Feldman et al.

, Principles of Neuropsychopharmacology, Sinauer Associates

Drugs and the Brain

Neurotransmitter Transporters 1
Drugs Bind to Neurotransmitter Transporters

2013 California Institute of Technology

To most experts, success of serotonin-selective reuptake inhibitors (SSRIs)

implies participation of serotonergic systems in the brain
Rostral System
Midbrain Raphe nuclei

Caudal System

Feldman et al., Principles of Neuropsychopharmacology, Sinauer Associates

3 classes of proteins transport ions across membranes:

(transporter)

Ion channels that flux

many ions per event

Ion-coupled
transporters

Alberts et al., Molecular Biology of the Cell, Garland Science

Active transporters
(pumps) that split ATP

First, Na+-coupled cell membrane neurotransmitter transporters

utilize the Na+ concentration gradient originally established by the Na+ pump
(the Na+, K+-ATPase)

Presynaptic
terminals

Na+-coupled
cell membrane
serotonin
transporter

Na+-coupled
cell membrane
dopamine
transporter
cytosol

Na+
NH3+

HO
N
H

outside

Na+
HO
HO

H2
C

C
H2

NH3+
4

Second, H+-coupled vesicular neurotransmitter transporters

utilize the H+ concentration gradient originally established by the proton pump
(the H+-ATPase)
ATP-driven proton pump
~ 100 nm

proton-coupled
neurotransmitter transporter
Synaptic vesicle contains
neurotransmitter
and
H+
ATP

neurotransmitter

(1,000 to 10,000 molecules

of each)
cytosol

Plasma Membrane Neurotransmitter Transporter Genes

1. For serotonin (SERT), dopamine (DAT), and norephinephrine/noradrenaline (NET)
2. For GABA, four genes (next miniLecture)
3. For glutamate (various abbreviations), 5 genes.
Ligands have no known clinical uses.

Plasma Membrane Neurotransmitter transporters have two major jobs

1. They terminate the action of the neurotransmitter
2. They replenish the supply of neurotransmitter in the presynaptic terminal,
where it can be loaded into synaptic vesicles.

MDMA or Amphetamines Release Transmitter into the External Medium

H2C

H
N

CH3

3,4-methylenedioxy-N-methamphetamine
(MDMA, ecstasy, XTC)
pKa ~ 8.5

CH3

ATP-driven proton pump

proton-coupled
vesicular
serotonin transporter

depleted
serotonin vesicle
vesicle
serotonin

cytosol

MDMA is a
false substrate
for two
transporters

H+
MDMA

MDMA-H+

MDMA-H+
MDMA

MDMA dissipates
the vesicles H+
store, preventing
the vesicle from
pumping serotonin

SERT
serotonin

Rudnick & Wall,

PNAS 1992 7

Electrochemical measurements allow experiments to detect dopamine by

oxidizing the adjacent hydroxyl groups

cytosol
synaptic
cleft & surrounding HO
carbon fiber

HO

H2
C

C
H2

NH3+
8

Amphetamine

In dopamine transporter knockout mice (DAT -/-),

presynaptic stimuli (^) lead to longer individual dopamine release pulses;
but amphetamine fails to release dopamine

Jones et al .,
J Neurosci , 19989

Na+-coupled cell membrane neurotransmitter transporters:

major targets for drugs of therapy and abuse
Antidepressants
(SSRIs =
Selective serotonin
reuptake inhibitors):

Attention-deficit
disorder medications:
amphetamines

Drugs of abuse:
MDMA

Drugs of abuse:
cocaine
amphetamines
Presynaptic
terminals

Na+-coupled
cell membrane
serotonin
transporter

Na+-coupled
cell membrane
dopamine
transporter
cytosol

NH3+

HO
N
H

outside
HO
HO

H2
C

C
H2

NH3+
10

Feldman et al., Principles of Neuropsychopharmacology, Sinauer Associates

Drugs and the Brain

End of miniLecture

2013 California Institute of Technology

11

Drugs and the Brain

Neurotransmitter transporters 2
Neurotransmitter Transporters as Molecules

2013 California Institute of Technology

12

The Alternating Access Model for ion-coupled transporters

alternating access
Alberts 4th 11-9
Garland

13

Fluoxetine Bound to LeuT, a Bacterial Homologue of Neurotransmitter Transporters

A Static, Atomic-Scale View

outside

inside

PDB file: 3GWW

14

SSRIs bind tightly to, and stabilize, intermediate state(s) of the serotonin transporter.
A Dynamic, Low-Resolution Model

Cao et al., J Neurosci 1997

15

The GAT1 inhibitor, tiagabine, is used for epilepsy.

Tiagabine prolongs GABAergic inhibition,

presumably decreasing the possibility of excess neuronal firing.

How do GABA transporters normally clear all the ~10,000 transmitter molecules
from a single synaptic vesicle, within just a few ms?
.
Hypothesis: GABA transporters are present at very high densities.

We measured this density.

16

Back to the cerebellum, rich in GABA inhibition

Molecular
layer
Purkinje
cell layer
Ganule
cell layer

White
matter

Kandel et al.,
Principles of Neural Science
McGraw-Hill Professional
Publishing

17

How to localize and quantify GABA transporters

NH2
A fusion protein:
GABA transporter (GAT1)-GFP

18

Brain of GABA Transporter-GFP Knock-in Mouse

cerebellum

19
Chiu et al., J. Neurosci 2002

Molecular layer
(basket cells)

Purkinje cell layer

pinceaux stain
heavily, showing
soma-hillock veto

Granule cell layer

20

GAT1-GFP expression in cerebellum:

basket cell terminals in molecular layer,
GABA transporter density is ~1000/(m2)

50 m

21

Drugs and the Brain

End of Unit on Neurotransmitter Transporters

2013 California Institute of Technology

22