1

Neurotransmitter TransportersSammanda Ramamoorthy, Ph.D

Department of NeurosciencesMUSC

rama@musc.edu792-3989

Recommended Reading on Neurotransmitter Transporters

1. Neurotransmitter Transporters: Structure, Function, and Regulation.Edited by Reith MEA. Humana Press: Totowa, NJ; 1997.

2. Neurotransmitter Transporters: Methods in Enzymology. Vol. 296Edited by Susan G. Amara. Academic PressSan Diego, CA; 1998.

Specific Transport Across Lipid Specific Transport Across Lipid BilayerBilayer

2

Action of Membrane Transport Proteins

Transport of Glucose from the Transport of Glucose from the IntestionalIntestionalLumen into the BloodLumen into the Blood

.

R RR

RR

RT T

T

T

RT

Postsynapticterminal

Glia

Presynapticterminal

Important Factors for Neurotransmission

Density of transporter sites

Affinity of substrates

Density of receptor sites

Location on neuron

Glial localization

Depolarization

ER

RR

secondm

essengers

T

TT

Affinity of ligands

Biosynthesis of NTs

Metabolism of NTs

3

.

Depolarization

R R R R R R

TT

TT

ER

T

Transporters Clear Neurotransmitters from Synaptic Spaces

Different Families of Neurotransmitter Transporters

4

Sodium and Chloride-Dependent Neurotransmitter Transporters

Structure, Function and Pharmacology of Na+/Cl-

-Dependent NTTs

12 TMD containing proteins of ~600 aa with 40-60% homology. Both N- and C-terminilocated intracellular with a large EL2. They cotransport 1Na+ and 1Cl- with theirsubstrates. Electrogenic in nature. Transport process generates substrate -coupled and-uncoupled currents.

Highly conserved N- terminal region (TMD1-TMD4)is important for the transport ofcommon ions, Na+ and Cl-. Divergent C-terminal region (TMD7-TMD12) is importantspecific substrate recognition and inhibitor binding. Km in nM-µM range.

Antidepressants and drugs of abuse like cocain and psychostimulants (amphetamines)act on monoamine transporters (DAT, NET and SERT) as inhibitors indicate their rolein neuropsychiatric diseases like depression and substance abuse.

Antiepileptic and anticonvulsive drugs act on GABA transporter (GAT1) as inhibitorswhich implicates the role of GAT in epilepsy and seizures.

K/Os for NET and SERT have no obvious phenotypes, but DAT K/Os show hyperactivephenotype.

Predicted Structures for Na/Cl-Dependent Transporters

5

GAT1/NET (SCL6)GAT1/NET (SCL6)Transporter Gene FamilyTransporter Gene Family

GABA (rGAT2)

GABA (rGAT3)

Taurine (hTaurine)

Creatine (rCreat)

GABA (hGAT1)

Norepinephrine (hNET)

Epinephrine (fET)

Dopamine (hDAT)

Serotonin (hSERT)

Proline (rProT)

Glycine (rGlyT1)

Glycine (rGlyT 2 )

Substrate (Clone)

Diminished Dopamine Clearance in DAT K/O MiceDiminished Dopamine Clearance in DAT K/O Mice

Important Structural Domains in Monoamine Transporters

6

NTTs Act Like Ligand-Gated Ion Channels.

NTTs generate stoichiometric currents which are not voltage-dependent intheir “classic transport” mode.

Because there is a concomitant flux of 5-12 charges per transport cycle, NTTs also generate nonstoichiometric currents which are voltage-dependent in a“channel-like” mode.

Whether “classic transport” mode and “channel-like” mode exist together ornot?

Transporters: Models for How They WorkTransporters: Models for How They Work

Sodium and Potasium-Dependent Neurotransmitter Transporters

7

Structure, Function and Pharmacology of NA+/K+

-Dependent GLUTs (EAATs)

Primary structure is still in controversy. 6-10 TMD containing proteins (523-573aa)of several subtypes. Divergent N- and C-termini and a large EL2. Highly conserved C- terminal region. Km in µM range.

*Unlike the other NTTs, EAATs are post-synaptically located, and cotransport 2-3Na+ and 1H + with countertransport of 1K+.

*Not only decrease the local concentration of Glutamate but provide this aa as a Substrate for the synthesis of GABA.

*EAATs (1-4) have Cl-conductance and are neuromodulatory. Neuronal EAATs 3&4 produce Cl-influx causing local hyperpolarization and thus prevent excessive excitation.

Implicated in neurological disorders like Alzeimers disease, Huntington’s choria and Amyotrophic Lateral Sclerosis. Competitive inhibitors for EAATs also act on GluRs.

K/Os for EAAT1, 2 but not 3 showed elevated extracellular Glu. All K/Os showed loco-motor impairment.

Predicted EAATCs Structure

Different Types of Glutamate Transporters

8

Cl- -Conductance In Glutamate Transporters

Vesicular- Neurotransmitter Transporters

Structure, Function and Pharmacology of VNTTs

12 TMD containing proteins with cytosolic N- and C-termini located outside the Vesicular lumen and the large EL-1 inside the lumen.

N- and C-terminal domains and EL-1 are divergent and involved in specific substraterecognition.

*Vesicular accumulation is an amplification step for overall clearance of NTTs fromextracellular space. Also protects NTs from leakage and metabolism.

*The neurotoxin MPP+ acts as a substrate for both VMAT and PM DATs. Implicated in neurological disorders like idiopathic Parkinson’s disease and Alzeimer-type dementias.

VNTTs depend on electrochemical gradient generate by ATP-driven proton-pump.Threefamilies VMAT/VAChT, VGAT/VIAAT and VGLUT differ in their requirement for electrochemical gradient. VMAT/VAChT predominantly on chemical component (∆pH) and VGLUT on electrical component (∆Ψ), whereas VGAT/VIAAT on both.

Drugs like reserpine and vesamicol are selective inhibitors for VMAT and VAChTsrespectively. VMAT-2 K/Os have deficit locomotor activity and feeding behavior, anddie shortly after birth.

9

Predicted VNTs Structure

Previously known protein, BNPI (brain-specific Na-dependent Pi transporter) recently identified astransporting Glutamate into SVs (VGLUT1). Unlike BNPI, VGLUT1 does not require Na gradient for vesicular transport but depends on ∆Ψ. Science 2000; 289 957-960.

Different Types Of VNTTs

Regulation

10

Transcription

Translation

activity

AntidepressantsPsychostimulants

Loca

lizati

on

PM-Rete

ntion

Func

tion

Glycos

ylatio

n, Pho

spho

rylati

on

Multim

eriza

tion, P

roteio

lysis

Protein

-Protein

Inter

actio

ns

Regulation of Amine Transporters:“New Reasons for Excitement”

NONO

Lipid rafts

“relay

stati

on”

Psychiatric DiseasesAddiction, AgingNeurodegenerative Diseases

SERT

Gene

TranscriptionFactor Second Messengers

Gene Expression

(+) /(-)

Long-Term Regulation

PKA cAMP

CREB

(+) /(-)

6.8 kb

4.9 kb

3.0 kb

1 2 3 4

SERT mRNAs

Serotonin Transporters

Translation, Trafficking,Localization

PKC

(-)

Ca2+/Cam(+)

PKG(+)

Second Messengers

Acute Regulation

SERT mRNAs

A B

6.8 kb

4.9 kb

3.0 kb

P

2 ndMessenger

PSS

ATP

ADP

Pi

?

CELLULARRESPONSE

Cation

CELLULARRESPONSE

Kinase

Phosphatase

11

Nucleus

Gene Transcription

.

Delivery

T

A

.

PT

Activity

.

PT

Endocytosis

P

.

TP P

TDegradation

.

T

Recycling

Recruitment

.

T

Golgi T

ER

Tranlation

TAP

PP2A

PP2A

PP2A

LocalizationSurface Retention

Functional Expression

TAP

TAP

TAP

TAPPP2A

PhosphorylationGlycosylationMultimerizationTAP(Transporter Asso. Proteins)Limited Proteiolysis

SignalsReceptors

Extracellular

IntracellularPIP2

Ins P3

Agonist

PLC

Receptor

.

ATP

ADP

ER

Ins

P3-S

en

sit

ive

Ca

-ch

an

ne

l

Ca-pump(Ca/ATPase)

Ca-sensitive Ca-channelCa2+

PKC

Ca2+

Ca2+

.

Interacting

P

Proteins

(+)

Na+, Cl-

5HT

SERT

K+

CocaineAntidepressants(-)

5HT, Amphetamine

.

SE

RT

SERT

SE

RT

SE

RT

SERT

Intracellular

SERT Pool

Calcium

Channels

Ca2+ Action potential

Ca2+

CaM KII

CaM KII P

autophosphorylationCa2+ indipendent

5HT

SERT

K+

P

Interacting

Proteins

SERT P

(+)

Keep Up for Next Action

Acute Modulation of Plasma MembraneNorepinephrine Transport

Acute Regulation Stimulus

Effect onUptake

Source

Electrical Stimulation Cat Atrium(Gillis 1965; Rorie et al. 1989 Dog Saphenous Vein Stjärne, 1992) Rat Tail Artery

Nitroprusside Rabbit Circulation(Eisenhofer et al. (1990)

ANF Rat Brain Slice(Fernandez et al. 1990; Rat Adrenal MedullaVatta et al. 1991)

Ang II Rat Adrenal Medulla(Vatta et al. 1992;Lu et al. 1996) Rat Brainstem Neuronal Cultures

Insulin Rat Brain Neuronal Cultures(Boyd et al. 1985)

ATP Rat PC 12 cells(Hardwick et al. 1989;(Hendley et al. 1988) Rat Brain Synaptosomes

Forskolin(Bunn et al. 1992) Bovine Adrenal Chromaffin Cells

8-Br-cAMP Bovine Adrenal Chromaffin(Bunn et al. 1992) Cells

12

Acute Regulation of Plasma Membrane

Serotonin Transport

Acute Regulation

Stimulus

ß-PMA

Con A Rabbit platelets

Genistein Human platelets

CGS9343B JAR cells

BeWo cells

8-Br-cGMP RBL-2H3 cells

NECA RBL-2H3 cells

Histamine Human platelets

Calmidazolium RBL-2H3 cells

JAR cells

BeWo cells

Human platelets

RBL-2H3 cells

JAR cells

Raphe culture (rat)

Rabbit perfused lung (in vivo)

Endothelial cells

Source Effect on Uptake

020406080

100120

020406080

100120

vehicle β-PMA α-PMA β-PDBu α-PDBu

vehicle β-PMA β-PMA staurosporinestaurosporine+

Treatments

Treatments

Spec

ific 5

-HT

Upt

ake

(% o

f con

trol

)Sp

ecifi

c 5-H

T U

ptak

e (%

of c

ontr

ol)

Kinase Activation DecreasesKinase Activation DecreasesSERT ActivitySERT Activity

XY-axis

Z-axis

Z-axis

13

The Journal of Biological Chemistry, January 8, 1999, 274(50):35794-35801

Regulated Trafficking of the Human Dopamine TransporterCLATHRIN-MEDIATED INTERNALIZATION AND LYSOSOMAL DEGRADATION IN RESPONSE TOPHORBOL ESTERS*Gwynn M. Daniels§ and Susan G. Amara§¶

WT L

V

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SEVKDGV V P T PA

CE D

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SAVPSPGAG

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EIPCGDIRLNAVHOOC

1 2 4 5 6 7 8 9 11 12

Indicates putative phosphorylation sites

Indicates potential N-linked glycosylation sites

E

F

Extracellular

Intracellular

I G

Y

Q

R

H

W

L

NDE

K

C

E

TN

F

S

Y

T

S H

NG

S

T

N

W

P

S

A

E

FYNT STTI

G

10

20

30

40

506070

80

90

100

110

120 130

140

150

160

170180

190

200

3

210220

230

240

250

260 270280 290

300

310

320

330

340

350360

370380

390

400

410

420430

440

450

460470

480

490

500 510

520

530

540 550560

570

580

590

600

610

620

630

A F

W

A Y

S

Potential Consensus Protein Kinase Sites on the Human Serotonin Transporter

Serotonin Transporters are Phosphorylated In Vivo

Std (kDa)

205116

80hSERT

[Cholera toxin] (ng/ml)10 100 250 500 10000

Std (kDa)205116

80hSERT

0 1 10 100 500 1000[ß-PMA] (nM)

Std (kDa)

205116

80

hSERT

0 250 500 750 1000[Okadaic acid] (nM)

14

The Journal of Neuroscience, December 1, 1999, 19(23):10193-10200

Glutamate Induces Rapid Upregulation of Astrocyte GlutamateTransport and Cell-Surface Expression of GLAST Shumin Duan, Christopher M. Anderson, Becky A. Stein, and Raymond A. Swanson

The Journal of Biological Chemistry, January 8, 1999, 274(2):889-895

Regulation of -Aminobutyric Acid (GABA) Transporters byExtracellular GABA* Eve M. Bernstein and Michael W. Quick

Regulated Trafficking of SERTRegulated Trafficking of SERT

P

P P

Internalization

PP

Phosphorylation

PP2Ac dissociates

R

P

P P

Dephosphorylation

P

P

Recycling

P

15

Activity Dependent Regulation ofActivity Dependent Regulation ofSERT TraffickingSERT Trafficking

R

PPhosphorylation

P P

PP2Ac dissociates

S

Internalization

PP

P

P

Recycling

Dephosphorylation

AntidepressantsPsychostimulants

.

R RR

TP

T

T

P

TT

TT

TT

T

RR

Dep

ola

rizatio

n

Activity

SERT internalization

Neurotransmitter released

Neurotransmitterclearance

Axon

Pre-synaptic terminal

SERT internalization

Post-synaptic terminal

5-HT SERT in EndosomeTTR ReceptorT SERTSecretory vesicle

PKC

R

R

PKC

RR

T

R R

R P

R

DesensitizationReceptor internalizationSecond

messengers

P

Phosphorylation-Dependent Co-Ordinate Trafficking of Pre-Synaptic Transporters and Post-Synaptic Receptor

Regulate Normal Neurotransmission

Intron/ExonBoundries

Regulatory Regions of HumanSerotonin Transporter Promotor

5HT Uptake

Polymorphism of Human Serotonin Transporter Genein Mental Illness

Mental Illness (Depression, Suicide, Alcholism)

ATG

ATG

16

Biogenic Amine TransporterContributions to Brain Disorders

SERT Obsessive-Compulsive DisorderDepressionSchizophreniaAnxiety DisordersAlcohol Abuse

DAT Attention Deficit/Hyperactivity Disorder

NET HypertensionDepressionSchizophrenia

EpilepsyOrthostatic Intolerance