Cloning, Pharmacological Characterization, and ... - CiteSeerX

ls)2t;-l95X/92/l):to:*t:3.llsSll:3.lx)/l) Copyright c by The American Society for Pharmacology Alt rights of reproduction in any form reserved. MOLECLIAR puARMAcoI,o(;v, 42:383-3911

ACCELERA

and

Experimental

Therapeutics

TED COMMUNICATION

Cloning, Pharmacological Characterization, and Chromosome Assignment of the Human Dopamine Transporter BRUNO MARC

GIROS, SALAH G. CARON

EL MESTIKAWY,

NATHALIE

GODINOT,

KEQIN

ZHENG,

HONG

HAN,

TERESA

YANG-FENG,

and

Departments of Cell Biology (B.G., S.E.M., N.G., M.G.C.) and Medicine (M.G.C.), Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710 and Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 (K.Z., H.H., T.Y.-F.)

Received

May 4, 1992;

Accepted

June 8, 1992

SUMMARY

We have probes

screened derived

from

a human

nigra cDNA library with

substantia

the rat dopamine

transporter.

A 3.5-kilobase

cDNA clone was isolated and its corresponding gene was located on the distal end of chromosome 5 (5p1 5.3). This human clone

codes

for a 620-amino

acid

protein

weight

of 68,51 7. Hydropathicity

with

analysis

a calculated

molecular

suggests

the presence

of 1 2 putative transmembrane domains, a characteristic feature of sodium-dependent neurotransmitter carriers. The rat and the human dopamine transporters are 92% homologous. When per-

manently

expressed

in mouse

fibroblast

Ltk

cells,

the human

clone is able to induce a saturable, time- and sodium-dependent, dopamine uptake. This transport is blocked by psychostimulant drugs (cocaine, I- and d-amphetamine, and phencyclidine), neu-

rotoxins

The

(6-hydroxydopamine

monoamine

and

DA plays

a key role in the

regulation

of

CNS functions (locomotor activity, positive reinforcement, spatial memory, higher cognitive functions, and neurohormone release) and in major neurological diseases, as well as psychiatric disorders (Parkinson’s disease, Tourette syndrome, schizophrenia, and bipolar affective disorder). DA mediates a variety

of functions

through

the

stimulation

of at least

subtypes of guanine nucleotide-binding proteincoupled receptors (1, 2). Subtypes of these receptors have been shown to be the target of neuroleptic drugs and, thus, could be involved in the etiology of schizophrenia (3, 4). Various subtypes of DA receptors may act in concert (5) to mediate the neurotransmitter role of DA in the CNS. Therefore, the acute or chronic concentration of DA in the five

line), and various uptake inhibitors (mazindol, 1 2909, and amfonelic acid). The rank orders

GBR 1 2783, GBR of the K values of

these substances at the human and the rat dopamine transporters are highly correlated (r = 0.998). The cloning of DNA human dopamine transporter gene has allowed establishment of a cell line stably

expressing

the human

dopamine

transporter

and,

for

the first time, an extensive characterization of its pharmacology. Furthermore, these newly developed tools will help in the study of the regulation of dopamine transport in humans and in the clarification of the potential role of the dopamine transporter in a variety

of disease

states.

N-methyl-4-phenylpyridine

various

such

(MPP))), neurotransmitters (epinephrine, norepinephrine, y-aminobutync acid, and serotonin), antidepressants (amitriptyline, bupropion, desipramine, mazindol, nomifensine, and nortripty-

distinct

This work was supported by Grants NS-15976, IP-53-NIH 40159 from the National Institutes of Health, Bethesda, MD.

44221,

and

MH-

CNS has crucial functional consequences. It is well established that the concentration of DA is acutely controlled in the intercellular space mostly by reuptake mechanisms (6). After its

release,

the

concentration

of DA

at

or around

the

synapse

is rapidly

reduced, due to the presence of uptake sites on the plasma membrane of presynaptic dopaminergic nerve endings. Consequently, the DAT plays a pivotal role in the regulation of pre- and postsynaptic dopaminergic transmission.

The DA uptake

site, aside

from

its physiological

role, is also

the target of a variety of psychoactive agents, such as cocaine, amphetamine, and PCP derivatives (7). It has also been hypothesized that neurotoxins like 6-OH-DA and MPP (the active derivative of MPTP) enter dopaminergic neurons by the route of the reuptake site (8). In the case of human Parkinson’s disease, the severe depletion of striatal DA is overcome by the administration of L-DOPA to the patients (9). Here again, it is generally believed that L-DOPA penetrates residual dopami-

ABBREVIATIONS: DA, dopamine; DAT, dopamine transporter; SSC, standard saline citrate; SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid; PKC, protein kinase C; CNS, central nervous system; PCP, phencyclidine; PCR, polymerase chain reaction; Kb, kilobases; 6-OH-DA, 6-hydroxydopamine; GABA, ‘y-aminobutyric acid; NA, norepinephnne; TM, transmembrane; PKA, cAMP-dependent protein kinase; NAT, noradrenaline transporter; DOPA, 3,4-dihydroxyphenylalanine; MPP, N-methyl-4-phenylpyndine; MPTP, N-methyl-4-phenyl-1 2,3,6tetrahydropyridine. 383

384

Giros

eta!.

nergic neurons and is subsequently decarboxylated into DA. However, it is not known whether, in the human brain, this uptake process involves the plasma membrane DAT. The implication of DA in the etiology of some aspects of depression (locomotor impairment, emotional withdrawal), although not clearly proven, has long been suspected (10). This is further illustrated

by the

patients

and

data,

DAT

incidence

action

sometimes

not

of depression

antidepressant

although

the

high

the only

controversial,

plays

appears

to be a critical

as drugs

widely

used

DA

transport

in the

but

a good

experimental

a pivotal

target in the

at the

In the

brain

has for

these

suggest

synapse

for substances

model

Thus,

strongly

role

clinic.

animal

in Parkinsonian

of L-DOPA.

that

but

of abuse,

past

been

the

regulation

intensively

human

DA

also

as well of

studied,

transport

has

so

far been missing. The neurotransmitter reuptake process is driven by the ergy of the sodium gradient generated by the Na,K-ATPase (11). The uptake neurotransmitters

is mediated by proteins and ions. Recently, cDNAs

and

human

transporter,

sites,

have

GABA been

of homology,

cloned

thus

advantage

defining

of this

They

a new

all share

family

information,

levels

oftransporters.

we and

others

rat

NA uptake

significant cloned

the

cDNA coding for the rat and the bovine DA and serotonin reuptake site (15-20). We now report the cloning of the DAT from a human substantia nigra cDNA library. The isolation of a cDNA and the establishment of a clonal cell line expressing the human DAT have allowed the determination, for the first time,

of the

detailed

pharmacology

of the

human

brain

DAT.

tamed Cell

Repository.

Procedures

amplification.

amplification 796

Two

of the

of

the

rat

rat

cDNA

DAT

sequence,

probes

(17).

was

Probe

obtained

ACGGAGTGCAGCTG-3’

(nucleotides

CACCTTCCCTGAGGT-3’

(nucleotides

with

the

tides

primers

1881

72’

rat

for library

filters

(BAS

42’

for

7.4,

4x

formamide,

overnight

sulfate,

in

SDS,

plaque in

42*

purified,

3.5-kb

fragment

,

lx

0.6

in the

SSC,

0.1%

(15

mm)

and

(Stratagene)

chromosomal

sequenced

in both

for

at

46’

(15

sequencing

orientations

by

the

20 was

dextran probe

were

then

clone

A

washed

assignment

studies,

two

probes

coding beginning

region (clone of the coding

ization

(see

Table

were

used,

and a 3.5-kb

2-4), and sequence

2), a mapping

somatic

a 1-kb (clone panel,

Stable

cell lines

by the

oratories).

The

chain

cell

hybrids.

comprising

was termi-

For these the

clone corresponding 1-4). For somatic consisting

full cell

length to the hybrid-

of 17 mouse-human

and

the

was

their

human

National

Repository

Institute

catalog.

For

nick-translated

excised

from

signal

human

in

with

pBluescript

resistant

Established

cell

inserted gene

and

of the

‘

(Ltki

cells

were

with

the

construct, respectively. Both with the selection plasmid

method was

to 0.4

mg/ml

stably

5

contained

or the rat DAT

fibroblast

uptake

lines

was

hormone

the human

mouse

phosphate

of DA

but

growth

expressing of

calcium

level

were

medium.

(Bethesda

then

Research

assayed

in the

geneticin

taking

(GIBCO)

up 0.78

and

Lab-

clonal

cell

in the

1 nmol

of [3H]

DA/min/105 cells for the rat and for the human DAT, respectively, were selected for pharmacological studies. [3H]DA uptake experiments. Cells were distributed in 24-well plates, at a density of 10 cells/well, and were grown for 48 hr, up to n2.5 x 10 cells/well. For uptake experiments, the Dulbecco’s modified Eagle medium was replaced by a buffer containing 5 mM Tris base, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KC1, 1.2 mM CaC12, 1.2 mM MgSO4, 1 mM ascorbic acid, and 5 mi D-glucose, (final pH, 7.1) (uptake buffer). end

of incubation, buffer,

gentle

the

cells

resuspended

shaking, For

for

uptake

were

in

washed

0.4

ml

of

1 hr. An aliquot

inhibition

studies,

three 1%

was cells

times

with

and

left

SDS,

taken were

0.5 at

ml 37’,

for scintillation preincubated

for

2

mm in the presence of compounds to be tested, then 15 nM [3HJDA was added in a final volume of0.5 ml, and the incubation was continued for 4-5 mm. In order to perform the most accurate comparison between pharmacological

with

values

were

120

To mM

done

obtained

the

determine

same the

day Na

for

the

rat

with

the

same

dependency,

and

human

DAT,

dilutions

NaCI

was

of drugs substituted

LiC1.

Molecular cloning of DAT. A human substantia

a eDNA coding nigra library was

for the human screened with two

probes derived from the rat DAT, which encompassed the first (TM-i) to the fourth (TM-4) TM domains (probe A) and the ninth TM domain (TM-9) up to the end of the coding sequence (probe

B).

found inserts,

to

prises

2-4)

was

construct or pCMV5-TS3 simultaneously cotransfected

pRSVNeo,

that

of the

transfection

pCMV5-HBT cell lines were

were

of probes

dideoxynucleotide

clone

signal by

clones

were subcloned

method.

Chromosomal

2-4

polyadenylation

in pCMV5.

pBluescript

(clone

in

consensus

a clear

SSC,

preparation

Mutant

nor

in 0.2x

Positive

length

pH

SDS,

10%

Filters

fragments and

A full

0.01%

translated

and

mm).

EcoRI

Tris-HC1,

containing

42

at

Hybridization

of DNA).

SDS,

assignment).

7.0),

nicked

2-4 x iO cpm/zg

in 2x

pH

mM

Cell

ob-

Sciences

Results

polyvinylpyrroli-

20

tRNA.

solution cpm/ml

clone

nigra

prehybridized

(0.1%

M NaC1,

same i0

54,

nitrocellulose

been

ficoll),

yeast

94’,

at

substantia

duplicate

had

Denhardt’s 0.1%

run

a human

onto

20 ig/ml

of 5 x

was

from

polyadenylation

experiments

and

hybrids

probe

two

was

vector pCMV5 (17), to provide the pCMV5fragment comprises neither a poly(A) tail

the

1357

these

cDNA

and

hybrids,

Medical

in detail

Mutant

3.5-kb

NA10567)

subcloned in the expression HBT construct. The cDNA

to be tested.

nucleotides

from

and the corresponding

the

nation

42

activity,

pBluescript

(for

5’-ACACB, amplified

of

(21).

that

albumin,

DNA,

presence

15 mm at

5’-CAGA-

and

amplification

transferred

Na:1 citrate,

at

the

or B (specific 0.1%

mM, sperm

to

of General

described

Sciences the

and GM07298)

[3H]dATP and [3H]dCTP, to a specific activity of 3.4 x 10 cpm/gg. Hybridization to human metaphase chromosome spreads, post-hybridization wash, emulsion autoradiography, and silver grain analysis were carried out as previously described (22). Stable cell line transfection and expression. A full length EcoRI

counting.

(nucleo-

as described

& Schuell)

serum

carried

for

were

Schleicher

(60

PCR 109

primers Probe

between

Plaques

in 35%

SSC

the

796-778).

PCR each),

bovine

salmon

twice

The (1-mm

(Clontech)

2 hr

nucleotides

109-126)

located

sequencing.

85;

0.1%

gig/ml

sequence.

and

cDNA

from

with

by

5’-TGGCAGCTGTCTCCTTCCACTTTA-3’ was

30 cycles

Cloning

done,

and 1881-1858),

of the

and

obtained

5’-CTGGCTACTTTCCTGCTGTCTCT-3’

1357-1379)

(nucleotides

A,

Medical

of uptake

were

are

hybridization,

with PCR

and

Institute

content

General

situ

NA10324,

(NA1O611

Characterization

chromosome of

NA09940,

from the National

At the

Experimental

NA09938,

hamster-human

lines

Taking

have

to

Chinese

obtained

that co-transport encoding the

as well as the human

(12-14).

en-

(NA09925

Among

108 clones,

hybridize of 1, 1.2,

with and 3.5

for sequencing

three

(1-4,

both probes kb, respectively,

and

15-1,

and

analysis.

The

larger

initiation

site, The

186i.

a poly(A) is not

and

ending

3’ untranslated

reading

tail, and a clear

apparent

(data

with

not

a TAG

stop

frame

polyadenylation shown).

The

1)

at position

terminated

signal putative

into

com-

(Fig. consensus

codon

is not

were

EcoRI

clone

an open reading frame of 1860 nucleotides with an ATG, which reasonably fits a Kozak

starting

2-4)

A and B. Their were subcloned

in this

protein

by

region encoded

cDNA is 620 amino acids long, with a calculated molecular weight of 68,517. Hydropathicity analysis shows a pattern in which 12 21-24-hydrophobic amino acid segments could represent TM domains (Fig. 1). When compared with the by

this

Human DAT Cloning M1I

CTCAACTCCcAGTGTGCCC

MG ,4.t

AG? Sr

5.G Ly.

AGC 5r

MA Lys

TOC Cy3

TCC Sec

GTG Vii

GGA Gly

CTC Lsu

ATG Mst

TCT S*r

TCC Ser

GTG Val

GTG VaX

GCC Ala

CCG Pro

OCT Al.

MC Lys

GAG Glu

CCC Pro

AAT Sari

0CC Ala

GTG Val

GOC Gly

CCG Pro

?.AG Lys

61 27

GTG

ACC Thr

AGC S.r

ICC S.r

ACC Thr

CTC L.u

ACC Thr

MC A3n

CCC Pro

CCC Arq

CAG AGC GinS.rPro

CCC

GTG V.1

GAO Glu

GCC Al.

CG Gin

CAT Asp

COG Arq

GAG Giu

163 61

GCT Ma

GTG Vai

GPC Asp

CTG Lu

CCC Aia

MC ksn

GTC Vai

TOG Trp

AGO Arg

TrC Ph.

CCC Pro

TAC Tyr

CTG L.u

TGC Cy.

TAC Tyr

MA Lya

MT an

GOT Giy

GGC Giy

285 95

TrC Ph.

TAC Tyr

ATG P4.t

GAG Glu

CTG Lu

0CC Pd.

CrC I..u

GGC Giy

CAG Gin

TFC Ph.

AAC Aan

AGO Arq

GM Giu

SOC Giy

CCC Ma

OCT Si.

387 129

GAG Glu

GTG Val

GAG Glu

CTC Iau

ATC us

CTT lao

GTC V.1

ATG Nt

GG Clii

CAG Gin

MC kin

GGP Gly

Vai

CAG Gin

CTC L.u

,.cc

roG

G Gly

SAG Lys

SAG Ly.

ATC Ii.

C kap

TTT Ph.

CTC Lu

CTG Lsu

TCC S.r

GYC Vii

ATT Ii.

GGC Giy

TTT Ph.

GGT Gly

GCcl TTc Ai.lPh.

CTG Iau

GTC Vi

CCC Pro

TAC Tyr

CTG L.u

CrC Lu

TTC Ph

ATG 61st

GTC Vai

ATr Ii.

OCT Ala

COG Giy

ATG N.t

CC Pro

CTT tu

GGT

GTC

TGG

MG

ATC

TGC

ccc

ATA

CTG

AAAIGGT

GTG

GGC

TTC

ACG

GTC

ATC

CTC

ATC

TCA

CTG

TAT

GTC

GGC

TTC

TTC

TAC

MC

GTC

ATC

ATC

CCC

TOG

0CC

489

Gly

V.i

Trp

Ly.

Ii.

Cyu

Pro

Ii.

tau

Ly.[y

V.i

Gly

Ph.

TSr

V.i

Ii.

Lu

Ii.

S#{149}r L.u

Tyr

Vai

Gly

Ph.

Ph.

Tyr

Aan

Vai

Ii

Ii.

Ala

Trp

Pd.

163

CTG I..,,

CAC at.

TAT

CTC

TCC Sr

TcC Sr

TTC Ph.

ACC Thr

ACG Thr

CrC

CCC

TGG

ATC

TCC

TOG

AAC

Pro

Trp

Ii.

Asn

Asn

Ser

Trp

Ass

AGC Sr

CCC Pro

MC

Iau

TGC Cys

AAC

Giu

CAC His

SAC

Lu

TIC Ph.

GAG

Tyr

Cys

TcG Ser

GST Asp

0CC Ai.

CST Hi.

cCT Pro

GGT Giy

GP.C Asp

TCC Ser

591 197

AGT

GG

GAC

AGC

T1G

GGC

CrC

MC

IIAC

ACT

iTT

GGG

ACC

ACA

CCT

OCT

CCC

IISG

TAC

TTT

GSA

COT

CCC

GIG

CrC

CAC

dC

CC

CAG

AGC

CAT

0CC

AlC

GAC

693

Asp

Sr

Sr

Giy

Lu

p Thr

Ph.

Giy

Thr

Thr

Pro

Ala

Ala

Glu

Tyr

Ph.

Glu

Arq

Giy

V.i

L.u

His

Leu

His

Gin

Sr

His

Giy

Ii.

Asp

231

TCA

SOC

AAG

GTG

795

Lys

Vai

265

385

TM1 ThrTrp

‘TM 2

*

S#{149}r Giy

*

sn

*

fl3

*

n

TM 4 GAC

CTG

GOG

cCT

CG

CGG

TOG

CAG

CTC

ACA

0CC

TGC

CTG

GTG

CTG

GTC

ATC

OrG

ccc

crc

TAC

TTC

AGC

CTC

1GG

MG

GGC

GTG

AAG

ACC

Asp

Lu

Giy

Pro

Pro

Arq

Tsp

Gin

L.u

Thr

Aim

Cys

L#{149}u Vai

L.u

Vsi

Ii.

V.i

L.u

L.u

Tyr

Ph.

S.r

Lsu

Trp

Lye

Giy

V.i

Lys

ThrS.rGly

GTA

TOG

ATC

ACA

GCC

ACC

ATG

CCA

TAC

GTG

GTC

CrC

ACT

CCC

CTG

CTC

CrC

COT

000

GTC

ACC

CTC

CCT

GGA

0CC

ATA

GAC

GGC

MC

AGA

OCA

TAC

CrC

AGC

897

v.1

Trp

Ii.

Thr

Ala

Thr

61st

Pro

Tyr

V.i

Vi

1su

Thr

Al.

L.u

L.u

L.u

Arg

up

v.i

Thr

Lu

Pro

Giy

Ais

Xis

Asp

Sly

Ii.

Arq

Ala

Tyr

Leu

Ser

299

TM S

TM 6 OTT

GAC

Trc

TAC

CGG

CrC

TGC

GAG

IXG

TCT

T

AlT

GAC

GCG

CCC

ACC

CAG

GTG

TGC

TTC

TCC

CTG

GGC

GTG

COG

TTC

COG

GTG

Cr0

ATC

CCC

TTC

TCC

999

vai

Asp

Ph.

Tyr

Arg

Iau

Cy.

Giu

Ala

S#{149}r Vi

Trp

Ii

Asp

Ala

Al.

Thr

Gin

V.1

Cys

Ph.

S.r

L.u

Gly

Vii

Giy

Ph.

Gly

Vai

L.u

Xl.

Ala

Ph.

Ssr

333

AGC

TAC

AAC

SAG

TIC

ACC

AAC

AAC

TGC

TAC

AGO

GAC

GCG

ATT

GTC

ACC

ACC

TCC

ATC

AAC

TOC

CTG

ACG

ACC

TTC

TCC

TCC

CCC

TTC

GTC

CrC

TTC

TCC

TTC

S.r

Tyr

Asn

Ly

Ph

Thr

Aan

Aso

Cys

Tyr

Arq

Asp

Ais

Ii.

V.1

Thr

Thr

S.r

Ii.

Asn

Cys

L.u

Thr

S.r

Ph#{149} S.r

S.r

Gly

Ph.

Vai

Vol

Ph.

Ssr

Ph.

367

CTG Lsu

GGG Giy

TAC Tyr

ATG Met

OCA Al.

CAG Gin

MG Ly.

CAC His

AG? Sr

GTG Vai

CCC Pro

ATC Ii.

COG Gly

GAC Asp

GTG V.1

0CC Ala

SAG Lys

GAC Asp

COG Gly

CCA Pro

CCC lip

CTG L.u

ATC Ii.

TTC Ph.

ATC Il.

MC Ii.

TAC Tyr

CCG Pro

GAA Glu

0CC Ala

ATC Ii.

CCC Al.

ACO Thr

CTC Leu

1203 401

ccT Pro

CTG Iau

rcc S.r

TCG S.r

GcC Ala

TOG Trp

GcG Ala

GTG V.1

GTC V.1

TTC Ph.

TTC Ph.

ATC Ii.

ATG M.t

CTG I..u

CTC ACC L#{149}u Thr

dO isu

GOT up

ATC ii.

GAC Asp

AGC S.r

0CC Ala

AIG Met

GOT Gly

OCT Gly

ATO Met

GAG Glu

ICA Oar

ITO V.1

ATC Il.

ACC TSr

CCC Gly

CTC Leu

AIC Il.

1305 435

GAT Asp

GAG Giu

TTC Ph.

CAG Gin

CTG Iau

CTG Iau

CAC His

AGA Arq

CAC His

CGT Arg

GAG Giu

CrC lao

Tic Ph.

ACG Thr

CTC 1.eu

TTC Ph.

ATC Il.

CTC V.1

CTG Leu

CCC Ala

ACC Thr

1TC Ph.

CTC t.u

C10 L.u

TCC Ser

CTG Tic 1..u Ph.

TiC Cys

GIC Vai

ACC Thr

MC Aso

GOT Gly

CCC Giy

ATC Ii.

I 407 469

TPC Tyr

GTC Vai

TTC Ph

ACG Thr

Cit Lu

CTG Iau

GAC Asp

CAT

IITT

GCA

0CC

GGC Giy

ACG Thr

TCC S.r

ATC Ii.

CTC L.u

Tfl Ph.

GGA Gly

0131 Vai

CTC Leu

ATC Ii.

GM Gb

GCC Ala

MC Ii.

COlA Giy

OrG Val

0CC Ala

TGG Trp

TTC Ph.

TAT Tyr

GOT Giy

OTT Vai

COG Giy

CAl Gin

1509 503

TTC

AGC Sr

GAC Asp

GAC Asp

ATC Ii.

CAG Gln

CAG Gln

ATG $.t

ACC Thr

GGG Gly

CAG Gin

COG Arg

CCC Pro

AGC Oar

Cli TAC L#{149}u Tyr

TOG Trp

CCC Arq

CrC Leo

Tic Cys

TOG Trp

SAG Lys

CTG Leu

Cit Val

AGC Ser

CCC Pro

TGC Cys

TTT Ph.

CTG L.u

TTC Ph.

GTG V.1

GTC V.1

GTG Val

1611 537

orc;l cc v.i Ssr_Il_V.lIThr

rrc Ph

AGPi Arq

CCC Pro

CCC Pro

CAC His

TAC Tyr

GGA Giy

CCC Ala

TAC Tyr

ATC 1i

TTC Ph.

CCC Pro

GAC Asp

TOG Trp

CCC Ala

MC Asn

CCC Ala

Crc Lsu

GGC GOp

TOG Trp

GIG Vsi

ATC Tie

0CC Ala

ACA Thr

TCC Oar

TCC Ser

ATG Met

0CC Ala

ATG M.t

GTG

AAG Ly.

TTC Ph.

TGC Cys

AGC Ser

CrC L.u

CC? Pro

COG Gly

TCC Ssr

ITT Ph.

CGA Arq

GAG Giu

AM Lys

CTG Leu

0CC Ala

TAC Tyr

0CC Ala

A1T Ii.

GCA Ala

CCC Pro

GAG Glu

SAG Lys

GAC Asp

COT Arg

GAG Glu

CrG Le

GTG V.1

GAC Asp

ACG Thr

CTC Lu

CCC Arg

CAC His

TOG Trp

CTC Lu

MG Lys

1860 GTG V.1 620

OTT

TM 7 1101

TM $

TM 9

*

*

Hi.IPh._Al._Ala

TMIO

*

TMII Ph.

_____________ GTC

AGC

CTC L.u

TM 12

ATT

v.1

CCC Pro

ATC Ii.

TAT Tyr

GCG Ala

GCC Ais

TAC Tyr

AGA Arq

GGG Giy

GAG Giu

GTG V.1

CGC Arq

CAG Gin

TTC Ph. -

TAG stop.

A000AGCAGAGACGAAGACCCCAGGAAGTCATCCCGCAATGGGAGAGACACGAACAAACCAAGGAAATCT

1713 571

1815 605

1 933

AAGTTTCGAGAGMAGGAGGGGCMCTTCThCTCTTCAACCTCTTACTGAAAACACP.AACCA

Fig. 1. Nucleic

.

3500

amino acid sequences of the human DAT cDNA clone. Nucleotides and amino acids are numbered (right side) start codon. The 1 2 TM domains are boxed. Senne and threonine residues existing in consensus phosphorylation motifs have been underlined (solid bars for PKA, open bars for PKC). ‘, Position of residues involved in the putative leucine/methionine zippers in TM-2 and -9. Potential asparagine glycosylation sites in the second extracellular loop are circled. consecutively

acid and deduced

from

the

putative

rat DAT, the human sequence possesses one extra glycine residue at position i99, in the third putative extracellular loop. The two transporters differ by 48 amino acid residues (Fig. 2), resulting in 92% overall identity. The bovine DAT has a 73amino acid residue longer carboxyl-terminal tail than the human DAT, and an overall homology of 84%. Compared with other recently cloned neurotransmitter uptake sites, the greatest homology is found with the NAT (66% and 75% with

tative sites for PKA and two for PKC are found (Fig. 1). Interestingly, there are no potential glycosylation sites on the proposed intracytoplasmic loops and no putative phosphorylation sites on the extracytoplasmic loops. This observation is well in line with the 12-TM segment model, in which both the carboxyl-terminal and amino-terminal domains are located in the intracellular space. Also noticeable is the periodic repeat of leucine residues at every seventh position in the putative ninth

conservative

TM

The

substitutions;

human

(asparagine extracellular According

DAT

possesses

see

Fig.

three

2).

potential

glycosylation

sites

residues 181, 188, and 205) in the large second loop, as compared with four in the rat DAT. to consensus phosphorylation sites (23), three pu-

segment.

“leucine

This type of structure (24). Interestingly, however, one of the leucine

zipper”

observed; a methionine. Biochemical

characterization

has been referred to as a in TM-2 a similar motif is residues is substituted by of

DA

uptake.

As

shown

386

Girosetal. TM

Hum

DA

M..SKSKCSNGLMSSVVAPA

EPNAVGPKE

ELILN.

C

‘JGV

LTSS

Rt

DA

M..SKSKCSJVGPMSSVVAPA

tSNAVGPRE

EIILIVJKE

NGV

LINS

Boy

DA

M.SEGRCSVjAl1MSSVVAI’A

FANAMGPKA

ELVLIVKE

NGV

LINS

MLLARMNPthJQPENNGADTGPEQPLRARKT

IIui, Nor

ELLVh

ERNGV

LI I

PRQSPVEA

RET

PPQTPVEA

RET

SK

RET

SK

LLNPPQSPTEA LLAPRDGD..A

....

K

PRET

IDFLLSVIGFA ADFLLSVGFA K

IDFLLS74

GFA

TM2

hum

DA

TM

VDLANVWRFPYLCYKNGGGAFt

p

LL[MV1A

PLFYMELALG

FjNREGAAJGJVWKICPI

KGV

FT

ILlS

Rat DA

VDLANVWR

p

LLFjMVIA

PLFYMELALG

FNREGAAGIVWKICPV

KGV

FT

ILISFYVG

Boo

VDLANVWRFI’YLCYKNGGGAFL

DA

Horn

Nor

VD

Hun,

DA

FPYLCYKNGGGAF

LAN

VWR

PYLFjP4MV

F I’ Y LCY

KNGGGAF

1P

A

TI4LIIA

FNREGAAGfrWK!CPI

RGV

Yl

ILISLYIG

PLFYMELALG

YNREGA4IjVWKICPF

KGV

YA

ILIALYVG

NVI1A%

LIfL.SSF

NVlIA%

L

YF

Boy

NVIlAi

L

YLLSSF

TELP

TICNHS

NSPRIcSILARAPN

LSSF

LNLP

TDjGII

NSPNT[PKLLNGS..VLG1HITJKYSKYKFjTPAAEIFYIERGVLHLFESSG1H

DA

NVlIAVSLY

Nor

7700

TM

II urn

ri

LGPPRVQI.T

DA

1Lt

L7

Bor

DA

ILLGPPR IELGP!’R

Iloor Nor

11-4NNS

s

GID

Il-cJNN

S

GID

LP

S...SGPrGJTjSR...1jTPAAEJYFERGVLHLI-ES

4

TM

CLVLIV

VQLLLCL

,

VLILYFSLWKGVKTSGKVVWI

TA

VLfLYFSLWKGVKTSGKVVWI

TA

-

VLT

P

V

P

LLLRGVftiIIA]ID

IRAYLS

DFYRL

E

SV

LLLRGVTLPGft4MD

IRAYLS

DF

RL

E

SV

VLF

LLLR

I

AYLS

DF

RL

E

SV

VLF

LLVIGVjTLPGASN

I

AYLHIDF

I

TM

6

GID

5

P

LP

Vj’.jVI7.LYFSLWKGVKTSGKVVWITA

TM

Y

TEL!’

QLTSCLVLIVI

[1GL1’

Y

M

CLVLNI\’LJLYPSLWKGVKTSGKVVWITA VQLT

IITLPGAVD

RLKE

V

7

lnDA

VlDAATfV

FSL

vjrjGvLIArssYNK

NNCYRDA1V[9j

SI

CLIS

SSGFV

FSFLGYMQK4SNPIGPV

RatDA

VlI)AA1V

PSI

VFGVLIAFSISYNK

NNCYRDAIIIT

SI

S

IS

SSGFV

FSFLGYMI6Q)4F4PIRDV

TEPGL

UovDA

VIDAAIIiI

FSL

4LIGVLIAFSISYNK

1jNNCYRDAII1j

v

S

IS

SSG

FSFLGYMQKj14SIPIGDV

KLPGL

IlumNor

lDAATjI

FSL

II

II1IyJLAJ

TIPLSS

IFIIYPEAI

TLPLSS

CITS

TM

DA

RtDA BoD.t

IJ1JYI)EAITlPLSS\

IINo

VFIIYPEAISTLS

IL

IYVITLL

D.. D.

1h)

D.

y\I:TLL

AVFFL A STF

VFF

9

1DS84MGGMESIVITGLI[EF

LL

RHRELF

LFIVL

TFLLSLP

Vf\GG

LL

L

IDS4MGGM[SIVITGLV4EF

LL

RHRELF

LGIVL

TFLLSLF

V1GG

RI-IRELF

LLVVL

TFLLSLF

RIIRKLF

FGVTFSTFLL

LL

L

IDSAMGGMESJVITGLAGEF

L

LDSSMGGiILAVITGLA[

L

I

L

10

TM

VIj.GG

LF

IjKGG

Ii

IFAAGTSILFCVII[A!G\4VFYG\G

rs

Dl DI’Q

QiT

PS

QI’ps1LYwRLcwKLvsrcrLLFvvvvsIv1iRil’I! QRPNLYWRLCWKLVSP FLLYVVVVSIVTIFRJPII’Il

PS

DI

Q.

RRPSILYWRLCWK

Q

PRPc4LYWRLCWKJ.VSP

LIG

4VPYGV

VS

FLL

VVVVSIATFRfrIPI1

FLL.VVVVSIINiKJPJLT

12

YGAYIPI

V,NALG\\\IATSSANVPIYA

Rut

YGAYIFI

V

YK

NALG#{188}II

SLPGSPRE

1SSiANVI’IYATYKI(SLIGSFRE

YGAYVFPEV

TALGD,IAASSiS\VI’IYA

lion, Nor

YD

%V\GS\

B’D.

RIVRKIIKSCIVVPSIOGI’GRGGI’PTl’SPRLACIITRAFP%VTGAPPVPRELTPPSICRCVI’PLVCAlIPAVESTGLCSVY

YIPPPV

GL

L

MLL

AVPF

IluinDA

DA

TE

IFAAGTSILI’GVLILAIG4VFYGQ

TM

Uo

AIFSILGYMAHEKhjNIEDV

LL

1Y\I1LLI1FAAGTSILIAVL.EAIG\SIVPYG\DIiPSNDI

D

SG

TM

VAVFFI

IPAAGTSILIGVL.

IYVF1LL

V

KD(PGL

8

TM

1Lt

3

YVG

PLFYMELALG

Rat DA

SSF

I

1DFLLSVGFA

I

YKL

SLPGSSRE

LSSiVLVPIVIYKLSI

LAY

I

PEKDRELNDRGEVRQFTLRIIWLKV

620

LA

I

PKKDIIQLVjDRGEVRQFTLRIIWLLL

619

LAY

I

PETEIIGRVjDSGGGAPVI-IAPPLARGVG

621

I

PENEIIIILNAQRDIRQFQLQIIWLAI

617

GSL%\ERLA

693

Fig. 2. Alignment of protein sequences from the human, bovine (20), and rat (17-19) DAT with the human norepinephrine transporter (1 4). The conserved regions are boxed. The putative transmembrane domains have been overlined. There is a possibility that the apparent longer carboxylterminal sequence for the bovine DAT might be due to a sequencing error, giving rise to a frame shift at residue 634 (T. Usdin and M. J. Brownstein, personal

communication).

on Fig. 3A, transfection of Ltk cells with the cDNA established in these cells a [H]DA uptake was

time

the

uptake

DA.

At

and

Na buffer

37#{176} and

accumulation plateau these

Replacement decreased

in the

was was

dependent. dramatically linear

reached,

particular

over lasting

of 7-8

at

least

30

of

mm mm.

and

the

ability

of various

always

determined

period.

observed in untransfected Determination of the formed

by increasing 3B). The Eadie-Hoftsee yielded a monophasic constant, K,,,, of 1.2

No

significant

in Ltk cells kinetic constants

the

±

concentration transformation curve, with 0.3 iM and cells,

under

a

(data

DA

in

then

uptake

not shown). of uptake was

a

for

of

able

drugs after

standard

assay

for

potency sine >

per-

mine, file

series

uptake.

to compete

of magnitude. site

of human

a large

[1HJDA

for

was

of

five

independent

of compounds the on

mazindol

=

cocaine

>

DA

transport,

Fig. 4B, comparison 1) for the rat and

the

of 0.998 for >10 compounds. Various “classical” tricyclic

all

the

>

the

in the

Furby

existence

were 2 orders

of a single rank

GBR129O9

order =

>

CNS.

of

nomifen1-ampheta-

pharmacological rat

ex-

to compete

about

d-amphetamine

classical

out

compounds

The =

uptake.

ability

within

transporter.

as defined of the human

4A,

ligand,

GBR12783

closely

DA

experi-

carried

for their

with

the

bupropion

matches

was

in Fig.

labeled

is consistent

drugs

which

of DAT

As shown with

This

these

error

characterization

characterization

was

condi-

standard

Pharmacological amining

of DA up to 30 tM (Fig. of the data (Fig. 3C) an apparent dissociation maximal velocity, V,,,00, of the

(average ± in triplicate).

ther

the

Because

was

tions ments,

[7H]

[1H]DA,

transport

incubation

nmol/min/1O

a period for

nM

Li of

properties,

a 4-5-mm

0.5

15

DAT that

by

uptake

[H]DA

with

±

of

of Na the

kinetic

to interfere

2.2

presence

human activity

pro-

As shown

in

K, values DAT

(from Fig. 4A and Table gave a correlation coefficient

antidepressant

molecules

have

a

Human

387

DAT Cloning

(I)

0 0 0

E C

Time

(mm)

(DAJ

Fig. 3. Characterization of the human DAT permanently (#{149}) or LiCl (0). B, Saturation of uptake by increasing triplicate.

i.tM

expressed in Ltk cells. A, Time course of [3H]DA (1 5 nM) uptake in the presence of NaCI concentrations of DA (0-30 MM). Results are the mean of three to five determinations, in

B

A 120

4

100

5 0

C 0 U

6

0

0

7

0 0.

8

9 i-

i-

i#{252}- 10.6 Inhibitor (M)

iO-5

9

10

8

7

6

Hum DAT

(-Log

5

4

Kl (M))

Fig. 4. Pharmacological characterization of the human DAT clone. A, [3H]DA uptake competition assay in Ltk cells permanently transfected with the human DAT. The cells (2.5 x 1 0 cells/well) were preincubated with various concentrations of the compounds and then with [3HJDA for 5 mm, as described in Experimental Procedures. B, Correlation between the K, values for different uptake inhibitors of the rat and human DAT expressed in Ltk cells. The K, values were determined using the EBDA-LIGAND program (34). All curves were best fitted to a single class of inhibitor binding site. The results presented here are the means of three different experiments with seven concentrations of compound for each value.

moderate to low affinity for the human DAT. Moreover, the major anti-Parkinsonian drug L-DOPA is able to block DA reuptake, with a K above 10 tM. Finally, the efficacy of various neurotransmitters to displace DA uptake has also been determined on Ltk cells transfected with the human and rat DAT (Table 1); they all had a low affinity for the DAT. Chromosome localization of the human DAT. In situ hybridization of the cDNA probe to normal human metaphase spreads revealed one specifically labeled site. Of 192 grains in 75 cells scored, 37 (19.3%) were found to be at the distal short arm of chromosome 5, band p15.2-15.3 (Fig. 5). No other chromosomal blot

analysis

sites of DNA

were

labeled from

above

19 rodent-human

background. somatic

fragments hamster

of 2.8,

2.4,

fragments

shown).

Thus,

guishable

1.9,

1.8,

of 2.8,

only

in the

1.5,

2.1,

the 3.6-kb

hybrids

and

1.7,

human

was

0.9

1.55,

kb,

and

five

and

0.9

kb

fragment

subject

that

have

human show

chromosome specific

hybridization

5 present

hybridization,

due

analysis.

human chroreported to

at a frequence possibly

not

was distin-

to discordance

The 3.6-kb fragment was found to segregate with mosome 5 (Table 2). One hybrid, GM/NA09930A, to

Chinese (data

of 0.12 to

the

failed limited

sensitivity.

Discussion

Southern cell

hy-

brids confirmed the chromosome assignment made by in situ hybridization (Table 2). The i-kb cDNA probe detected three EcoRI fragments of 3.6, 2.8, and 0.9 kb, six mouse-specific

Because included transporter proteins

of its

structural

features,

in the 12-TM domain family. Although have

been

characterized

sodiumthe first

the

can

be

and chloride-dependent members of this group

of

only

human

recently,

DAT

the

family

388

Giros eta!.

TABLE

1

already

Pharmacological characterization of human DAT and rat DAT permanently expressed in LtIC cells competitmon experiments were performed, as described for Fig. 4, in the presence of 15 nu [3H]DA. All K, values were determined using the EBDA-LIGAND program and represent

the average

determinations,

of three

to five independent

experiments

in triplicate

with a standard error of 10,000 >10,000 1,220 >10,000 >10,000 >10,000 >10,000 >60,000

Bupropion

Pimozide PCP d-Amphetamine

Nortriptyline Amitryptiline Zimelidine Desipramine I-Amphetamine

6-OH-DA MPP#{176} DA(Km)

L-DOPA GABA NA Serotonin Adrenaline

p

I1!E:i

17.5 22.5 22.5 16.8 60 109 322 521 667 677 2,160 3,400 8,000 25,000 12,000 22,600 >10,000 >10,000 890 >10,000 >10,000 >10,000 >10,000 >400,000

I..

comprises

homologs, nephrine,

the

bovine

have

human large

transporters consensus

PKC

and

one

site

for

sites are

are also somewhat

never

been

shown

with

of the different

Another

zipper

TM-2 As

pointed

most

in TM-2

in the

motifs protein

by

amino-terminus,

in loop

tail.

rat,

Most

of

and

leucine

is replaced et al. (24),

TM-2.

In

all cloned

and

bovine

of two

case

of the

methionine

is the

The

zipper

leucine

transporters the

one

DAT.

except

in TM-9

Leucine

and

certain

the

by a methionine.

to leucine.

whereas

the

treatment

is the presence

TM-9

site,

after

or inhibitors.

feature

identified

of

cells

activators

originally

bonding

for

is found

zippers

implicated

are

in protein-

DNA-binding

between

of DAT

efficiency of the

proteins

(24).

Because

ration

of reliable

have

been

21

and

a complete

I

22

formed.

More

jecting

Xenopus

could

theoretically

process

at the

that

has

obtained

from

of the

experimental

human

made

In

uptake

knowledge

been

case,

greatly

on rat

The time,

about

human

substantia the

prepaattempts

in humans

were

of

DA

synapto-

in the

has

attempts

difficulty

improve

a few

transport

study

with

the

a

level.

striatal

only

DA

such

extensive characterization. DAT allows, for the first

homoloIf such

difficulties

to characterize

oocytes

for

synaptic

synaptosomes,

recently,

-9 or

accumulated

studies

pharmacological

this

and

polymerization.

clusters of DAT might form on especially at the synapse. Such

molecules

been

somes.

(27).

motifs, endings,

of the

has

TM-2

intermolecular

for these nerve

0

II U H

puand

intracytoplasmic

in transfected

among

or heterologous

Most

15

the

PKA

by phosphorylation/dephosprospect will be to study

residue

interactions

uptake

14

on

Two

carboxyl-terminal

Landschulz

uptake

were

In addition,

found

However, it should be pointed out that the leucine zipper in TM-2 is interrupted by two prolines (amino acid residues, 101 and 112). The presence in the DAT proteins of one or two leucine zippers suggests that they might be used either for

the

13

in

human,

that

in the

on

common

proteins. are

second

structural

second

serotonin

clustering

31

kinase

alternative

role exists dopaminergic

23

protein

is conserved

rat

only

DAT

out

suitable

the

human

the

The sites

a feature

proteins.

found

in the

to be regulated an interesting

motifs

motif,

for TM

in the

interesting

leucine

loop,

of these

and

These

in common.

sites

are

rat

25).

conserved in the rat DAT. These observaunexpected, because DA transport has

Thus,

activity

gous

12

PKA

these tions

phorylation.

classical

site

the

(12-20, glycosylation

second

sites PKC

norepi-

recently,

external

domains

to one

species

human

characteristics

phosphorylation

consensus

addition

more

potential and

counting the

transporters

structural

cytoplasmic

one

GABA,

and,

extracellular

several

members,

human

rat proline

three

putative

intramolecular

14 13

never

(26),

been

performed

perby

nigra

in-

mRNA

did not allow ofthe human of the phar-

assay

molecular cloning direct assessment

32

macological

33 34

In order to have the most accurate determination of the pharmacological profile of human versus rat DA uptake, we estab-

35

lished

5 Fig.

has

to these tative

eight

and

serotonin,

the several

DAT

than

rat

rat

DA and

proteins the

more

i. e., the

5. In situ chromosomal assignment. Silver grain distribution along chromosome 5 after in situ hybridization with human DAT probes, illustrating the localization of a positive signal within the region 5p1 5.3. Three typical examples of positive labeling of chromosome 5 spreads are shown on the right.

as

and

characteristics

stable

similar

same

comparison,

cell as

clonal DAT DAT

ment

the

expressing (i.e.,

eukaryotic

and human for the rat with

lines

possible,

of DA

lines

both

same

cell line, cell

uptake

Ltk

cells).

expressing

published

DAT

human

under

expression

were chosen. The expressed in Ltk values

in the

similar

the

studies

pCMV5,

purpose

levels

values that cells are all in

conditions

vector,

For

using

CNS.

of the

of rat

were obtained in good agreerat

striatal

Human DAT Cloning TABLE

389

2

Segregation

of human

sequences

detected

by human

DAT probe

with

human-rodent

somatic

hybrids

Human

chromosome distribution between the various hybrid cell lines was analyzed by Southern blot, using a partial cDNA coding hybridization signal corresponds to a reactive EcoRl human DNA fragment distinguishable from any mouse or hamster DNA fragment. which the considered human chromosome is present at a frequency of 0.1 or more.

Presenceof sequence/ presence of chromosome

for the human DAT. A positive Informative hybrids are those in

Human chromosomes i

2

3

4

5

6

7

8

9

2 9

3 8

3 5

4 5

8 9

7 6

4 4

5 410

0

+1-

6

5

4

3

0

1

3

2

-/+

2

3

6

5

1

5

6

5

8

810

8

1

6979664788878683494

Concordant +/+ -/-

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

1

3 7

7 7

2 7

6 4

4 4

1

8

4

7

9

4

7

3

5

7

5

0

3

7

5

8

8

610

8

3

4

1

4

1

3

7

0

1

3

2

3

3

7

5

1

7

4

3

2

1

2

8

4

4

3

6

2

2

1

0

Discordant

Totaldiscordant hybrids Totalinformative hybrids % Discordant

synaptosomes

(Table

investigate

It

the

has

long

preferred

ment came

19

19

18

17

18

19

17

16

19

14

16

18

16

18

16

18

19

19

16

18

18

17

15

17

42

42

56

47

6

32

53

44

47

43

38

22

44

44

50

44

37

42

38

44

17

24

60

14

1), thus

validating

human

DAT

characteristics

been

suspected

that

endogenous

target

in humans (28). from the fact that

the

the

present

attempt

in these

cells.

human

responsible

for

A major drawback in rats the affinity

accumulates

into

symptoms

DAT

cocaine

to

is the

Using for

reinforce-

to this hypothesis of cocaine is higher

very

the

ability

cloned

value

synaptosomes

for

the

cloned

human

NAT

is 320

nM

(14);

this makes the human DAT the better target for cocaine, thus providing stronger support for the “DA hypothesis” of cocaine effects in humans. However, these differences are not important enough

to exclude

a dual

interaction

of cocaine

at both

trans-

porters. It has been observed that amphetamine can elicit schizophrenic behavior in nonpsychotic individuals or enhance schizophrenic syndromes in psychotic patients (for review, see Ref. 29). This is associated with a marked stereoselectivity, with damphetamine being more potent than its 1-stereoisomer. Despite some early controversies, the same stereoselectivity is observed in the inhibition of DA uptake in rodents, whereas the same potency is found for both enantiomers for the inhibition of NA uptake (29). From these observations, it was concluded

that

the

“amphetamine

quence of DA rather than confirm the stereoselectivity phetamine in the blockade 2),

confirming

likely

the

that

result

was

a conse-

NA

uptake inhibition. We of d-amphetamine versus of the DA uptake in humans

human

of an

psychosis”

amphetamine

effect

on

DA

psychosis

uptake.

hereby 1-am(Table is more

We

also

show

directly that PCP, which is thought to act primarily on a opioid receptors, can block DA uptake via the human DAT (7). Some

recently

developed

nomifensine,

and

human

and

DAT

hand,

bupropion, NAT

it is confirmed

nortriptyline

human for the

antidepressant

and

have

(Ref.

here

that

because

depression

symptoms

DAT

also

might

In humans,

high

(iO)

monkeys,

and

tricyclic

the

doses

be blocked

Table a low times

human are

used

it should

affinity 1).

On

DAT. in the

be

affinity more

other

like for the selective

Despite clinic

these

to alleviate

that

discrepancy

nation,

obtained cell lines has

for

rat

only

with

L-dopa,

sibly

be

involved

disorders for

that

also

etiology

the

gene

most

distal

diseases.

much

attention

possible

However,

this

been

with

linkage, long

sets gene

such of

of families

(31, encountered

a human different polymorphic,

phisms

have

cDNA

different

sets

members.

will alleles

of well

established

It already

appears

and

been

a likely

found

restriction for

restriction

I

Pifi,

B. Giros,

and

M.

Cs.

Caron,

manuscript

on the

Interestingly, 5, because been

with not studies search

a seen.

probes

been

rec-

confirmed

illustrate the for a single

multidimensional

respect,

the

permit

us

of

human

the

look with

the

fragment

DAT

length

enzymes in preparation.

for

DAT

families

that

dis-

characteriza-

to

of these

MPP

poten-

determined its mapping reveals

has

discovered

In this

to the

with

is localized

These in the

pos-

approach

5pl5.3.

5, has

32).

for

DAT

of the

phrenic

was

as schizophrenia.

segregation

at

liM

could

diseases have

the

1.2

psychiatric

to chromosome

of chromosome

responsible

DAT major

DAT

is

like

with

schizophrenia

which

arm

some

5,

devoted

cloned model

for which

of the

human

familial

difficulties

candidate

within

the

the

substances itself,

gene, we chromosomal

DAT, brain.

compared

of these

chromosome

has

the

in different

tion

of

linkage

ognizing

order

encoding

of

uptake

As an initial

or association

part

DA

of

the

com-

in the

between DA

nM,

that

subtype

is present

with

100

expla-

either

transported

tial polymorphism of the DAT chromosome localization. In situ

that

trivial

an essential

another

an impairment the

with the obtained.

et al. can be using rat striatal

when

in affinity

but

any

suggest

MPP,

is

in

a linkage

here,

putatively

or neurological

search

to

EC,,

striatal

of Javitch

synaptosome

or NA,

the

in rat

are missing

shift

striatal

K,,, value in synaptosomes (Table 1). As mentioned above,

highly

the

or

affinity

(8, 30).

that

transfected been

due

findings

uptake

not

6-OHDA,

used

MPP

the

observed

not

DAT

a similar

and

cells

produces

of 10 zM have

These

with

found

transport

to those

a better

Interestingly, DAT

Ltk

the conditions used.’

then disease

have

DA

values

similar

transfected for

which

DAT,

are

(8)

to inhibit

results

under

ponent

et al.

is presumably

because

potential

for the the

considered drugs. the MPTP metabolite

by most

mice,

mazindol,

antidepressants,

possess are 10-100

DAT and, therefore, human NAT than

like

comparable

14 and

desipramine,

differences,

drugs,

or human

and

of Parkinson’s

is 170 nM. Using

rat

This

Javitch

of MPP

synaptosomes

terminals

to those

synaptosomes,

for NA than DA uptake sites. In the present study we demonstrate that cocaine has a higher affinity for the human than for the rat DAT (50 versus 320 nM, respectively; Table 1). The K, of cocaine

dopaminergic similar

like

cogene

schizo-

gene

is

polymor-

TaqI

and

390

Giroseta!.

MspI.

Interestingly,

are known chromosome 5p

monosomy

severe

underlies

condition

Thus,

the

as

a unique

understanding

the

of this

and

the 5 will

that

potentially

chat”

the

important

and

genetic

its

(33),

system

host for

18.

for 19.

cells,

a better char-

cloning

corresponding

to further

a

a cDNA

into

pharmacological

the

17.

retardation. of

model

Moreover,

of

mental

expression

valuable

way

syndrome

and its

K. Prince,

assignments

5, the most frequent at 5p14-5pl5 (33). This

du

biochemical

localization

few gene

characterization

as

process.

open

“cri

and

well and

of

acteristics

mosome

fact

malformations

isolation,

DAT,

represent

the

with

cloning,

human

DAT

the

despite

on the short arm ofchromosome deletion in humans occurs

of the gene

20.

human on

chro-

characterization

of this

21.

marker. 22.

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reprint Medical Durham,

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