ring system is in the outer vestibule and the dimethyl-aminopropyl side chain
reaches into the substrate binding site. Other inhibitors (i.e., SSRI's) must bind in distinct
modes in this region to account for the observation that binding of paroxetine and
carbamazepine was not mutually exclusive. The presence of two binding communicating
54
sites can also be inferred from the increase in affinity and blocking efficacy seen with
bivalent phenethylamines (Schmitt et al., 2010).
My approach also allowed for probing the effect of amphetamines on the
conformation of the binding pocket. Amphetamines are subject to inward transport by
SERT (and the other monoamine transporters). They alter the conformation of the
transporter in a way that allows for substrate efflux (Sitte and Freissmuth, 2010). Earlier
experiments suggested counter-transport mechanism contingent on the oligomeric
arrangement of transporters (Seidel et al., 2005) and an important role of
serine/threonine-kinase-mediated phosphorylation of residues in the N-terminus of DAT
and SERT (Fog et al., 2006). Recent experiments indicate that the N-terminus may
possibly function as a lever (Sucic et al., 2010) in a manner analogous to that seen in
BetP (Ressl et al., 2009). Thereby, it affords the communication between the individual
moieties within the transporter oligomers. The issue is to understand how the N-terminus
senses that an amphetamine – rather than serotonin – resides within the substrate binding
site and the permeation pathway. My observations show that carbamazepine cannot be
accommodated by SERT in the amphetamine-bound state. The conformation of the
amphetamine-bound state can also be inferred to be distinct from the inward-facing
conformation: the latter is stabilized by ibogaine (Jacobs et al., 2007). The ibogaine-
bound conformation, however, allowed for simultaneous binding of carbamazepine to a
site that may correspond to the second, low-affinity imipramine binding site (Schloss and
Betz, 1995; Sur et al., 1998). Thus, in the presence of amphetamine, neither the inner nor
the outer vestibule is accessible to carbamazepine. This conformation is therefore
fundamentally different from the serotonin-bound state. It will be interesting to explore
how the change in the inner vestibule is transmitted to the N-terminus of SERT (and of
other monoamine transporters).
An important aspect to note here is the strength and nature of mutual interaction
of the above mentioned ligands with monoamine transporters in the presence of
carbamazepine. This can be judged from the point of intersection of the two lines
obtained in the presence or absence of carbamazepine. The addition of carbamazepine
increases the slope for the substrates or inhibitors. Farther the point of intersection from
the origin of the axes in the rectangular Cartesian coordinate system, weaker is the
55
strength of interaction. As observed in the case of amphetamines, by definition of
parallelism, the intersection point of the two curves (in presence and absence of
carbamazepine) is considered at infinity.
Among the inhibitors, ibogaine depicted the strongest case of mutual interaction
with carbamazepine. This is because, the point of intersection (in the third quadrant) was
very close to the origin of the axes in the rectangular Cartesian coordinate system, as
recapitulated also in the human dopamine transporter (DAT). The avalaible evidence
supports a model where, at equilibrium, carbamazepine and ibogaine simultaneously bind
to SERT and DAT in the inward-facing conformation. This is consistent with the
presence of a second low affinity binding site for the tricyclic ring system of
carbamazepine in the inner vestibule of the transporters.
As regards amphetamines, I remark that the substrate-bound and amphetamine-
bound conformations of SERT are fundamentally different. Structural rearrangements
that occur during ligand binding shed light on the nature of the conformational switch
associated with transport. This provides an insight into the translocation process and into
the putative channel mode into which these transporters can switch. The channel mode is
presumably important to understand the action of amphetamines and its congeners. The
medical relevance associated with the substrate permeation pathway can also be
appreciated by considering mutations that affect the transporter in a subtle way and that
are for instance known to occur in ADHD patients. The fact that amphetamine works in
clinical settings such as ADHD has always been difficult to understand. It may be
surmised that amphetamines (that would intuitively be thought of to aggravate the
situation) act in the long term by depletion of monoamines stores (in particular dopamine)
contained in synaptic vesicles in the brain. It may however also be that the action of
amphetamines is due to mutations (Mazei-Robison et al., 2008) in the transporter or to a
change arising from differences in associated proteins (e.g. syntaxin etc.) (Carvelli et al.,
2008).
Overall, I have been able to substantiate the validity of LeuTAa as a model to
ascertain the basis of TCA inhibition in monoamine transporters. The conclusions derived
from Dixon plots with respect to ibogaine and amphetamines are of particular interest: (i)
they underscore (i) the internal symmetry of the vestibules that can both accomodate a
56
tricyclic ring as a low-affinity ligand and (ii) the fundamemtal difference between the
amphetamine- and the substrate-bound conformation.
57
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6. APPENDICES
STRUCTURES OF AMINO ACIDS
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STRUCTURES OF CHEMICAL COMPOUNDS
carbamazepine serotonin (5-HT)
imipramine paroxetine
ibogaine MDMA (Ecstacy)
PCA
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7. CURRICULUM VITAE Personal details Birth November 24, 1985, Alipurduar, India Citizenship Indian Basic education 1990 – 2003 : primary, secondary and higher-secondary school, Miramar, India 2003 – 2007 : studies in engineering and biotechnology, Ranchi, India Languages English, Hindi (fluent), French (advanced), German (Level 2), Bengali (only speak) Scientific publications Sarker, S., Weissensteiner, R., Steiner, I., Sitte, HH., Ecker, GF., Freissmuth, M., Sucic,
S. (2010) The high-affinity binding site for tricyclic antidepressants resides in the outer vestibule of the serotonin transporter (in revision).
Sarkar, A., Ray, D., Shrivastava, AN., Sarker, S. (2006) Molecular biomarkers: their
significance and application in marine pollution monitoring Ecotoxicology 15: 333-340.
Gaitonde, D., Sarkar, A., Kaisary, S., D’Silva, C., Dias, C., Rao, D.P., Nagarajan, R., De
Souza, S.N., Sarker, S., Patil, D. (2006) Acetyl cholinesterase activities in marine snail (Cronia contracta) as a biomarker of neurotoxic contaminants along the Goa coast, West Coast of India Ecotoxicology 15: 353-358.
Meeting abstracts Sarker, S., Steiner, I. (2008) Do the binding sites of substrates and tricyclic
antidepressants overlap on the human serotonin transporter? BMC Pharmacol.8 (Suppl.):A9. 14th Scientific Symposium of the Austrian Pharmacological Society (APHAR), Innsbruck, Austria, November 21-22, 2008.
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Creative writing Sarker, S. (2005) Pollution: A menace posed by mankind. An International Interactive
Internet Project sponsored by the IEARN-US. (2000 - 2007). Global Project facilitator.
Sarker, S. (2002) Conversions bring dissentions and not brotherhood. In: ‘Vasant’ (Ed)
Vanaja Mudaliar, Padmashri Vasantrao Dempo Higher Secondary School of Arts and Science, Miramar, Goa, pp, 21-22.
Sarker, S. (2002) 50 years on – have we made the most of our freedom? In: ‘Vasant’
(Ed) Vanaja Mudaliar, Padmashri Vasantrao Dempo Higher Secondary School of Arts and Science, Miramar, Goa, pp, 28-29.
Sarker, S. (2002) Gravity and beyond – is it the ultimate theory of light and matter? In:
Souvenir of the Fourth Annual Convention of the Indian Association of Physics Teachers (IAPT), Goa Regional council, V-A (Ed) F.X. Borges, held at DCT’s Dhempe College of Arts and Science, Panaji, Goa. pp, 43-52.
Sarker, S. (2002) Gravity - beyond the universe. In: The Navhind Times, Science &
Technology – Section December 7, 2002, pp, 14 Sarker, S. (2001) The scourge of India. In: The Herald, junior section, Vol.1. No.29,
Sunday, March 18, 2001 pp, 12. Sarker, S. (2001) Help save your planet. In: The Herald, junior section, Vol.1. No.31,
Sunday, April 1, 2001 pp, 1 & 12. Sarker, S. (2001) Our national priorities should be. In: The Herald, junior section,
Vol.1. No.35, Sunday, April 29, 2001 pp, 1. Sarker, S. (2001) Global spiritualism – An unaccomplished dream. In: Laws of Life
Conversations. Pub: IEARN –USA. (Ed) Sarah Lucas. Vol. III, pp, 17. Sarker, S. (2000) My Perception of Life. In: Laws of Life Conversations. Pub: IEARN –
USA. (Ed) Sarah Lucas. Vol. II, pp, 162.
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Conferences/meetings Organised the 3rd International Workshop on ‘Cell Communication in Health and
Disease’, General Hospital (AKH), Medical University of Vienna, Austria, February 17-18, 2010.
Participated in the 15th Scientific Symposium of the Austrian Pharmacological Society
(APHAR) Graz, Austria, November 19-21, 2009. Participated in the 2nd SFB-35 Symposium on ‘Transmembrane Transporters in Health
and Disease’, Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria, September 4-5, 2009
Organised the 2nd International Workshop on ‘Cell Communication in Health and
Disease’, General Hospital (AKH), Medical University of Vienna, Austria, February 11-12, 2009.
Participated in the 14th Scientific Symposium of the Austrian Pharmacological Society
(APHAR) Innsbruck, Austria, November 21-22, 2008. Participated in the 1st SFB-35 Symposium on ‘Transmembrane Transporters in Health
and Disease’, Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria, September 26-27, 2008
Organised the 1st International Workshop on ‘Cell Communication in Health and
Disease’, General Hospital (AKH), Medical University of Vienna, Austria, February 20-21, 2008.
Represented India at the 8th Annual I*EARN Conference and 5th Annual World Youth
Summit on ‘Africa Connects’, University of Cape Town, Upper Campus, Cape Town, South Africa, July 8-14, 2001.
Represented India at the 7th Annual I*EARN Conference and 4th Annual World Youth
Summit on, ‘Sharing and Understanding Tele-Education in the 21st Century’, China Hall of Science & Technology, Beijing, China, July 10-16, 2000.
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International projects An International Education and Resource Network (I*EARN) project entitled, ‘Pollution
- A menace posed by mankind’ at the Global Environment Forum – I*EARN, 2000-2007.
A Global Youth Action Network (GYAN) representative for India, 2001-2007. Honors and awards Cell Communication in Health and Disease (CCHD) Doctoral Fellowship 2007 by the
Medical University of Vienna, Austria co-sponsored by the Austrian Science Fund (FWF) to work towards a PhD with Univ. Prof. Dr. Michael Freissmuth.
Rhodes Scholarships (University of Oxford, UK) 2007, shortlisted for preliminary
interviews, October 14, 2006 at Tata Sons, Mumbai, India. Commonwealth Scholarships (UK) 2007, shortlisted for preliminary interviews,
December 13, 2006 at the Indian Institute of Technology (IIT), Delhi, India. Indian Academy of Sciences Summer Fellowship Programme 2006 to work on, ‘Analysis
of IκBα Protein Expression in Oral Precancer and Cancer Tissue Biopsies’, with Dr. B.C.Das, Director, Institute of Cytology and Preventive Oncology, (Indian Council of Medical Research), Noida, India.
Best Student Award for the academic year 2002-03, Padmashri Vasantrao Dempo Higher
Secondary School of Arts and Science, Miramar, Goa, India. I*EARN-USA Scholarship 2001 for the global web-based interactive project, ‘Pollution
– A Menace Posed by Mankind’ and the essay, ‘Global Spiritualism – An Unaccomplished Dream’.
I*EARN-USA Scholarship 2000 for the essay, ‘My Perception of Life’, the Laws of Life
Essay Project (I*EARN). Science Talent Scholarship 2000-01 and 1998-99 awarded by the State Institute of
Education, Government of Goa, India. Merit Certificate under National Scholarship Scheme in recognition of the high rank
secured in the Goa Board Secondary School Certificate Examination, 2001.
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Extracurricular activities Excellence Award - Shield for Outstanding Performance in Extra Curricular activities for
2000-01 from The Rosary High School, Miramar, Goa, India. Certificate of Excellence to a Black Belt in Shotokan Karate (license to the rank of SHO-
DAN) awarded by the Japan Karate Association (JKA) Tokyo, Japan (Regd. No. Ind 1-0328; dated August 2, 1997).
Gold Medal at the First All Goa Shotokan Karate Championships, May 28, 1995, Peddem
Indoor Stadium, Mapusa, Goa, India.
