IDENTIFICATION OF DOMAINS IN THE DOPAMINE TRANSPORTER INVOLVED IN THE INTERACTION WITH G-BETA GAMMA SUBUNITS: ROLE IN DOPAMINE

MEDIATED EFFLUX

By

GABRIELA MARIE HIDALGO

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2018

© 2018 Gabriela Marie Hidalgo

To my family, loved ones, and all those who have supported my dreams

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ACKNOWLEDGMENTS

I am forever indebted to the University of Florida for being my home throughout

my undergraduate and graduate education. I thank the chair and Department of

Pharmacology and Therapeutics for allowing me to contribute to the breadth of

knowledge and works being produced. I am grateful to the members of my committee,

Dr. Urs and Dr. Harrison, for their guidance and contribution to my learning and

furthering of my studies hence presented. I deeply thank Dr. Torres for welcoming me

into his lab, for being such a wonderful mentor, and for all his patience, honesty, and

support. I thank every member of the Torres lab, especially Dr. Jose Pino and Marisol

Quiroz, for making this journey an incredible experience and for a close-knit community.

Most importantly, I thank my family and my loved ones for their unwavering support and

encouragement without which I could not have gotten to where I am.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF FIGURES .......................................................................................................... 7

LIST OF ABBREVIATIONS ............................................................................................. 8

ABSTRACT ..................................................................................................................... 9

CHAPTER

1 INTRODUCTION .................................................................................................... 11

Dopamine System .................................................................................................. 11 Drug Addiction and Amphetamine .......................................................................... 12

Dopamine Terminal ................................................................................................ 15 Dopamine Transporter ............................................................................................ 17 Protein-Protein Interactions .................................................................................... 20

Hypothesis .............................................................................................................. 26 Aims ........................................................................................................................ 26

2 MATERIALS AND METHODS ................................................................................ 36

Cell Culture, Transfection, and Treatments ............................................................ 36

[3H]-DA Efflux Assay .............................................................................................. 37 [3H]-DA Uptake Assay – Function and Kinetics ...................................................... 38 [3H]-DA Uptake Assay for Efflux Normalization ....................................................... 38

Lysate from Adherent Cells and Protein Quantification Assay (Using Bio-Rad Dc Assay).................................................................................................................. 39

Biotinylation Assay .................................................................................................. 40 Western Blot ........................................................................................................... 40

Co-Immunoprecipitation .......................................................................................... 41

3 RESULTS ............................................................................................................... 43

Establishing the Conditions to Measure DAT-Mediated Efflux in Heterologous Cells..................................................................................................................... 43

Effect of Alanine Substitution of hDAT Residues 587-590 (FREK) on DAT-mediated DA Efflux .............................................................................................. 44

Expression of DAT Wt and Mutants in Stable Cell Lines ........................................ 44

Effect of Alanine Substitution of hDAT Residues 587-590 (FREK) on DA Uptake .. 46 [3H]-DA Efflux Induced by AMPH and mSIRK in Cell Lines Expressing DAT WT,

DAT F587A, DAT R588A, and DAT E589A ......................................................... 49

Immunoprecipitation of DAT and Co-immunoprecipitation of Gβ ............................ 51

6

4 DISCUSSION/CONCLUSION ................................................................................. 65

LIST OF REFERENCES ............................................................................................... 75

BIOGRAPHICAL SKETCH ............................................................................................ 82

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LIST OF FIGURES

Figure page 1-1 The two primary pathways of the dopamine system are the mesolimbic. .......... 27

1-2 Psychostimulants target DAT and disrupt dopamine regulation. ........................ 28

1-3 Dopamine neurotransmission. The process of DA neurotransmission. .............. 29

1-4 Dopamine transporter (DAT) structure. The DAT is a 12. ................................... 30

1-5 Physical interaction between DAT and Gβγ confirmed through. ......................... 31

1-6 GST-fusion pull-down assays indicate that Gβγ binds only to the DAT. ............. 31

1-7 Activation of Gβγ subunits reduces DAT activity in striatal ................................. 32

1-8 mSIRK promotes DA efflux in heterologous cells through activation. ................. 32

1-9 Treatment of DA neurons with mSIRK activate Gβγ and induces DA. ............... 33

1-10 Characterization of residues in the Gβγ binding motif within the C. .................... 34

1-11 A schematic representation of the effect of Gβγ subunit activation. ................... 35

3-1 Effect of different conditions on DA efflux in transient transfection of WT. ......... 54

3-2 The effect of different conditions on DA efflux in transient transfection .............. 55

3-3 Expression of DAT WT and DAT mutants of residues 587-590 .......................... 56

3-4 Functionality and kinetics of DA uptake in stably transfected CHO cell .............. 57

3-5 The observed effect of alanine substitution of hDAT residues 587-590 .............. 58

3-6 Concentration response mediated [3H]-DA efflux induced by AMPH .................. 59

3-7 Concentration response mediated [3H]-DA efflux induced by AMPH .................. 60

3-8 Concentration response mediated [3H]-DA efflux induced by AMPH. ................. 61

3-9 Immunoprecipitation of DAT and co-immunoprecipitation of Gβ using. .............. 62

3-10 Observation of the effect the R588A mutation has on physical .......................... 63

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LIST OF ABBREVIATIONS

AMPH Amphetamine

D2R D2 dopamine receptor

DA Dopamine

DAT Dopamine transporter

dST Dorsal striatum

Gβγ G-beta gamma subunits

NAcc Nucleus accumbens

PFC Prefrontal cortex

SNc Substantia nigra pars compacta

VMAT2 Vesicular monoamine transporter 2

VTA Ventral termental area

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

IDENTIFICATION OF DOMAINS IN THE DOPAMINE TRANSPORTER INVOLVED IN

THE INTERACTION WITH G-BETA GAMMA SUBUNITS: ROLE IN DOPAMINE MEDIATED EFFLUX

By

Gabriela Marie Hidalgo

December 2018

Chair: Gonzalo E. Torres Major: Medical Sciences

Dopamine (DA) is an essential catecholamine implicated in many physiological

pathways and diseases. It is primarily involved in locomotor activity via the nigrostriatal

pathway and emotion/reward regulation via the mesolimbic pathway. DA homeostasis is

maintained via a reuptake processes through the dopamine transporter (DAT). DAT has

been identified as a target of psychostimulants such as cocaine and amphetamine. It

has been found that amphetamine induces reversal in DAT function resulting in DA

release via efflux. DAT can also carry out this efflux mechanism physiologically through

G-beta gamma activation. G-beta gamma acts on the dopamine transporter by

interacting with the intracellular C-terminal domain. The amino acid residues 587-590

within the C terminus were identified as critical for G-beta gamma and DAT binding. The

purpose of this study was to determine the residue(s) essential for DAT efflux function.

Single-point mutations, exchanging the wildtype (WT) residues 587-590 for an alanine

and subsequent functional experiments revealed that mutating residues R588 and K590

respectively resulted in a decrease and elimination of DAT-mediated efflux. There was

no effect on DAT efflux in the F587A and E589A mutants, thus further studies focused

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on the R588A mutant. Efflux assays on cells transfected with DAT WT and DAT R588A

using AMPH and the G-beta gamma mSIRK in increasing concentrations were

performed. These results lead to the conclusion that the effect of efflux was reduced in

the R588A mutation and was more sensitive to mSIRK. The precise role of these and

other amino acids have yet to be fully elucidated.

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CHAPTER 1 INTRODUCTION

Dopamine System

Dopamine (DA) is a monoamine or catecholamine neurotransmitter produced

predominantly in the brain though also found in multiple other regions of the body

(Carlsson et al. 1957, Carlsson & Waldeck, 1958; Montagu, 1957). Also known as 3-

hydroxytyramine, DA is a precursor for noradrenaline and adrenaline. DA is involved in

regulating movement, motivation, emotion and reward. It is implicated through excess or

deficiency in many diseases including schizophrenia, Parkinson’s Disease, ADHD, and

drug addiction among others (Carlsson et al., 2001; Nestler, 2005; Iversen and Iversen,

2007). Within the central nervous system dopaminergic neurons are located in and

project to various regions of the brain, thus dopamine has multiple functions depending

on the location at which it is synthesized and released (Moore & Bloom, 1978). There

are two primary locations at which dopaminergic neurons can be found: the substantia

nigra pars compacta (SNc) and the ventral tegmental area (VTA) (Bjorklund and

Dunnett 2007). The dopaminergic neurons from the SNc and VTA project to various

regions of the brain via two pathways respectively the nigrostriatal pathway and the

mesolimbic (mesocorticolimbic) pathway (Figure 1-1).

Axons from dopaminergic neurons in the nigrostriatal pathway project from the

substantia nigra to the dorsal striatum (dSTR), which includes the putamen and the

caudate nucleus (Alexander, Crutcher, & DeLong, 1991). Dopamine synthesized and

released within the dorsal striatum is responsible for controlling motor activity

(Alexander, Crutcher, & DeLong, 1991). Thus, the nigrostriatal pathway is implicated in

Parkinson’s Disease as a loss of dopaminergic neurons from the SNc results in

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decreased dopamine synthesis and release at the axon terminals in the dSTR,

ultimately affecting and causing loss of motor control and function (Spina & Cohen,

1989). Although understanding the role of DA and dopaminergic neurons within the

mesolimbic pathway is of high importance, for the purposes of understanding the

implication of DA and its transporter (DAT) as targets for psychostimulants, attention

must be focused on dopaminergic neurons found in the mesocortical/ mesolimbic

pathway (Robbins & Everitt, 1996).

Dopaminergic neurons which originate in the VTA, project to multiple brain

regions thus leading to the more popularly known roles of DA including but not limited to

mood, reward, and motivation (Robbins & Everitt, 1996). These neurons project from

the VTA to the amygdala, prefrontal cortex (PFC), hippocampus, nucleus accumbens

(NAcc), olfactory tubercle, and hypothalamus (Heimer et al., 1997; Bjorklund and

Dunnett 2007). Altogether, the function of dopamine in the mesocorticolimbic pathway

regulates emotion, reward, and cognitive function. Due to vast role of dopamine

understanding its biochemical implications and the function of its transporters is critical

to uncovering therapeutics for diseases.

Drug Addiction and Amphetamine

Drug addiction can be defined as a compulsion to intake a licit or illicit substance

to reach a state of euphoria or relaxation without control over how much of a substance

is used. The DSM, Diagnostic and Statistical Manual of Mental Disorders, categorizes

mental disorders on the basis of research and observation and is used by clinicians,

researchers, and physicians. The most recent version, the DSM-V, combined

dependence and abuse as a single classification- substance use disorder. While these

terms can be used interchangeably, there are distinctions and particular characteristics

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that define drug addiction. In 1993, Nestler et al. stated that chronic drug administration

resulted in changes that could be identified as tolerance, sensitization, withdrawal, and

dependence of which the accumulation of these and their effects result in addiction

(Nestler, Hope, & Widnell, 1993). As a result of gradual changes induced by long-term

drug use and the typically exhibited increase in amount of drug administered in order to

arrive at the same effects, it has been suggested that drug addiction modulates

changes in neural plasticity (Nestler et al., 1993).

Early pharmacological studies using animal models identified that due to the

adaptive changes and reinforcing tendencies of addictive drugs, the mesolimbic

dopamine system was the target of drugs such as opiates, cocaine, nicotine, ethanol,

and amphetamine (Bozarth, 1989; Wise, 1990; Kuhar et al., 1991; Brown, Robertson, &

Fibiger, 1992; Koob, 1992; Vrana, Vrana, Koves, Smith, & Dworkin, 1993). Further

identifying and characterizing the specific targets and mechanisms of actions of such

drugs have been the objective of drug addiction research. A well-established notion is

that a characteristic effect of addictive drugs is the increase of dopamine concentration

both in the VTA as well as the regions to which VTA dopaminergic neurons project (Di

Chiara and Imperato, 1998, Nestler, 2005). Findings from several studies have

suggested that addictive drugs take over the mechanisms involved in integrating

synaptic changes induced by experiences which in turn alter synaptic transmission in

the mesocortical/mesolimbic dopamine systems (Lüscher and Malenka, 2011).

Of the many drugs tested in the laboratory setting, cocaine and amphetamine

have been understood to interact with and affect monoamine systems specifically

monoamine transporters, though the research presented here will focus on the

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dopamine system. Self-administration studies in rats and using rat striatum found that

cocaine had a competitive, inhibitory effect on the dopamine transporter (Ritz, Lamb,

Goldberg, & Kuhar, 1987). More specifically, cocaine inhibition of dopamine reuptake

through the dopamine transporter increases extracellular concentrations of dopamine

thus prolonging dopaminergic signaling (Amara & Sonders, 1998). The impact of

cocaine on the dopamine transporter and the resulting disregulation of dopamine

signaling has been found to induce locomotor activity in the dorsal striatum and affect

the mesocorticolimbic pathways of reinforcement (Amara & Sonders, 1998).

Amphetamine, on the other hand, plays a more complicated role in psychostimulant

targeting of monoamine transporters such that its precise mechanism of action is not

fully understood (Figure 1-2).

First synthesized in 1887 (Edeleano, 1887), amphetamine and amphetamine-like

substances have been used to treat illnesses such as obesity, ADHD, and narcolepsy.

Amphetamine belongs to the phenethylamine class and has varying effects on the

human body. Although it has been used in a positive manner for medical treatments,

amphetamine is more notoriously known for its addictive tendencies and role in reward-

mediated drug addiction. Chronic use of amphetamine has been linked to altered gene

expression in the mesocorticolimbic system (Robison & Nestler, 2011). Excessive use

of amphetamine readily leads to tolerance which promotes greater self-administraztion

and drug-sensitization which is likely due to its interaction with the dopamine system,

specifically the mesocorticolimbic pathways responsible for reward and behavior. The

precise mechanism of amphetamine was unclear as to whether it functioned at the

15

plasma membrane or vesicular level, however it was determined that it predominantly

acts at the vesicles (Sulzer et al., 1995).

Although the target variability and precise mechanisms of each addictive drug is

distinct, the primary actions of have been studied at great lengths and are important

potential therapeutic targets (Lüscher and Malenka, 2011). The following subsections

will introduce and explain the dopamine system on which addictive drugs acts, so as to

better understand the regulation and dysregulation of dopamine.

Dopamine Terminal

In order for DA to exert its function, DA must first be synthesized and then

released into the synaptic cleft. To understand the synthesis of dopamine it must be

stated that DA is catecholamine meaning it is formed by a benzene ring with two

hydroxyl groups forming the catechol, and an ethyl-amine side chain (PubChem

Compound Database). The primary synthesis of DA begins with L-Phenylalanine which

is converted via phenylalanine hydroxylase, oxygen (O2) and other cofactors into L-

Tyrosine. The enzyme tyrosine hydroxylase (TH) and cofactors then act on L-Tyrosine

to form the direct DA precursor L-DOPA. DOPA decarboxylase then converts L-DOPA

into DA which is then ready to be packaged into vesicles and released from the

presynaptic neuron upon the firing of an action potential (Figure 1-3) (Musacchio, 2013).

The aforementioned synthesis of dopamine occurs within the presynaptic

terminal, yet there are many components involved in the productions, release, recycling,

degradation, and overall homeostasis of DA. The bioenergetics and biophysical

characteristics of the vesicle monoamine transporter are driven by a proton gradient and

is sensitive to both transmembrane potential and pH (Kanner and Schuldiner, 1987;

Njus, Kelley, & Harnadek, 1986; Johnson, 1988). The vesicle monoamine transporters

16

(VMAT) move cytosolic monoamines to vesicles and function as a result of the vacuolar

ATP-dependent H+ pump (V-ATPase) which generates the H+ electrochemical gradient

(Kanner and Schuldiner, 1987; Njus, Kelley, & Harnadek, 1986; Johnson, 1988). Two

isoforms of VMAT have been identified: VMAT1 and VMAT2. VMAT1 is involved with

the peripheral nervous system and is expressed in endocrine/paracrine cells while

VMAT2 is involved with the central nervous system and expressed in neurons;

(Nirenberg, Chan, Liu, Edwards, & Pickel, 1996). The human VMAT2 was cloned in

1993 by two groups (Surratt et al. 1993; Erickson and Eiden, 1993). The VMAT2 is

responsible for facilitating DA uptake into synaptic vesicles.

Additionally, dopamine receptors of which there are 5 (1-5), are seven

transmembrane G protein-coupled receptors (GPCRs) and have diverse functions

(Ford, 2014). The dopamine D2 receptors (D2R) are highly important to the

maintenance and control of dopamine neurotransmission. D2Rs are located on DA

nerve terminals, on postsynaptic neurons, and DA neuron bodies, as well as non-DA

neurons regulating many functions (Bolan et al., 2007). Specifically, D2R activation was

found to inhibit DA synthesis, cell firing, and DA release. D2Rs function via feedback

mechanisms by which DA activates them which signals for a reduction of DA release

from any continuously firing action potentials (Paladini, Robinson, Morikawa, Williams, &

Palmiter, 2003). D2Rs have also been found to interact with the dopamine transporter,

increasing its function and the amount of extracellular DA re-uptaken only during

excessive stimulation and D2R activation (Bolan et al., 2007). It is important to note

that while there is an apparent interaction between overly activated D2R and increased

DAT function, DAT does not depend on D2R as indicated by experiments exhibiting loss

17

of D2Rs (Dickinson et al., 2002; Schmitz, Schmauss, & Sulzer, 2002). Based on such

findings, the role of D2Rs is selective in DAT-mediated DA uptake (Ford, 2014).

Following action potential activity and activation of postsynaptic receptors,

extracellular DA that is not re-uptaken and recycled in pre-synaptic vesicles is degraded

and metabolized by monoamine oxidase (MAO) (Singer et al, 1979; Bach et al., 1988)

and catechol-O-methyltransferase (COMT) (Axelrod, Senoh, & Witkop, 1958). The

process of DA synthesis, neurotransmission, and regulation is complex involving many

receptors, signals, effectors, and mechanisms of which many new details are being

uncovered.

Dopamine Transporter

Focusing more specifically on the components of the dopamine terminal, the

dopamine transporter (DAT) is crucial for controlling the concentration of dopamine both

inside and outside of the cell. In 1991, the NET protein was encoded via the isolation of

a cDNA clone which opened up the possibilities within monoamine transporter studies

(Pacholczyk, Blakely, & Amara, 1991). Due to conserved amino acid residues found

among DAT, SERT, and GABA transporters compared to NET, the genes that encode

these and other transporters have been identified which allowed for further studies into

the function of theses transporters among others (Torres et al., 2003). The DAT is a 12-

fold transmembrane protein whose gene (SLC6A3) is found on chromosome 5p15.3

and is divided into 15 exons (Figure 1-4) (Torres et al., 2003; Vandenbergh et al., 1992).

The human DAT (hDAT) protein is made up of 620 amino acid residues at which the

least conserved residues respective to the NET and SERT proteins are found at the N-

terminal and the C-terminal (Torres et al., 2003).

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Upon determination of the residues and structure of DAT, researchers sought to

find the location of the residues at which the aromatic dopamine ring interacts with DAT.

In order to study the interaction between DAT and DA, residue specific mutations were

made in which a phenylalanine was replaced with an alanine in transmembrane domain

3(TMD3) ultimately causing a decrease in DA affinity (Chen, Vaughan, & Reith, 2001;

Lin, Wang, Kopajtic, Revay, & Uhl, 1999). Post-translational modifications, such as N-

linked glycosylation, have also been found to be critical in the function of DAT and other

monoamine transporters. Specifically, modulation of transporter activity via

phosphorylation has been of interest and several studies have shown that protein

kinase C (PKC) activation down-regulates transporter activity and transporter

internalization is similar to that of G-protein coupled receptors (Torres et al., 2003; Zhu,

Kavanaugh, Sonders, Amara, & Zahniser, 1997).

Prior to discussing how DAT function can be affected by intracellular proteins,

signals, or mediated by drugs, it is critical to have a general understanding of its primary

mechanistic function. It is well known that Na+ is essential for reuptake through

monoamine transporters. DAT reuptake involves the substrate and co-transport of two

Na+ ions and one Cl- ion(Torres et al., 2003). It has also been found that DAT as well as

the other monoamine transporters, display channel-like activity which in DAT has been

shown to be driven by chloride conductance (Torres et al., 2003). The anionic currents

exhibited by DAT potentially regulate DA release and dopaminergic neuron excitability

and are highly involved in neuronal firing in midbrain (Ingram, Prasad, & Amara, 2002).

Thus, DAT regulation of DA release and neuron excitability plays a significant role in

reward-mediated behaviors.

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Due to the importance of DAT in maintaining dopamine homeostasis, it has

become paramount to understand how its role could be altered by drugs or implicated in

different disease states. As mentioned earlier, DAT is a target of psychostimulants

which ultimately results in DA imbalance. Amphetamine and cocaine are two examples

of psychostimulants that directly affect DAT function. Cocaine has been shown to act as

a direct competitive inhibitor which blocks DA reuptake leading to excessive dopamine

stimulation since released dopamine remains in the synaptic cleft (Giros, Jaber, Jones,

Wightman, & Caron, 1996). The role of amphetamine in altering DAT function is more

complex and to date has yet to be entirely understood. However, it has been well

established that amphetamine promotes catecholamine release and blocks uptake thus

enhancing the effect of catecholamines at the synaptic cleft (Glowinski et al., 1966;

Carlsson et al., 1966; Besson et al., 1971). It has been suggested that due to its effect

on dopaminergic and catecholamine systems amphetamine may induce a neuronal

feedback inhibition (Bunney, Aghajanian, & Roth, 1973). A study by Sulzer et al. found

that amphetamine increases cytosolic DA possibly by affecting the vesicular gradient

thus preventing DA packing (Sulzer et al., 1995). It was suggested that the cytosolic

increase of DA by amphetamine could alter synaptic transmission and facilitate reverse

transport of DA through DAT (Sulzer et al., 1995). The results of these studies with

AMPH seem to indicate that efflux is an alternative mechanism of neurotransmitter

release (Levi and Raiteri, 1993).

To further understand the role of DAT and why the effects of psychostimulants

are so important to understand, a series of experiments were carried out in which DAT

was removed. Relative to wild-type mice, DAT knockout mice took 100 times longer to

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clear extracellular dopamine and had significantly more spontaneous locomotor activity

(Giros et al., 1996). These findings further indicated the highly important role of DAT in

DA regulation. Within the same study, 10 μM of AMPH were administered to both wild-

type and DAT knockout mice (Giros et al., 1996). It was found that after 30 minutes, the

concentration of extracellular dopamine increased in wild-type mice, but there was no

change in DAT knockout mice (Giros et al., 1996). These results thus determined that

the role of amphetamine in the dopaminergic system is to reverse the role of DAT which

is concurrent with previous findings. The fact that AMPH-stimulated increase in

extracellular DA concentration is dependent on DAT suggested that perhaps the efflux

mechanism induced by AMPH through DAT could be an intrinsic characteristic of DAT

itself.

In order to determine the mechanism of DA efflux through DAT, a manner by

which to activate DA release without the use of psycho stimulants had to be employed.

Thus, the intracellular signals and/or protein-protein interactions potentially involved in

DAT efflux needed to be identified and studied so as to understand the intrinsically

driven efflux mechanism of DAT.

Protein-Protein Interactions

Previous research indicated the significance of the dopamine transporter as a

regulator of dopamine homeostasis and as a target for psychostimulants. As mentioned,

DAT not only has a reuptake function, but also has the ability to have outward flow of

dopamine into the synaptic cleft. It was critical thus to examine the mechanisms by

which the DAT carry-out efflux occurs and which types of interactions are involved. To

understand this, specific protein interactions needed to be identified and further studied.

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Protein-protein interactions are some of the most fundamental and prominent

ways by which physiological processes occur. G-protein coupled receptors (GPCRs)

are among the most important class of proteins as they play a role in almost every part

of human health and disease. Due to their pervasive role in biochemical and

physiological processes, studying GPCRs is not only critical to understanding specific

medicines but also for potentially developing therapeutics targeting specific GPCRs.

There are approximately 826 GPCRs that have been identified and are classified into

five major families (Clapham & Neer, 1997). Currently, 43 of the over 800 known

GPCRs are therapeutically targeted.

G-protein coupled receptors share a common architecture of an extracellular N-

terminal followed by 7-transmembrane helices and an intracellular C-terminal (Clapham

& Neer, 1997). GPCRs are unique not only with regard to the vast family sizes, but also

in the diversity of structure and modularity within the different subfamilies. The variability

of the ligand-binding sites is what primarily distinguishes each GPCR. The diversity

among ligand-binding sites accompanied by helical differences, secondary structures,

and disulfide bond patterns create a large repertoire of GPCRs which have yet to be

fully understood (Katritch, Cherezov, & Stevens, 2012). The GPCR interaction of

interest for the purposes of the thesis study later detailed is that of GPCRs and G-

proteins.

A subset of G-proteins are identified as a heterotrimeric complexes consisting of

a G-alpha (Gα) and G-beta gamma (Gβγ) subunits as well as an effector (Clapham &

Neer, 1997). Within these subunits, there are variations resulting in different pairings of

subunits further adding to the multiple combinations of interactions that can

22

physiologically occur. While not all location/process specific G-proteins are known, the

mechanism by which they function is well comprehended. The G-proteins are located

on the intracellular side of the plasma membrane and are loosely attached via covalent

interactions with lipids. Upon ligand binding to the GPCR, GTP replaces the GDP

normally bound to the Gα subunit of the G-protein(Clapham & Neer, 1997). This

exchange induces conformational changes which weaken the interaction between the

Gα and Gβγ subunits. Upon separation each subunits interacts with other intracellular

proteins and components which ultimately carry out effector functions and/or signaling

cascades (Clapham & Neer, 1997). It is important to understand that GPCRs and G-

proteins do not exert simple on/off mechanisms rather a spectrum of outputs dependent

on their locations, ligands, and specificities of interactions.

Previous studies found that G-proteins, specifically synaptic vesicle-associated

G-protein Gαo2, regulate DA uptake into synaptic vesicles mediated by VMAT2 (Ahnert-

Hilger et al, 1998). In 2006, it was found that VMAT2 mediated vesicular monoamine

uptake was driven by the amount of monoamines within vesicles (Brunk et al., 2006).

Subsequent site-directed mutagenesis studies revealed that the first luminal domain of

VMAT2 functioned as a monoamine sensor regulated by Gαo2 subunits which was

ultimately responsible for G-protein down-regulation. Due to the relationship between

DAT mediated DA reuptake and VMAT2 driven vesicular DA uptake, it was suggested

that DAT could be regulated by G-proteins (Garcia-Olivares et al., 2013). In fact, G-

proteins and other modes of protein-protein interactions have been identified as being

involved in the regulation of the dopamine transporter thus affecting DAT function and

23

DA release or reuptake (Eriksen, Jørgensen, & Gether, 2010; J. D. Foster, Cervinski,

Gorentla, & Vaughan, 2006; Garcia-Olivares et al., 2013).

In order to determine that DAT was regulated by G-proteins, Garcia-Olivares et

al., conducted a series of biochemical and functional experiments. Initially, a physical

interaction between DAT and G-proteins was found through co-immunoprecipitation.

More specifically, using three different approaches - two DAT epitope antibodies,

striatum versus cerebellum and DAT-KO striatum, and stable expression of hDAT in

heterologous cells lines (MN9D and HEK293) – it was found that DAT antibodies co-

precipated Gβ, thus determining the interaction between DAT and Gβγ subunits(Figure

1-5) (Garcia-Olivares et al., 2013). Co-immunoprecipitation studies using a Gα pan

antibody revealed that there was no interaction between Gα and DAT (Jennie Garcia-

Olivares et al., 2013).

It was then critical to determine the location at which Gβγ interacts with DAT.

GST-fusion pull-down assays with three purified GST-fusion proteins of the amino

terminus (N-terminal), first intracellular loop, and carboxy-terminus (C-terminal) revealed

that interaction between Gβγ and DAT occurred at the C-terminal of the DAT protein

(Figure 1-6) (Garcia-Olivares et al., 2013).

To examine the effect of G-proteins on DAT function, Gα and Gβγ were

overexpressed in HEK293 cells stably transfected with DAT (Garcia-Olivares et al.,

2013). Gα i2 overexpression had no effect on DAT function, however overexpression of

Gβ1γ2, Gβ4γ2, and Gβ5γ2 were each found to cause a reduction of [3H]-DA uptake

(Garcia-Olivares et al., 2013). These results revealed two things: one that Gβγ

overexpression reduced DAT function and two that this effect was not specific to any Gβ

24

isoforms which exist in the brain (Garcia-Olivares et al., 2013). In addition, the effect of

endogenous Gβγ regulation on DAT function was tested in native systems in which Gβγ

was activated by the peptide mSIRK (Garcia-Olivares et al., 2013). The mSIRK peptide

used was a synthesized cell-permeable myristoylated peptide which was found to

specifically bind to Gβγ subunits (Smrcka et al., 2003; Smrcka et al., 2008). Upon

binding to the Gβγ subunit, mSIRK is believed to release Gβγ thus exposing the

signaling surfaces (Smrcka et al., 2003; Smrcka et al. 2008). Experiments in striatal

synaptosomes and in vivo in the striatum respectively revealed a dose-dependent

inhibition of DAT function with mSIRK (Garcia-Olivares et al., 2013). On the other hand,

incubation with scb-SIRK showed no effect of the uptake function of DAT (Figure 1-7).

Altogether, this study concluded that Gβγ subunits act endogenously to regulate DAT

function and maintain DA homeostasis (Garcia-Olivares et al., 2013).

To further determine the role of Gβγ in DAT regulation, specifically in DAT efflux,

Garcia-Olivares et al., expanded upon the previous findings. Initially, amperometry was

performed on CHO and CHO-DAT cells after application of mSIRK(10 μM), scr-SIRK,

and cocaine (20 μM) followed by mSIRK (10 μM) (Garcia-Olivares et al., 2017).

Treatment with mSIRK (10 μM) in CHO-DAT cells resulted in a greatly increased

amplitude, indicative of an increased DA current (Garcia-Olivares et al., 2017). The

scb-SIRK (10 μM), cocaine (20 μM) and mSIRK (10 μM), and CHO cells with mSIRK

(10 μM) did not display an oxidative DA currents (Figure 1-8) (Garcia-Olivares et al.,

2017). The results indicated that mSIRK activation of Gβγ subunits induces DA efflux

through DAT (Garcia-Olivares et al., 2017).

25

In a functional experiment, an efflux assay was run using 0.02 μM [3H]-DA; the

data confirmed the amperometric results (Garcia-Olivares et al., 2017). A time-

dependent treatment of CHO-DAT cells with mSIRK (20 μM) induced efflux reaching

saturation at 10 minutes, while scb-mSIRK (20 μ M) and control resulted in only basal

release of [3H]-DA (Garcia-Olivares et al., 2017). Primary cultures of rat mesencephalic

DA neurons were used to test the dose dependent effect of mSIRK in native systems

(Garcia-Olivares et al., 2017). Consistent with previous experiments, mSIRK induced a

significant amount of efflux relative to scb-mSIRK and control, but the effect of mSIRK

was inhibited by pretreatment with the Gβγ inhibitor Gallein (10 μM) and the DAT

blocker GBR 12935 (0.5 μM) (Figure 1-9) (Garcia-Olivares et al., 2017).

Since it was previously determined that Gβγ directly interacts with the C-terminal

of DAT, the next step was to determine the precise region within the C-terminal at which

this interaction occurs (Garcia-Olivares et al., 2017). Through a series of in vitro binding

assays using synthesized peptides, it was determined that the interaction took place at

the amino acid sequence S582 through A596 (Garcia-Olivares et al., 2017). More

specifically, alanine substitutions to residues 587-590, residues 583–586, and residues

591– 594 in DATct1 were done to determine the precise Gβγ-binding site (Garcia-

Olivares et al., 2017). It was found that only the mutations at residues 587-590

eliminated the interaction between DAT and Gβγ; mutations at residues 583-586 and

residues 591-594 showed no effect (Figure 1-10) (Garcia-Olivares et al., 2017).

Ultimately the findings of this recent study in heterologous cell lines revealed the role of

Gβγ in DAT modulation via activation of Gβγ by a peptide mSIRK (Garcia-Olivares et

al., 2017). It was found that mSIRK activation of Gβγ was able to induce release of DA

26

through DAT and suggest that the reverse function of DAT, efflux, may be intrinsic and

an mechanism by which physiological dopamine homeostasis is maintained (Garcia-

Olivares et al., 2017).

Hypothesis

Previously, it was determined that activation of Gβγ subunits inhibited DAT-

mediated DA uptake (Garcia-Olivares et al., 2013). Furthermore, it was found that the

peptide mSIRK led to the activation of the Gβγ subunits which bound to residues 587-

590 on the C-terminus of DAT and induced DA efflux through DAT (Garcia-Olivares et

al., 2017). Based on these findings, it was essential to determine the role that residues

587-590 (FREK) played in the interaction between Gβγ subunits and DAT (Figure 1-11).

It was hypothesized that the Gβγ binding motif within the C-terminus of DAT promotes

DA efflux through DAT and that changes to the residue(s) (587-590) of Gβγ binding

motif could alter DA efflux through DAT.

Aims

In order to address the proposed hypothesis, the study presented focused two

specific aims.

Aim 1: to define the residues within the Gβγ binding motif (FREK) that play a role in DAT function. This was addressed using site-directed mutagenesisto create alanine substitutions at each residues site generating four mutant DATs. These mutants were transfected along with the DAT wildtype (WT) in Chinese hamster ovarian (CHO) cells and biochemical and functional uptake and efflux experiments were run.

Aim 2: to determine the impact any of the mutantions had on the physical interaction between Gβγ subunits and DAT using immunoprecipitation and co-immunoprecipitation techniques.

27

Figure 1-1. The two primary pathways of the dopamine system are the mesolimbic

(light blue) and nigrostriatal (yellow) pathways. The dopaminergic neurons of the mesolimbic pathway begin at the ventral tegmental area (VTA) and project to the amygdala, hippocampus, nucleus accumbens (NAcc), hypothalamus, prefrontal cortect (PFC), and the olfactory tubercles. The mesolimbic pathway is responsible for mood, reward, and cognitive function. The nigrostriatal pathway initiates at the substancia nigra pars compacta (SNc) and projects to the dorsal striatum. The nigrostriatal pathway is involved in the regulation of locomotor activity and movement.

28

Figure 1-2. Psychostimulants target DAT and disrupt dopamine regulation. A) Cocaine

has been identified as a DAT blocker, preventing DA from being reuptaken, and increasing the duration and amount of DA present in the synaptic cleft (Amara & Sonders, 1998). B) Amphetamine acts as a substrate for DAT and competes with DA for entry into the presynaptic neuron. The mechanism of AMPH is complex, but it is predominantly understood to decrease vesicular stores of DA, increase cytosolic DA, and act on DAT to promote reverse transport – efflux (Sulzer et al., 1995).

A

Cocaine

B

Amphetamine

29

Figure 1-3. Dopamine neurotransmission. The process of DA neurotransmission begins

with synthesis of DA from tyrosine to L-DOPA to DA. These conversions are catalyzed respectively by the enzymes tyrosine hydroxylase and L-amino acid decarboxylase (AADC). VMAT2 facilitates DA incorporation into vesicles which are then fused to the plasma membrane as the result of action potential depolarization. DA is then released into the synaptic cleft where is activates psosynaptic receptors. Extracellular DA is reuptaken by DAT and D2-Receptors regulate DA release via feedback mechanisms. Catechol-O-methyltransferase (COMT) or by monoamine oxidase (MAO) metabolize excess DA.

30

Figure 1-4. Dopamine transporter (DAT) structure. The DAT is a 12 transmembrane

protein comprised of an N-terminus, 6-extracellular loops, 5-intraceullar loops, and a C-terminus. A) The 2-dimmensional primary structure of hDAT shows each of the 620 amino acid residues (Giros and Caron, 1993; Lene Norregaard et al. EMBO J. 1998; 17:4266-4273). B) The figure on the left is a schematic view of the 12 transmembrane domains and the triangles indicate folds. The figure on the right is an X-Ray crystal structure of DAT with DA bound to it [PDB code 4XP1 (Wang et al., 2015)]. The colors of the α-helices correspond with the colored transmembrane domains from the image on the left (Grouleff, 2015).

A

B

31

Figure 1-5. Physical interaction between DAT and Gβγ confirmed through co-

immunoprecipitation using different methods. A) IP-IgG rat antibody was used as a negative control and two different DAT antibodies were tested. DAT and Gβγ interaction was observed most strongly with the DAT1 antibody. B) Co-IP using DAT-KO striatum and cerebellum as negative controls identified DAT and Gβγ interaction only in WT rat striatum. C) Co-IP of DAT and Gβγ observed in heterologous cell lines stably expressing hDAT, MN9D-DAT, with mock transfected cells as controls. D) The same results in (C) were observed using HEK293-DAT cells (Garcia-Olivares et al., 2013).

Figure 1-6. GST-fusion pull-down assays indicate that Gβγ binds only to the DAT C-

terminal (DATC). A) Assay performed using mouse striata. B) Assay performed using purified Gβγ from bovine brain (Garcia-Olivares et al., 2013).

B A

A B

C D

32

Figure 1-7. Activation of Gβγ subunits reduces DAT activity in striatal synaptosomes. A)

[3H]-DA (final concentration, 0.1 µM) was added to each sample and allowed to preload for 8 min. Increasing concentrations of mSIRK and scb-SIRK were then added and samples were pre-incubated for 10 min at 34°C. B) Treatment with vehicle or 5 µM mSIRK was used to perform an uptake assay and a kinetic analysis of the synaptosomal [3H]-DA uptake, n = 3. **p<0.01 (Garcia-Olivares et al., 2013).

Figure 1-8. mSIRK promotes DA efflux in heterologous cells through activation of Gβγ.

A) Amperometric current for CHO-DAT cells treated with mSIRK (10 μM) was highest indicating that mSIRK promotes DA efflux. B) Maximum DA currents in CHO-DAT cells show a significant increase in the oxidation of DA, representative of DA efflux, in mSIRK (n=9) treated cells relative to scb-SIRK (n=3) and mSIRK after cocaine (n=3) treated cells (Garcia-Olivares et al., 2017).

B A

A B

33

Figure 1-9. Treatment of DA neurons with mSIRK activate Gβγ and induces DA efflux.

A) A dose-response curve using mSIRK concentrations (in μM: 0.01, 0.1, 1, 10, and 100) show an increase in [3H]-DA efflux at 10 μM and 100 μM. B) Treatment with mSIRK (10 μM) and scb-SIRK (10 μM), show results consistent with other experiments. Treatment of mSIRK (10 μM) in the presence of the DAT blocker GBR12935 (0.5 μM) or the Gβγ inhibitor gallein (10 μM) negate the effect of mSIRK and prevent efflux (Garcia-Olivares et al., 2017).

A

B

34

Figure 1-10. Characterization of residues in the Gβγ binding motif within the C-terminus

of DAT. A) Schematic of the amino acid residues of the C-terminus of hDAT. B) Visual representation of the in vitro binding assay used to determine the effect of alanine substitutions in the amino acid residues (583-586, 587-590, and 591-594) on Gβγ interaction with DAT. C) DATCt peptides and mutated peptides were immobilized on an avidin-coated microplte and purified Gβγ was added to determine the relative interaction between the peptide and Gβγ. Bound Gβγ was detected using the following: a Gβ pan-antibody, Protein A-HRP-conjugated antibody and QuantaBlu kit was used. It was found that alanine substitutions to the 587-590 region resulted in no interaction between that peptide and Gβγ. All results are shown as relative binding to group without Gβγ (n=3). *P<0.05, **P<0.01(Garcia-Olivares et al., 2017).

A

B

C

35

Figure 1-11. A schematic representation of the effect of Gβγ subunit activation and

binding to DAT. The illustration on the left shows that Gβγ subunit activation inhibits the uptake function of DAT. The illustration on the right shows that this Gβγ subunit activation and binding to the residues 587-590 (FREK) on the C-terminus of DAT induces DAT-mediated DA efflux.

In

Out

Gβγ N

C

UPTAKE

In

Out

Gβγ N

C

EFFLUX

Gβγ

36

CHAPTER 2 MATERIALS AND METHODS

Cell Culture, Transfection, and Treatments

Chinese hamster ovary (CHO) cells were purchased from American Type Culture

Collection. CHO cells were cultured in Ham's F-12 medium supplemented with 10%

fetal bovine serum (FBS), 1 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml

streptomycin. The human DAT cDNA was cloned into pEYFP-C1 vector and used to

transfect CHO cells with Lipofectamine 2000 (Invitrogen). For stable transfections, DAT-

expressing single clones were selected with G418 (Gibco), verified by DAT immunoblot,

and maintained in Ham’s F-12 media containing 0.5 mg/ml G418. Site–directed

Mutagenesis (QuickChange IITM) was used to create single amino acid changes

(alanine substitutions) on the DAT C-terminal region between residues 587-590 (FREK)

and DAT function was analyzed. Dopamine uptake and efflux experiments were

conducted 24–48 hr after transfection in 24-well plates. CHO-DAT cells were plated in

24-well plates coated with poly-d-Lysine. Cells were incubated with different drug

conditions (500 μL) to test the effects on DAT-mediated uptake and efflux.

Concentrations of amphetamine ranging from 0.1-100 μM, mSIRK concentrations

ranged from 1.0-100 μM (myr-SIRKALNILGYPDYD) (EMD Chemicals), scr-mSIRK 40

μM (myr-SLYRLISLAPRGDYD) (NeoBioScience), and mSIRK + GBR 10 μM were

prepared from individual 10 mM stocks in efflux buffer (-Ca2+). The control cells were

incubated with efflux buffer treated with 16 μL of 0.1% DMSO or simply efflux buffer, if

not testing with mSIRK. The efflux buffer was prepared using the same quantities and

components as for the uptake buffer however there was no CaCl2 added.

37

[3H]-DA Efflux Assay

The conditions to test DAT-mediated efflux in CHO-DAT cells have been

described previously (Garcia-Olivares et al 2017). Briefly, cells plated in 24-well plates

were loaded (20 min, 37 °C) with 250 μL of 0.06 μM of [3H]- (3,4-[7-3H]

dihydroxyphenylethylamine) (DA, 27.8 Ci/mmol; Perkin Elmer, Waltham, CA, USA) in

uptake buffer (4 mM Tris base, 6.25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.25 mM

CaCl2, 1.25 mM MgSO4, 0.57 mM ascorbic acid, 5.6 mM glucose, 1 mM tropolone, pH

7.4). After loading with [3H]-DA, cells were washed with 1mL ice-cold efflux buffer (4

mM Tris base, 6.25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 0.57 mM

ascorbic acid, 5.6mM glucose, 1mM tropolone, pH 7.4), and incubated at 37°C with 500

μL of efflux buffer in the absence or presence of different drugs. After 10 min incubation,

the released [3H]-DA was collected from the extracellular medium and transferred to

scintillation vials filled with 4mL scintillation counting fluid (RPI Bio-safe IITM), and

counts per min (cpm) were obtained using a LS-Counter scintillation counter (Beckman

Coulter, Brea, CA, USA). Additionally, efflux experiments with CHO cells expressing

either hDAT WT or mutated hDAT with an alanine substitution at the C-terminal

residues 587-590 (FREK) were performed to test the role of the C-terminal FREK region

on DAT efflux function. In each plate, [3H]-DA Efflux measurements were normalized to

the total [3H]-DA uptake after the 20min preload period, and the fractional [3H]-DA efflux

([3H]-DA efflux / [3H]-DA uptake) was used to study the functional effects of mutations

on C-terminal of DAT. Efflux is expressed as a percentage, relative to baseline levels of

extracellular [3H]-DA in the absence of treatment.

38

[3H]-DA Uptake Assay – Function and Kinetics

Unlabeled DA and [3H]-DA were used to determine the uptake functionality and

kinetics of DAT WT and the DAT mutants (587-590) transfected in CHO cells.

Approximately 24-48 hr after transfection, 5 μL of increasing concentrations of

unlabeled DA (0.0 µM, 0.1 µM, 0.3 µM, 1.0 µM, 3.0 µM, 10.0 µM, and 30.0 µM) were

added to cells in a 24-well plate. The unlabeled DA was prepared in uptake buffer via

serial dilutions beginning with the stock of 26.4 mM (5 mg DA/ 1 mL uptake buffer). The

nonspecific uptake was determined using the prepared stock of 26.4 mM unlabeled DA.

The cells were incubated for 5 min with the unlabeled DA after which 50 μL of [3H]DA

(3,4-[7-3H] dihydroxyphenylethylamine) (stock 35.6 μM; 27.8 Ci/mmol; PerkinElmer)

were added to the side of each well and left to preload for 5 min. Cells were washed

with 1 mL of 1X wash buffer (190 mL MQH2O, 10 mL 20X wash buffer: 80 mM Tris

base, 125 mM HEPES, 2.4 M LiCl, 100 mM KCl, pH 7.4), were solubilized in 0.4 mL of

1% SDS, and incubated at room temperature for 1 hr. The radioactivity was then

measured by liquid scintillation counting (Beckman Coulter, Brea, CA, USA).

[3H]-DA Uptake Assay for Efflux Normalization

[3H]-DA uptake measurements were used to normalize DA efflux relative to the

total amount of intracellular DA after the 20min preload period. Briefly, after the 20min

preload with [3H]-DA, CHO cells transfected with hDAT WT or mutated hDAT (residues

587-590) in a 24-well plate were washed with 1mL ice-cold efflux buffer and 4 wells

were solubilized in 0.4 mL of 1% SDS and incubated at room temperature for 1 hr. The

remaining 20 wells were used in the [3H]-DA efflux experiments. After incubation, the

solution containing the intracellular [3H]-DA was collected and transferred to scintillation

vials filled with 4mL scintillation counting fluid (RPI Bio-safe IITM), and counts per min

39

(cpm) were obtained using a LS-Counter scintillation counter (Beckman Coulter, Brea,

CA, USA).

Lysate from Adherent Cells and Protein Quantification Assay (Using Bio-Rad Dc Assay)

After CHO cells were transfected and were incubated for 72-96 hr until they

reached confluence. Media was aspirated and the plates were placed on ice. The cells

were washed three times with Buffer D (20 mM HEPES, 125 mM NaCl, 10 mL 10%

Glycerol, 1 mM EDTA, 1 mM EGTA, pH 7.6). Homogenization buffer was made by

adding 100X protease inhibitor diluted to 1X in Buffer D (1:1000 μL). Homogenization

buffer was added to each plate (500 μL) and cells were scraped off the plate into the

homogenization buffer. A stock of 10% Triton X-100 in PBS (50 μL) was added to a final

concentration of 1% to each plate. The homogenate was then pipetted into centrifuge

tubes and incubated with rotation for 1-2 hr at 4°C. Insoluble material was removed by

centrifugation at 16,000 x g for 15 min 4°C. The supernatant was then saved in

separate centrifuge tubes. Samples are stored at - 80°C

All solutions and the assay were respectively prepared and performed at room

temperature. Samples were taken out of the -80°C and left to thaw on ice. Solution A’

was prepared using 20 μL of Bio-Rad Dc Assay Reagent S and 980 μL of Bio-Rad Dc

Assay Reagent A. The total amount of Solution A’ needed was determined by adding

the number of samples plus the number of standards and multiplying by 25 μL. The

Solution A’ was heated to 37.5 °C in a dry bath. In a 96-well plate, 5 μL of the BSA

Standards (in mg/mL: 0, 0.156, 0.3125, 0.625, 1.2, 2.5, and 5) were added in duplicates

and 5 μL of each sample were also added in duplicates. Solution A’ (25 μL) was added

to each sample well followed by Bio-Rad Dc Assay Reagent B (200 μL). The samples

40

were then incubated for 15 min at room temperature. The samples were read at 750 nm

on the Spectrophotometer. Protein concentrations obtained can then be used for other

experiments.

Biotinylation Assay

CHO cells were washed with PBS and then incubated with gentle agitation for 30

min at 4°C with 1 ml of 1.5 mg/ml sulfo-NHS-SS-biotin prepared in Biotinylation buffer

(in mM: 150 NaCl, 2 CaCl2, 10 triethanolamine, pH 7.8). The reaction was quenched by

incubating the cells for an additional 10 min with 50 mM glycine in PBS. Cells were then

washed with PBS and incubated at 4°C for 1 hr with Lysis Buffer D (20 mM HEPES, 125

mM NaCl, 10 mL 10% Glycerol, 1 mM EDTA, 1 mM EGTA, pH 7.6) with added

Tritonx100 1% and protease inhibitor (PI) 1X. The cell samples (500 μL) were then

divided into three aliquots: 100 μL one for protein measurement, 80 μL for SDS page

total fraction sample, and the remaining 320 μL for biotinylation avidin purification. The

biotinylation aliquot was treated with ultralink-immobilized avidin beads to determine the

surface membrane proteins (Pierce). The biotinylation protein lysate was incubated for 1

hr at 4°C with 40 μL of the avidin beads. Samples were subsequently centrifuged at

2500 g for 2 min, supernatant was discarded, and beads were washed with Lysis Buffer.

Sample Buffer (SDS-4X LB) 20 μL was added to the total protein fraction and Sample

Buffer (SDS-2X LB) 40 μL was added to the biotinylated protein sample in order to

analyze protein expression by SDS-page 10% and Western blotting.

Western Blot

The protein samples (40 μg) were run in an SDS-page 10% gel electrophoresis

and subsequently transferred to nitrocellulose or PVDF membranes. The membrane

was blocked for 1 hr with 5% milk in TBST (Tris/TrisHCl 25mM, NaCl 0.13M, KCl

41

0.0027M, 0.2% Tween20). To identify DAT, the anti-DAT MAB369 (rat) antibody was

used (1:1000, 2 hr at room temperature, Millipore). Membranes were washed with TBST

3 times by hand and 3 times for 5 minutes with agitation. An α-Rat HRP-conjugated

secondary antibody (1:5000, 1 hr at room temperature) (Jackson Immunoresearch Lab)

was used to detect α-DAT. Membranes were washed with TBST 3 times by hand and 3

times for 5 minutes with agitation. Immunoreactive bands were visualized using Clarity

Western ECL Substrate (1705060, Bio-Rad Laboratories) and Clarity Max Western ECL

Substrate (1705062, Bio-Rad Laboratories) and were exposed for increasing amounts

of time to obtain the highest quality image.

Co-Immunoprecipitation

CHO cells transfected with DAT WT and the DAT R588A mutant were lysed

using Lysis Buffer D (20 mM HEPES, 125 mM NaCl, 10 mL 10% Glycerol, 1 mM EDTA,

1 mM EGTA, pH 7.6) containing Protease Inhibitor Cocktail Set I diluted to 1X (53913,

Calbiochem: 500 μM AEBSF, 500 μM Hydrochloride, 150 nM Aprotinin, 150 nM Bovin

Lung, 150 nM Crystalline, 1 μM E-64 Protease Inhibitor, 0.5 mM EDTA, 0.5 mM

Disodium, 1 μM Leupeptin, 1 μM Hemisulfate). After homogenization, 1% Triton X-100

was added, to each sample. Cell lysate samples were then and centrifuged at 16000g

for 15 min at 4°C to remove cellular debris. Protein concentration was determined using

the Dc Protein Assay kit (Bio-Rad Laboratories). Then 50 μL of the each lysate was

saved separately for later use as the total input. Three different antibodies were used for

immunoprecipitations (IP): DATC (431-DATC, PhosphoSolutions), DATEL2 (434-

DATEL2, PhosphoSolutions), and HA (ab9100, abcam). A fourth antibody normal rabbit

IgG (sc-2027, Santa Cruz Biotechnology) was used as a negative control. The IP

antibodies were added to each respective lysate (0.5 ml at 1 mg/mL; 500 μg total) at a

42

dilution of 1:200. Cell lysates were incubated for 20 h with rotation at 4°C. Protein A and

Protein G-coupled Sepharose beads were washed 3 times with Buffer D containing PI

1X and subsequently centrifuged at 2500g for 2 min. The Protein A and Protein G-

coupled sepharose beads were then added to each IP lysate sample and sample beads

were incubated for 2 hr with rotation at 4°C. Sepharose beads were washed 3 times

with Buffer D containing PI 1X and 1% Triton X-100 and then centrifuged at 2500g for 2

min, carefully removing the supernatant per wash. Sample Buffer 4X (40 μL) was then

added to each sample following removal of the supernatant. Prior to loading the entire

sample volume (40 μL) into an SDS-Page 10%, they were incubated for 30 min at 37°C

and centrifuged at 10000g for 2 min. Immunoblotting was performed with an anti-DAT

antibody (Mab369, Millipore) following the previously stated western blot method. Co-

immunoprecipitation of Gβ was done using the Gβ pan-antibody (T-20, Santa Cruz

Biotechnology).

43

CHAPTER 3 RESULTS

Establishing the Conditions to Measure DAT-Mediated Efflux in Heterologous Cells

In order to confirm the functionality of DAT efflux in DAT wild-type (WT) transient

cell lines, efflux assays were run under variable conditions. The values of [3H]-DA

released were analyzed using a P<0.05 ANOVA with Tukey’s multiple comparison test

and the results are expressed as the mean ± SEM (N= 12) (Fig. 3-1). Five different

conditions were tested: control, 40 μM AMPH, 40 μM mSIRK, 40 μM scr-mSIRK, and 10

μM mSIRK + GBR. Amphetamine 40 μM, mSIRK 40 μM (myr-SIRKALNILGYPDYD)

(EMD Chemicals), and scr-mSIRK 40 μM (myr-SLYRLISLAPRGDYD) (NeoBioScience)

were prepared from individual 10 mM stocks (16 μL in 4 mL efflux buffer (-Ca2+). The

control cells were incubated with efflux buffer treated with 16 μL of 0.1% DMSO.

Consistent with findings of previous studies, there was a significant difference of [3H]-

DA released respective to the control condition as compared to the treatment with

AMPH and mSIRK. There was no significant difference in the amount of [3H]-DA

released when comparing the treatments with AMPH and mSIRK. The conditions of

treatment with the negative control scr-mSIRK and mSIRK with the DAT inhibitor GBR

(mSIRK+GBR) showed no significant difference in [3H]-DA release compared to the

control and observed values were, in fact, similar. [3H]-DA release induced by treatment

with mSIRK was significantly greater than with scr-mSIRK or mSIRK + GBR.

44

Effect of Alanine Substitution of hDAT Residues 587-590 (FREK) on DAT-mediated DA Efflux

The effect of site-directed mutagenesis on DAT mediated DA efflux was

examined using the same conditions described for the efflux assay in only the WT cell

line. The [3H]-DA efflux values obtained were analyzed with a P<0.05 ANOVA with

Dunnett’s multiple comparison test and the results are expressed as the mean ± SEM

(N= 4). As was found with the efflux assay run on the transient WT DAT transfection, for

each mutant as well as the wildtype, the same trend was observed when comparing

efflux under control conditions relative to treatment with AMPH or mSIRK. Similarly, for

each cell line, there was a significant increase of [3H]-DA release under treatment with

mSIRK compared to both the scr-mSIRK and the mSIRK + GBR conditions (Figure 3-2).

Not only were there expected differences among the different treatment conditions, but

there was also a notable difference in [3H]-DA efflux when comparing each cell line.

Relative to the WT, the F587A and E589A mutants appeared to have similar [3H]-DA

efflux, suggesting that these residues are not implicated in DA efflux. The R588A and

K590A mutant lines, however, had a notable reduction of [3H]-DA efflux compared to the

WT as well as the F587A and E589A mutants.

Expression of DAT Wt and Mutants in Stable Cell Lines

Efflux assays were previously run in the presence or absence AMPH and mSIRK

to induce [3H]-DA to determine the effect of alanine subsitutions at each residue in the

DAT C-terminus region 587-590 (FREK). Results from these experiments showed that

AMPH and mSIRK both induced efflux. The F587A and E589A mutants showed higher,

but similar levels of efflux compared to the WT, but the R588A and K590A mutations

resulted in a significant reduction of [3H]-DA efflux. Based on these observations we

45

proposed three possible reasons for the recuction of efflux: first, the efflux defective

mutants were not expressed at the membranes of transfected CHO cells, secondly, the

mutants were expressed at the membrane, but were incapable of uptaking [3H]-DA, or

third, the mutants were expressed and able to uptake [3H]-DA but could not efflux. In

order to distinguish between these three possible explanations for the efflux results

observed biotinylation assays followed by immunoblotting were performed.

Chinese hamster ovarian cells were stably transfected with the DAT WT, F587A,

R588A, E589A, and K590A. The cells were lysed and prepared according to the

biotynilation methods detailed earlier. Both biotynilated and total fractions of the cell

lysates were run on an SDS-page 10%. The expression of DAT proteins were detected

following the western blotting methods and using the anti-DAT antibody MAB 369

(Millipore). The intensity of revealed immunoreactive bands provided insight to the level

of expression of each DAT type. A sample of CHO cells not transfected was used as a

negative control. Transfected DAT was labeled with YFP to visualize transfection of

cells using blue fluorescent light, therefore the immunoreactive bands were observed at

100 kDa rather than 75 kDa where DAT is typically seen. In both biotinylated and total

protein fractions, the DAT WT band was more intense which was indicative of higher

DAT WT protein expression at the cell membrane and among the total proteins (Figure

3-3). The DAT F587A and DAT R588A bands were less intense than the DAT WT which

revealed that there was less expression in the cells. The specific difference between the

bands was not quantified. There were weaker bands observed for the DAT E589A

mutant which suggests that this DAT mutant was not strongly expressed at the surface

of the cell membranes. Surprisingly, no band was present in the lanes where the DAT

46

K590A was loaded, suggesting that the DAT K590A mutant was not expressed at the

membrane. However, there were bands seen around 60 kDa in the biotinylated and

total protein fractions for only the K590A mutant. The presence and location of these

immunorective bands suggested that the K590A mutation could affect DAT maturation

or that it is trafficking defective. This result provided a reasonable explanation for the

basal levels of efflux observed in the DAT K590A even when treated with AMPH and

mSIRK.

Effect of Alanine Substitution of hDAT Residues 587-590 (FREK) on DA Uptake

Following efflux assays and determination of DAT expression at the surface of

the cell membrane through biotinylation assays for each DAT type, it was determined

that the K590A mutant did not efflux [3H]-DA and was not expressed at the cell

membranes. The F587A, R588A, and E589A mutants were all found to be expressed at

the membrane, thus leaving two questions unanswered. Knowing that these mutants

were expressed, did the mutations affect the ability to uptake [3H]-DA and if not, does

the mutation affect the ability of DAT to efflux? In order to test the uptake function of the

DAT mutants, uptake assays were performed on CHO cell lines expressing stable

transfections of the DAT WT and DAT 587-590.

Initially, uptake experiments to determine the uptake function and kinetics of DAT

WT and DAT mutants were done in a dose-dependent manner with unlabeled DA. The

increasing concentrations of unlabeled DA (0.0 µM, 0.1 µM, 0.3 µM, 1.0 µM, 3.0 µM,

10.0 µM, and 30.0 µM; non-specific 26.4 mM) were preloaded into cells for 5 min

followed by a 5 min preload with [3H]-DA (100 nM). The unlabeled DA and [3H]-DA

competed for entry into the cells through the DAT present resulting in data resembling a

Michaelis-Menten kinetic saturation curve. Following preload, the cells were washed

47

and solubilized using 1% SDS and incubated for 1 hr. The amount of [3H]-DA uptaken

was determined as CPM using LS-Counter scintillation counter (Beckman Coulter, Brea,

CA, USA). The results obtained show the transfected DAT WT was able to maximally

uptake [3H]-DA reaching saturation at 30 µM of unlabeled DA. The transfected DAT

mutants (F587A, R588A, and E589A) appeared to be reaching saturation at 10 µM

unlabeled DA and respectively uptook less [3H]-DA compared to the DAT WT. The DAT

K590A had [3H]-DA uptake levels near zero suggesting that the mutant affects the

ability of the transporter to uptake. The overall results of this uptake study indicated that

the mutations of DAT at residues 587-590 affected the amount of [3H]-DA the mutants

were able to uptake compared to DAT WT, resembling a decrease in Vmax (Figure 3-4).

These findings when compared to the DAT WT suggested that DAT F587A, R588A, and

E589A have, in order from least to greatest effect, an impact on DAT function as seen

by the reduced CPM of [3H]-DA uptake. The results of DAT K590 were consistent with

those observed from the efflux and biotinylation assays. However, because these

uptake assays were performed with only 5 min of [3H]-DA preload and in the presence

of competing unlabeled-DA the effects of the mutations on DAT uptake may not be

conclusive based on these uptake assays alone.

In order to determine the effect of the alanine substitutions at residues 587-590

on DAT function, uptake experiments using only [3H]-DA and a longer preload period

were performed. The cells were preloaded with [3H]-DA (100 nM) for 20 min and were

solubilized using 1% SDS and incubated for 1 hr after which the CPM uptaken [3H]-DA

by the DAT WT and each mutant type was determined. Results were analyzed using a

P<0.05 ANOVA with Dunnett’s multiple comparison test and expressed as the mean ±

48

SEM (N= 4) (Figure 3-5). The DAT F587A cell line showed a slight but not significant

increase in [3H]-DA uptake compared to the DAT WT. Uptake observed in the DAT

R588A was significantly less than the DAT WT, but the [3H]-DA CPM values were

relatively similar. In contrast, the DAT E589A mutant lines showed a significant

reduction of preloaded [3H]-DA uptake compared to the DAT WT. Concurrent with the

results of the efflux and biotinylation assays, the DAT K590A cells showed essentially

no [3H]-DA uptake comparative to the DAT WT and DAT F587A, R588A, and E589A

mutants.

The results of the uptake assay determined that the mutation of F587A had no

effect on the uptake function of DAT while there was no uptake observed with the DAT

K590A. The uptake results of the K590A correlate with the lower value of [3H]-DA

release under both AMPH and mSIRK conditions observed in initial efflux assays

(Figure 3-2) and the lack of DAT expression seen in the biotinylation immunoblot

(Figure 3-3). The results of [3H]-DA uptake seen with the DAT R588A mutant

suggested that this mutation had an almost normal but slightly affected uptake function.

These findings indicated that the reduction of efflux previously observed was a result of

the mutation and not a lack of [3H]-DA uptaken into the cells. On the other hand, the

DAT E589A mutant showed that it had a significant reduction of uptake compared to the

DAT WT, suggesting the mutation may affect uptake function, but may enchance the

efflux ability of DAT based of the efflux results (Figure 3-2). The amount [3H]-DA

uptaken by the cells was later used to normalize the amount of [3H]-DA effluxed as a

fraction over total amount uptaken for the DAT WT and each DAT mutant (F587A,

R588A, and E589A).

49

[3H]-DA Efflux Induced by AMPH and mSIRK in Cell Lines Expressing DAT WT, DAT F587A, DAT R588A, and DAT E589A

The previous uptake and efflux assay results indicated that the DAT K590A

mutant did not uptake [3H]-DA, thus there was essentially no efflux to be observed.

These findings were supported by the biotinylation experiments which revealed that

DAT K590A was not expressed at the cell surface, however a band was observed at a

lower molecular weight suggesting that the K590A mutation was trafficking defective.

Based on these results, the DAT K590A mutant was excluded from further experiments

since no efflux would be able to be observed. The previous results showed that the DAT

F587A mutant was able to efflux and uptake [3H]-DA at slightly higher but similar

amounts as the DAT WT. The DAT R588A mutant displayed a significant reduction of

[3H]-DA efflux compared to the DAT WT, yet its uptake was significantly different but not

much lower than that of the DAT WT. Lastly, the E589A mutant expressed opposite

results to the R588A mutant. The E589A mutant had slightly higher but similar [3H]-DA

efflux results relative to the DAT WT, but it had significantly less uptake of [3H]-DA. In

light of these results, it was decided that the sensitivity of DAT efflux to these mutations

should be observed at different concentrations of AMPH and mSIRK. To determine the

more specific effects of these mutants of [3H]-DA efflux through DAT, efflux assays were

run on CHO cells transfectedwith the DAT WT, F87A, R588A, and E589A mutants. The

cells were treated with AMPH and mSIRK in a concentration-dependent manner. The

different concentrations of AMPH, in μM, used were 0.0, 0.1, 1.0, 10, and 30 and the

concentrations of mSIRK, μM, of 0.0, 1.0, 10, 30, and 100. Cells were incubated in the

presence or absence of each condition for 10 minutes and a row of wells was left only

for [3H]-DA preload uptake.

50

The results for the WT and each mutant type are expressed as fractional [3H]-DA

efflux where by the CPM of [3H]-DA efflux was divided over the CPM of [3H]-DA uptaken

by the cells. The data was analyzed by a two-way ANOVA with Sidak's multiple

comparisons test and the results are expressed as the mean ± SEM (N= 6). When

treated with increasing concentrations of AMPH, no difference was observed between

the DAT WT and DAT F587A (Figure 3-6). The treatment of mSIRK on DAT WT and

DAT F587A showed that there was similar efflux between the two, except at the

concentration of 30 μM. However, at 100 μM mSIRK there was no significant difference

between the DAT WT and F587A. These results suggested that the DAT F587A does

not have a negative effect on DAT-mediated efflux, but may require more stimuli to

promote Gβγ-mediated DA efflux through DAT.

The DAT R588A mutation appeared to have an effect on efflux consistent with

previous results (Figure 3-7). There was a significant decrease of [3H]-DA efflux in DAT

R588A cells compared to DAT WT when treated with AMPH at concentrations of 1.0

μM, 10 μM, and 30 μM. When treated with mSIRK, there was also an observed

reduction of [3H]-DA efflux in the R588A cells compared to the WT cells most notably at

higher concentrations (30 μM, and 100 μM). Comparision of the efflux results between

the AMPH and mSIRK concentration-dependent treatments indicated that there was

observably a larger reduction of [3H]-DA efflux when the DAT R588A cells were treated

with mSIRK. Due to these results, it could be suggested that DAT R588A affects the

DAT efflux function and may likely affect the interaction between DAT and Gβγ since

less efflux was observed with the Gβγ activator mSIRK.

51

The efflux assays run using CHO cells expressing DAT E589A under

concentration-dependent treatment with AMPH showed that there was no difference in

[3H]-DA efflux between the DAT WT and E589A mutant. Similarly, treatment with

mSIRK also illustrated that there was no difference in DAT-mediated [3H]-DA efflux

between the WT and E589A. Altogether, these concentration-dependent efflux assays

induced by AMPH and mSIRK revealed that neither the F587A nor the E589A had

significant effects on the ability of DAT to efflux [3H]-DA (Figure 3-8). Contrarily, the

R588A mutant resulted in a significant decrease of DAT-mediated [3H]-DA efflux

induced by both AMPH and mSIRK. Due to the greater efflux reduction observed with

mSIRK treatment, the R588A mutant may have an effect on the ability of Gβγ and DAT

to interact and promote DA efflux.

Immunoprecipitation of DAT and Co-immunoprecipitation of Gβ

The functional experiments revealed that the DAT mutants had varying effects on

DAT-mediated efflux. It was determined that the K590A mutant was not viable to use for

further studies because the mutation likely affected DAT transport to the membrane and

DA was not uptaken into cells, thus no efflux could occur. Treatments with AMPH and

mSIRK in the F587A and E589A mutants revealed that these mutations did not

significantly affect the DAT efflux function as both mutants had efflux results similar to

those of DAT WT under the same treatment conditions. Consistent with early efflux

studies, treatment with varying concentrations of AMPH and mSIRK showed that the

R588A mutant had significant reduction in efflux compared to the DAT WT. Based on

these findings, continuing studies focused on further characterizing the role of the R588

residue in DAT-mediated efflux using the DAT R588A mutant expressed in CHO cell

lines. To observe the effect of the R588A mutant on the physical interaction between

52

DAT and Gβγ, immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) techniques

were utilized. In order to determine which antibody would be best for

immunoprecipitation and identification of DAT and co-immunnoprecipitation of Gβ, three

different antibodies were used in initial IP experiments. CHO cells and CHO DAT WT

cells were lysed and prepared as stated in the materials and methods. The antibodies

added to each CHO and CHO DAT WT sample (0.5 ml at 1 mg/mL; 500 μg total) for the

IP (dilution 1:200) were α-DATC (431), α-DATEL2 (434), and α-HA. An IgG (rabbit)

antibody was used as a negative control and total CHO and CHO DAT protein samples

were used as the inputs. Prepared samples were eluted at 37°C for 30 min, centrifuged,

and loaded into an SDS-page 10%. Following gel electrophoresis, the proteins were

transferred onto a PVDF membrane and immunoblotting was preformed to detect DAT

and co-immunoprecipitation of Gβ. To detect DAT, the α-DAT antibody MAB 369 (rat;

dilution 1:1000) (Millipore) was used as the primary antibody and an α-Rat HRP

antibody (dilution 1:5000) was the secondary antibody. Revelation of immunoreactive

bands showed that IP with the α-DATC (431) antibody produced a higher intensity, thus

greater detection of DAT compared to the IP with α-DATEL2 (434), and α-HA. There was

no detection of DAT in the CHO samples or in the IP IgG (rabbit) samples as they were

negative controls. To detect Gβ, the Gβ pan-antibody (T-20, Santa Cruz) (dilution

1:1000) was used as the primary antibody with α-Rabbit HRP (dilution 1:5000) as the

secondary antibody. Immunoprecipitation of DAT with α-DATC (431) was able to co-

precipitate Gβ better than the other IP antibodies that were used (Figure 3-9). As a

result, further co-immunoprecipitation studies were carried out using α-DATC (431) and

53

α-DAT MAB 369 for detection of DAT and α-Gβ T-20 for co-precipitation and detection

of Gβ.

Subsequently, the effect of R588A on the interaction between DAT and Gβγ

compared to DAT WT was observed using the IP and Co-IP techniques mentioned. Two

different IP protocols were used to determine which method was most efficient: one with

a 30 min incubation period, the second with a 2 hr incubation period. CHO cell samples

not transfected with DAT WT or DAT R588A were used as a negative control as were

DAT WT samples immunoprecipitated using IgG (rabbit). CHO, CHO DAT WT, and

CHO DAT R588A lysates were immunoprecipitated using α-DATC (431) and detected

with α-DAT MAB 369 and α-Rat HRP. No signal was detected in the CHO or CHO DAT

IgG (Rb) samples. Immunoreactive bands revealed that there was a higher intensity and

greater detection of DAT in DAT WT samples compared to DAT R588A. Using the Gβ

pan-antibody (T20), Gβ was co-precipitated with more intensity in the DAT WT samples

compared to the DAT R588A (Figure 3-10). When comparing the two IP methods used

there was no observed difference in efficacy. Though there appeared to be less

interaction between Gβγ and DAT in the DAT R588A mutant, thee results are not

conclusive as they may be a result of overall less protein expression in the transfected

cells relative to DAT WT.

54

Figure 3-1. Effect of different conditions on DA efflux in transient transfection of WT

DAT. P<0.05 ANOVA with Tukey’s multiple comparison test. Results are expressed as the mean ± SEM (N= 12). [3H]-DA release shown as counts per minute CPM*10min-1*well-1 was significantly different in the control condition compared to [3H]-DA release induced by 40 μM AMPH and 40μM mSIRK. There was a significant difference in the amount of [3H]-DA released by 40 μM mSIRK compared to the conditions of scr-SIRK 40 μM and mSIRK + GBR 10 μM. Relative to the efflux of the control condition, treatment with scr-SIRK 40 μM and mSIRK + GBR 10 μM showed no difference or significant difference above basal levels.

contr

ol

AM

PH 4

0µM

mSIR

K 4

0µM

scr-m

SIR

K 4

0µM

mSIR

K+G

BR 1

0µM

0

10000

20000

30000

40000

50000

[3H

]-D

A E

fflu

x

(CP

M*1

0m

in-1

*well

-1)

CHO-DAT cells

**

55

Figure 3-2. The effect of different conditions on DA efflux in transient transfection of

hDAT WT and alanine substitution of hDAT residues 587-590 (FREK). P<0.05 ANOVA with Dunnett’s multiple comparison test. Results are expressed as the mean ± SEM (N= 4). Each DAT type exhibits a similar trend of [3H]-DA release under the various conditions. The F587A and E589A mutants display greater [3H]-DA efflux relative to the WT, though no significance is indicated. The mutations of R588A and K590A appear to have the most effect of the DAT efflux function as both show much less [3H]-DA release.

WT F587A R588A E589A K590A0

10000

20000

30000

40000

50000[3

H]-

DA

Eff

lux

(CP

M*1

0m

in-1

*well

-1)

control

mSIRK 40µM

scr-mSIRK 40µM

mSIRK+GBR 10µM

AMPH 40µM

**

*

*

**

*

*

**

56

Figure 3-3. Expression of DAT WT and DAT mutants of residues 587-590 as measured

by biotinylation and compared to total protein fractions. The primary antibody used to detect DAT was α-DAT MAB369 (rat), 1:1000, 2 hours at room temperature and the secondary antibody, to observe a signal, was an α-Rat HRP, 1:5000, 1 hour at room temperature. The blot shows varying intensities of the amount of DAT found at the cell surface. The intensities of the biotinylated fraction correspond to those of the total fraction. The DAT WT protein had the highest intensities indicative of grater expression while the DAT K590A protein displayed band intensity similar to the negative control, CHO, indicating no expression at the cell membrane.

Biotinylated Fraction

Total Fraction

150

100

75

50

57

Figure 3-4. Functionality and kinetics of DA uptake in stably transfected CHO cell lines

expressing hDAT WT and hDAT mutants (FREK). The [3H]-DA preload uptake was expressed as counts per minute (CPM)*5 min-1* well-1. The DAT WT uptake was highest and reached saturation at 30 μM. The DAT F587A and R588A showed great reduction in [3H]-DA uptake compared to the DAT WT. The DAT E589A mutant took in less [3H]-DA than both the F587A and the R588A. The DAT K590A showed no [3H]-DA uptake and was the most distinct from the DAT WT. Overall, the mutations resulted in a reduction of uptake compared to the WT. Due to the mechanism of the assay and the competition for uptake between Cold-DA and [3H]-DA the reduction of uptake observed in the DAT mutants (F587A, R588A, and E589A) resemble a decrease in Vmax.

58

Figure 3-5. The observed effect of alanine substitution of hDAT residues 587-590

(FREK) on DA uptake in stably transfected CHO cell lines. P<0.05 ANOVA with Dunnett’s multiple comparison test. Results are expressed as the mean ± SEM (N= 4). The DAT WT and DAT F587A show the highest amounts of [3H]-DA preload uptake expressed as counts per minute (CPM)*20 min-1* well-1. The DAT R588A showed a significant reduction in [3H]-DA preload uptake compared to the DAT WT. The DAT K590A had close to no [3H]-DA uptake and was the most significantly distinct from the DAT WT. The DAT E589A mutant took in less [3H]-DA than both the F587A and the R588A, however the amount of uptake is not consistent with the [3H]-DA efflux observed in transiently transfected cell lines.

WT F587A R588A E589A K590A0

20000

40000

60000

80000

100000

[3H

]-D

A p

relo

ad

up

take

(CP

M*2

0m

in-1

*well

-1)

*

****

****

59

Figure 3-6. Concentration response mediated [3H]-DA efflux induced by AMPH and

mSIRK in stable cell lines expressing DAT WT and DAT F587A. P<0.05 paired t-test. Results are expressed as the mean ± SEM (N= 6-5). Samples were pre-incubated for 20 min with [3H]-DA followed by incubation for 10 min with various concentrations of AMPH (A) or mSIRK (B) at 35°C. A) There was no significant difference in efflux induced by AMPH between the WT and F587A. B) mSIRK activation of Gβγ induced DA efflux through DAT. At 30 μM there was a significant difference in the efflux between DAT WT AND DAT F587A, but there was no difference at 100 μM.

0 1 10 30 1000

100

200

300

400

500

mSIRK [mM]

Fra

cti

on

al [3

H]-

DA

eff

lux

(% o

f co

ntr

ol)

WTF587A

*

0 0.1 1 10 300

100

200

300

400

500

AMPH [mM]

Fra

cti

on

al [3

H]-

DA

eff

lux

(% o

f co

ntr

ol)

WTF587A

A

B

60

Figure 3-7. Concentration response mediated [3H]-DA efflux induced by AMPH and

mSIRK in stable cell lines expressing DAT WT and DAT R588A. P<0.05 paired t-test. Results are expressed as the mean ± SEM (N= 6-5). Samples were pre-incubated for 20 min with [3H]-DA followed by incubation for 10 min with various concentrations of AMPH (A) or mSIRK (B) at 35°C. A) At higher concentrations of AMPH (1.0, 10.0, and 30.0 μM) there was a significant reduction of efflux in the DAT R588A cells compared to DAT WT. B) Gβγ activation via treatment with different concentrations of mSIRK induced [3H]-DA release via DAT. At 30 μM and 100 μM of mSIRK there was a significant difference in the efflux between DAT WT AND DAT R588A.

0.0 0.1 1.0 10.0 30.00

100

200

300

400

500

AMPH [mM]

Fra

cti

on

al [3

H]-

DA

eff

lux

(% o

f co

ntr

ol)

WT

R588A

*

* *

A

0 1 10 30 1000

100

200

300

400

500

mSIRK [mM]

Fra

cti

on

al [3

H]-

DA

eff

lux

(% o

f co

ntr

ol)

WT

R588A

*

*

B

61

Figure 3-8. Concentration response mediated [3H]-DA efflux induced by AMPH and

mSIRK in stable cell lines expressing DAT WT and DAT E589A. P<0.05 paired t-test. Results are expressed as the mean ± SEM (N= 6-5). Samples were pre-incubated for 20 min with [3H]-DA followed by incubation for 10 min with various concentrations of AMPH (A) or mSIRK (B) at 35°C. A) Treatment with AMPH revealed no difference in efflux between DAT WT and DAT E589A. B) Gβγ activation via treatment with different concentrations of mSIRK induced [3H]-DA efflux. Similarly to the AMPH efflux results, there was no difference between the DAT WT and DAT E589A of mSIRK induced DAT-mediated [3H]-DA efflux.

A

B

62

Figure 3-9. Immunoprecipitation of DAT and co-immunoprecipitation of Gβ using

different IP antibodies. CHO and CHO DAT cell lysates (500μg total) were prepared and incubated with different antibodies to determine which was best of immunoprecipitation of DAT. At a dilution of 1:200, the antibodies used were IgG (IgG Rabbit) as the negative control, IPDAT1 (IPDATC (431)), IPDAT2 (IPDATEL2 (434)), and IPDAT3 (IPHA). Immuoblot detection of DAT using α-DAT MAB 369 followed by α-Rat HRP revleaed that IPDAT1 was the best antibody for the immunoprecipitation of DAT. This was indicated by the greater intensity of the DAT band for IPDAT1 compared to IPDAT2 and IPDAT3 samples. The ability of these DAT antibodies to co-precipitate Gβ was determined using the Gβ pan-antibody T20. Revelation of the immunoreactive bands showed that IPDAT1 was the best DAT antibody for co-immunoprecipitation of Gβ as observed by the slightly more intense band.

250

150

100

75

37

CHO-DAT CHO

IB DAT

MAB 369

Input

IB Gβ

T20

63

Figure 3-10. Observation of the effect the R588A mutation has on physical interaction

between DAT and Gβ using IP and Co-IP techniques. CHO, CHO DAT WT, CHO DAT R588A cell lysates (500μg total) were prepared and DAT was precipitated using the antibody DATC (431). The antibody IgG Rabbit was used as a negative control as were the CHO cell samples. A) Immuoblot detection of DAT using α-DAT MAB 369 (rat, 1:1000) followed by α-Rat HRP (1:5000) revleaed that there was more DAT WT expression than DAT R588A. Gβ was then co-precipitated using the Gβ pan-antibody T20. Revelation of the immunoreactive bands showed that DAT WT was better able to co-

100

75

B

30 min IP Protocol 2 hr IP Protocol Input

250

150

100

75

50

Co-IP of Gβ-T20

A

64

immunoprecipitation Gβ than DAT R588A as observed by the slightly more intense band. These results suggest that the R588A mutation results in a lesser interaction between DAT and Gβ. B) Immunoblotting of total protein input for CHO, CHO DAT, and CHO DAT R588A showed that there was less expression of DAT WT protein in R588A samples compared to CHO DAT WT. This observation suggested that the decreased expression of co-precipitated Gβ by DAT R588A may be a result of less protein expression overall.

65

CHAPTER 4 DISCUSSION/CONCLUSION

The dopamine transporter is central to maintaining dopamine homeostasis. The

effect of psychostimulants, such as cocaine and amphetamine, on DAT revealed its

crucial role at the dopaminergic neuron terminals. In early studies, it was thought that

the role of DAT was limited to dopamine reuptake, however studies using amphetamine

revealed that DAT could also conduct reverse transport. This reverse transport, also

known as efflux, would result in the presence of more DA in the synaptic cleft, thus

leading to increased signaling and excitation of DA neurons. Amphetamine was found to

induce this process by decreasing the availability of vesicular DA and increasing

cytosolic DA driving the outward flow via DAT rather than by exocytosis. Interest in the

efflux function of DAT led to more studies and in 2013, it was found that Gβγ activation

by the peptide mSIRK reduced DAT-mediated re-uptake (Garcia-Olivares et al., 2013).

These findings prompted further studies into the effect of mSIRK activation of

Gβγ on the function of the dopamine transporter. Amperometric studies revealed that

treatment with mSIRK caused a spike in DA currents, suggesting more release of DA

(Garcia-Olivares et al., 2013). Functional efflux experiments with [3H]-DA were

completed using mSIRK (10 μM) in the presence or absence of DAT blocker GBR12935

(0.5 μM) or the Gβγ inhibitor gallein (10 μM) and scb-mSIRK as a negative

control(Garcia-Olivares et al., 2013). The data showed that treatment with mSIRK alone

resulted in a significant increase of DA efflux while efflux in the presence of GBR 12935

and gallein were at levels similar to the vehicle and scr-mSIRK (Garcia-Olivares et al.,

2017). These findings were critical in suggesting that the reverse transport of DAT is a

mechanism that could be endogenously activated and is not solely observed in the

66

presence of psychostimulants. Knowing that the interaction between DAT and Gβγ

occurred at the C-terminus of DAT (DATC1), the precise site of interaction was

characterized through a series of site-directed mutagenesis experiments (Garcia-

Olivares et al., 2017). Native amino acid residues were substituted with an alanine in

the following residue regions, 583-586, 587-590, and 591-594 (Garcia-Olivares et al.,

2017). Binding assays performed revealed that substitutions in the 587-590 residues

abolished the interaction between DAT and Gβγ, while changes to the other regions of

residues did not have any effect on the interaction (Garcia-Olivares et al., 2017).

The question then remained as to which amino acid residue(s) within the 587-

590 region were essential to the Gβγ mediated efflux through interaction with DAT. It

was hypothesized that changes to the residue(s) of the Gβγ binding motif within the C-

terminus of DAT would result in altered DAT efflux function. In order to test the

hypothesis, site-directed mutagenesis at the residues 587-590 was performed and

native residues (FREK) were individually substituted for an alanine (A). Biochemical and

functional experiments were then carried out using CHO cell lines in which the DAT WT

and each of the four generated mutants (F587A, R588A, E5899A, and K590A) were

transiently and stably transfected.

In initial efflux assays using only CHO cell lines transiently expressing DAT WT,

DA efflux through DAT was tested in the presence or absence of efflux inducing

conditions. The significant increase of [3H]-DA efflux observed when treated with AMPH

was concurrent with previous findings of the effect of AMPH on DAT and DA release. It

therefore confirmed that in the heterologous systems used, the effect of AMPH could be

observed through the transfected DAT WT. Furthermore, mSIRK mimicked the effect of

67

AMPH and induced DAT-mediated efflux. Since the heterologous system used does not

conatin the specific components of DA neurons other than DAT, the findings confirm

that mSIRK induced DA efflux occurs as a result of the interaction between activated

Gβγ subunits and DAT. Additionally, it was established that the [3H]-DA efflux in cells

was mediated by DAT as the effect of mSIRK was attenduated when incubated along

with the DAT blocker GBR. The findings using the CHO DAT WT system affirmed that

efflux could be observed and induced through DAT by both AMPH and mSIRK. The

results of each condition for each DAT type (WT or mutant) exhibited the same general

trend seen in the efflux assay using only DAT WT. When analyzed holistically, the

F587A and E589A mutations had no diminutive effects on DAT mediated efflux when

treated with AMPH or mSIRK. Contrarily, a significant reduction in [3H]-DA efflux was

observed in DAT R588A and K590A relative to DAT WT. These findings raised three

important considerations on the effects of the mutations at residues (587-590) on DAT-

mediated efflux. First, the mutations caused a lack of DAT protein expression at the

surface of the transfected CHO cells, thus preventing uptake and efflux. Second, the

DAT proteins were expressed at the membranes, but the mutations affected the ability

of the DAT to uptake of [3H]-DA. Third, the mutations affected neither cell membrane

expression nor uptake, but did impact the efflux function of DAT.

Following the efflux experiments and moving to find answers to the concerns

presented, biotynilation assays were performed to determine whether or not the

mutations had any effect on DAT membrane expression that could be attributed to

reduced DA uptake or efflux. Comparison of revealed immunoreactive bands on an

immunoblot between total DAT protein fractions and biotinylated fractions showed that

68

overall there was less expression of mutant (FREK) DAT in the cell membranes relative

to DAT WT expression. The DAT WT bands were the most intense indicating that more

protein was present and expressed. The F587A and R588A had less intense bands

than the WT, but appeared to have realtively abundant expression compared to the

E589A. Most notably, there appeared to be a lack of DAT K590A expression at the

membrane in both biotinylated and total fractions compared to DAT WT and the other

DAT mutants. However, a band visible around 60 kDa was observed only in the K590A

lanes indicative of a un-glycosylated, immature form of DAT.

The biotinylation experiments addressed the issue concerning the effects of the

mutations on DAT expression at the cell membrane, thus two points still remained

unresolved. Individual uptake experiments were done to determine the effect of the

alanine substitutions on the DAT uptake function. Uptake assays with unlabeled DA and

[3H]-DA incubated for 5 min were performed to analyze the functional and kinetic effects

of the mutations on DAT. Consistent with the previous efflux and biotinylation assays,

the DAT K590A mutant did not uptake [3H]-DA. Analysis of the DAT F587A, R588A, and

E589A mutants pre-loaded with unlabeled DA in a dose-dependent manner and with

[3H]-DA revealed a reduction of [3H]-DA uptake compared to the DAT WT. Each of

these mutants displayed saturation curves resembling those of Michaelis-Menten

enzyme kinetics. The decreased uptake of [3H]-DA observed in DAT F587A, R588A,

and E589A and relative saturation at 10 μM unlabeled DA compared to the DAT WT at

30 μM unlabeled DA, resembled a decrease in Vmax observed in enzyme-subtrate

interactions.

69

Since the previous uptake experiment was run with only a 5 min [3H]-DA preload

time and in the presence of unlabeled DA, it was suggested that perhaps the cells did

not have enough time to uptake high levels of [3H]-DA. In turn, another set of uptake

assays were performed using a 20 min preload for [3H]-DA in transiently tranfected cells

expressing DAT (WT, F587A, R588A, E589A, and K590A). Relative to the WT, there

was no difference observed in the DAT F587A uptake. The R588A mutant displayed

significantly less [3H]-DA uptake than DAT WT. Despite the slightly significant decrease

in uptake seen with the DAT R588A, it can be inferred that this cannot be used to

explain the reduction of efflux, since the amount of [3H]-DA uptaken was still observably

similar to the WT. Thus, reduced R588A efflux cannot be attributed to the reduced

uptake. Contrarily, E589A mutants did display an even greater reduction in [3H]-DA

uptake compared to the DAT WT despite no observable differences in efflux. It can be

suggested that the E589A mutation may cause a reduced uptake function, but it

enhances DAT-mediated efflux. As expected based on the earlier results, no uptake

was noted with the DAT K590A. Uptake assays were then used to normalize the

amount of efflux relative to total amount of [3H]-DA uptaken by cells during a 20 min

preload in subsequent expriments.

With two points of interest concering the effect of alanine substitutions in

residues 587-590 on DAT-mediated efflux resolved, further experiements focused on

addressing the effects the mutants both expressed at the membrane and with functional

uptake had on efflux. Following the mentioned experiments, the K590A mutant was not

used for the purposes of these studies due to the overall lack of observed expression,

uptake, and respective significantly reduced efflux. Uptake and efflux results for the

70

K590A mutant showed that there was essentially no uptake in stably transfected cells

and a significantly dramatic reduction in efflux in transient transfected cells. The results

of K590 relative to WT remained consistent with the observed immunoreactive bands

which revealed the membrane protein expression following the biotinylation assay. It

showed that there was no band intensity for K590A at 100 kDa where DAT would be

expected. However, a band was observed in both total and biotinylated fractions around

60 kDa where immature DAT is typically observed. Altogether, the results for K590A

suggest that this residue is critical for DAT protein maturation, trafficking, or both. Since

the focus of this study was to determine the residues involved in Gβγ-DAT mediated

efflux, further studies focused on the F587A, R588A, and E589A mutants.

To determine the sensitivity of DAT efflux to the F587A, R588A, and E589A

mutations, efflux assays were run in a concentration-dependent manner using AMPH

and mSIRK. These assays were done in CHO cells stably transfected with DAT WT,

F587A, R588A, and E589A. When comparing the DAT WT to DAT F587A there was no

significant difference when treated with AMPH. However, there was reduced efflux

observed for the F587A mutant at 30 μM mSIRK, but none at 100 μM. Based on these

results, it could be concluded that the F587A mutation does not negatively affect DAT-

mediated efflux though it may have modulatory effects. Efflux experiments with DAT

E589A showed that its ability to efflux [3H]-DA was similar to the DAT WT. Consistent

with previous efflux experiments, a significant reduction of DAT mediated [3H]-DA efflux

was observed in R588A cells relative to WT cells for both AMPH and mSIRK

experiments. Results were normalized over the amount of [3H]-DA uptaken and

expressed as a percentage of the control. Significance in the AMPH efflux assay was

71

observed at 1.0, 10, and 100 μM, with greater observable differences corresponding to

increased concentration. Similar results were observed with mSIRK with the greatest

significant decrease of efflux in R588A was at 100 μM mSIRK. Analysis of the data

suggests that the effect of the R588A mutation on efflux was more sensitive in mSIRK

induced efflux. Keeping in mind that mSIRK is a Gβγ activator, it could be inferred that

the R588A mutation alters either the mechanism or physical interaction between DAT

and Gβγ subunits.

Based on these results and the proposed role of the R588 residue in the

interaction between between DAT and Gβγ subunits and the effect on DA efflux, the

impact of the R588A mutation on this interaction was studied. Using

immunoprecipitation and co-immunoprecipitation techniques the ability of DAT R588A to

co-precipitate Gβ compared to DAT WT was studied. IP and Co-IP experiments using

only CHO and CHO DAT WT cells determined that the antibody DATc (431) was most

effective at immunoprecipitation DAT and co-precipitating Gβ and as such was used in

the Co-IP experiments comparing DAT WT and DAT R588A. Results and analysis of

immunoreactive bands revealed that the R588A mutant was less able to co-precipitate

Gβ compared to DAT WT which may have indicated that it affected the interaction

between DAT and Gβγ subunits. However, immunoblotting of the total protein (inputs)

showed that overall there was less DAT R588A proteins. Since there was less DAT

R588A compared to DAT WT, the reduced physical interaction between DAT R588A

and Gβ could not be attributed to these studies alone.

Our findings indicate that single-point mutations within the DATCT1 of specific

residues 587-590, cause an inhibition of the DAT efflux function. The mutation of F587A

72

had no overall effect on either DA uptake or DAT mediated efflux suggesting it is not

essential to either of these two functions. The E589A mutant on the other hand showed

a significant reduction in DAT uptake but similar amounts of [3H]-DA efflux compared to

DAT WT, which indicated that the E589A mutation slightly inhibits DAT uptake yet

enchanes DAT-mediated efflux. The findings of the K590A mutant were important in the

inference that the K590 residue may be essential for DAT trafficking to the cell

membrane. Most important in relation to the premise of these studies, taking into

consideration that the R588A mutant displayed a significant decrease in DAT-mediated

efflux despite the fact that it was expressed at the cell membrane and had similar

uptake relative to the wild type, it can be strongly suggested that the R588 residue

within the DAT C-terminus plays a critical role in the DAT efflux function and possibly in

the interaction with Gβγ. Since the precise role of the R588 residue remains

inconclusive, further investigation should focus on characterizing the responsibility of

this residue within the C-terminus of DAT. The effect the R588A mutation has on

physical interaction between DAT and Gβγ subunits remains elusive and an important

point for future research. New or more precise assays for determining protein-protein

interactions should be employed in order to obtain concrete results on this question.

Additionally, future studies on the K590 residue in DAT should emphasize determining

the precise role of this amino acid residue in the processing, trafficking, and function of

DAT.

The efflux mechanism of DAT and all players involved in causing this reverse

transport of DA have yet to be entirely determined or understood. While residues in the

C-terminus of DAT that are involved in Gβγ mediated efflux have been successfully

73

identified, other components and effectors of DAT should be studied. A potential site

and mechanism for consideration is the N-terminus of DAT and phosphorylation (James

D. Foster & Vaughan, 2017). Studies involving the phosphorylation of the N-terminal of

DAT suggested that this phosphorylation facilitates AMPH mediated DA efflux through

DAT (James D. Foster & Vaughan, 2017). It was also found that truncating the first 22

amino acid residues of the N-terminus in an hDAT construct eliminated phosphorylation

by protein kinase C (Khoshbouei et al., 2004). Specifically, there are 6 serine residues

within the first 22 amino acid residues of the N-terminus that were suggested to be

possible phosphorylation sites on DAT (Khoshbouei et al., 2004). Based on these

findings, future studies of DAT efflux could focus on determining which serine residues

are essential for the phosphorylation involved in AMPH-induced DAT-mediated DA

efflux. This could be done via site-directed mutagenesis of one or multiple serine

residues within the N-terminus followed by biochemical phosphorylation studies and

AMPH efflux assays. Further investigation of the efflux function of DAT could help to

elucidate the mechanisms of DA-reinforced drug addiction and of psychostimulants

such as AMPH. Determining the amino acid residues critical to aspects of DAT function

and efflux could be beneficial to better understand defects and diseases in which DAT is

known to be implicated, including ADHD and schizophrenia. The knowledge of critical

amino acid residues for DAT efflux could be incorporated with the understanding of DAT

mutations in illnesses to develop targeted medications that can modulate the functions

of DAT. This may reduce the excess exciteability that is generally observed due to high

DA signaling. Typically, individuals with ADHD are treated with doses of AMPH and

methylphenidate which acts on the DA system, specifically DAT. Elucidating the

74

mechanism by which AMPH interacts with and affects DAT could provide better

approaches to treating individuals with ADHD, by using more DAT specific therapeutics.

In this manner, either more effective drugs can be developed and used or doses of

current medications could be tailored to the type of mutation found in individuals.

Furthermore, understanding DAT function can help address the DA reinforcement

aspect of drug addiction and therapeutics aimed at inhibiting DA efflux or excessive DA

signaling could be developed. Ultimately, the importance of the findings of these studies

lies in the possibility that one day they could be translated into therapeutics of DA and

DAT related disorders.

75

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BIOGRAPHICAL SKETCH

Gabriela Marie Hidalgo was born and raised in Miami, Florida. After graduating

from Our Lady of Lourdes Academy, she began her undergraduate education at the

University of Florida in August 2013. She received a Bachelor of Arts with a major in

visual art studies and a Bachelor of Science with a major in biology from the University

of Florida in May 2017. She chose to continue her education at the University of Florida

and in August 2017 entered the Biomedical Sciences Program in pursuit on a Master of

Science with a major in medical sciences.