Dopamine Transporter: Are Psychostimulants in Your Neighborhood Forcing You to Move?

Dopamine Transporter:

Are Psychostimulants in Your Neighborhood Forcing You to Move?

“Brain & Mind” Magazine

“Brain & Mind” Magazine

“Brain & Mind” Magazine

“Brain & Mind” Magazine

“Brain & Mind” Magazine

AMPH-STIMULATED DA EFFLUX

“Brain & Mind” Magazine

AMPH-INDUCED hDAT TRAFFICKING

“Brain & Mind” Magazine

AMPH-INDUCED hDAT TRAFFICKING

AMPH-STIMULATED DA EFFLUX

AMPH-INDUCED hDAT TRAFFICKING

REGULATION OF AMPH-INDUCED hDAT TRAFFICKING

UPTAKE

OUT

IN

EFFLUXES

OUT

IN

OUT

IN

DA

AMPH

Facilitated Exchange

OUT

IN

AMPH

NaDA

AMPH INCREASES [Na]i

rest 0 s 15 s 30 s 45 s 60 s 75 s

AMPH

AMPEROMETRY

Electrode Amperometric

700 mV

Patch Electrode

Dopamine

2e-

700 mV

DA

2e-20

0 pA

500 msec

500 msec

0.1

pA

0.2 pA

CTR

0.2 pA

AMPH

1 sec

0.2 pA

COC

BA

1

2

Patch Clamp

C **

*

200 msec

0.3 pA

0.1 pA

200 msec

*

B-20 mV

+40 mV

+100 mV

0.2 pA

0.4 pA

0.4 pA

0.2 pA

200 msec

0.2 pA

0.4 pA

*

*

C

STEP

2 sec

0.1 pA

Amperometry+700mV

A

DA

DA

1

2

Patch Clamp

*

**

AMPH-STIMULATED DA EFFLUX

AMPH-INDUCED hDAT TRAFFICKING

0 255

Total

Biotin.

CTR

AMPH Ins+AMPH0

50

100

150

*

% o

f c

on

tro

l

AMPH

Ins+

AMPH

AMPH

AMPH

Patch-Clamp and Confocal ImagingNoise Analysis of Steady-State Currents Transient and Steady-State Current AnalysisTransient and Steady-State Current Analysis

-160 mV-20 mV-20 mV

50 pA

250 msec

Transient Current(hDAT Number) Steady State Current

(hDAT Activity)

AMPH-induced hDAT current

NT

Cell-Surface

Total

CTR 5 10 15 20 30

hDAT

25 msec

-120 mV-20 mV

5 10 15 20

0.5

1.0

1.5 Biotinylation

Transient Charge (pC)

30

Time (min)

% o

f C

on

tro

l

-5 -10 -15 -20

-50

-100

-150

-200 r = 0.88p < 0.001slope = 8.35 ± 1.5

I (p

A)

Q (pC)

AMPH (min)

10

0 p

A

10

0 p

A

AMPH-induced hDAT currentWhole-cell Patch Clamp

AMPH

-160 -120 -80 -40 20 60 100

-2

-1

1

2

3

4

5

6

INITIAL

St e

ad

y S

tate

Cu

r re

nt

( I

/ I –

16 0

mV)

Membrane Potential (mV)

FINAL

AMPH-induced hDAT current

Final

Final

Tra

ns

ien

t C

ha

rge

Q /

Q -1

60

mV

-160 -120 -80 -40-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

Initial"ON"

Initial"OFF"

Membrane Potential (mV)

Whole-cell Patch Clamp

AMPH

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0

1.2

Ste

ad

y S

tate

Cu

rre

nt

I /

I m

ax

Time (min)

0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0

1.2

Tra

ns

ien

t C

ha

rge

Q /

Q I

ma

x

Time (min)

0.2 0.4 0.6 0.8 1.0 1.2

0.2

0.4

0.6

0.8

1.0

1.2 r = 0.87

Tra

ns

ien

t C

ha

rge

Q /

Q I

ma

x

Steady State CurrentI / I max

AMPH

AMPH

AMPH

Is AMPH-Induced Transporter Activity Responsible for hDAT Trafficking?

Basal 10 min 20 min

Y335A-DATAMPH

0 255

Y335A-DATAMPH+Zinc

0 10 20 30 40 50 600.5

1.0

1.5

2.0

2.5

3.0

3.5 WT

CTR

AMPH

Inte

rna

l F

luo

res

ce

nc

e(%

of

Co

ntr

ol)

Time (min)

0 10 20 30 40 50 600.5

1.0

1.5

2.0

2.5

3.0

3.5

CTR

AMPH

Y335A

Inte

rnal

Flu

ore

scen

ce(%

of

Co

ntr

ol)

Time (min)

0 10 20 30 40 50 600.5

1.0

1.5

2.0

2.5

3.0

3.5

WT

AMPH + Zn2+

Inte

rna

l F

luo

res

ce

nc

e(%

of

Co

ntr

ol)

Time (min)

0 10 20 30 40 50 600.5

1.0

1.5

2.0

2.5

3.0

3.5

Y335A

AMPH + Zn2+

Inte

rna

l F

luo

res

ce

nc

e

(% o

f C

on

tro

l)

Time (min)

AMPH-induced channel like activity AMPH-induced channel like activity supports DA Effluxsupports DA Efflux

hDAT is active until removed from hDAT is active until removed from the plasma membranethe plasma membrane

The activity of hDAT is required for The activity of hDAT is required for AMPH-induced traffickingAMPH-induced trafficking

CollaboratorsCollaborators

Christine SaundersJonathan Javitch

Bill ClarkeKelly Berg

NIDANIDA

James Lechleiter

Toni Shippenberg

Brandon LuteTeri Wei

Greta GalliErika Galli

Hongwei Wang

Kris KahligConcetta DipaceFrancesca BindaHabibeh KhoshboueiEmily SchwartzOliver Luo

Lyn Daws

Charles FranceUlrik Gether