Molecular Studies of Neurotransmitter Transport 
and Regulation of Neural Gene Expression 
Susan G. Amara, Ph.D. — Assistant Investigator 
Dr. Amara is also Assistant Professor of Pharmacology at Yale University School of Medicine. She received 
her B.S. degree in biological sciences from Stanford University and her Ph.D. degree in physiology and 
pharmacology from the University of California, San Diego, where she worked with Michael Rosenfeld. Dr. 
Amara began her postdoctoral studies at San Diego, in the Eukaryotic Regulatory Biology Program. She 
continued these studies at Yale as a Fellow of the Life Sciences Research Foundation. 
COMMUNICATION between neurons depends 
on precise chemical signals that are released 
from one neuron and interpreted by adjacent neu- 
rons. This transfer of information is based on the 
synthesis of chemical neurotransmitters by the 
presynaptic cell, their release into the synaptic 
cleft, and their recognition by specific receptor 
proteins on the membrane of the postsynaptic 
cell. Our research focuses on the structure and 
expression of genes encoding proteins with im- 
portant roles in neurotransmission and synaptic 
function. 
We have been interested in a family of mem- 
brane proteins that play a key role in synaptic 
function — the sodium-dependent neurotransmit- 
ter transporters. For precise and efficient neuro- 
transmission, the release of the neurotransmitters 
into the synaptic cleft must parallel the rise and 
fall of presynaptic excitation. Inactivation of the 
majority of classic neurotransmitters usually is 
achieved by rapid reuptake into the presynaptic 
terminal or surrounding glial cells by specific, 
high-affinity neurotransmitter transporters. Neu- 
rotransmitter entry is coupled directly to the 
transmembrane sodium gradient, which drives 
the uphill transport. Neurotransmitter transport- 
ers not only have a central role in synaptic trans- 
mission, but they are also the site of action for a 
wide range of drugs with both therapeutic and 
abuse potential. Clinically important drugs that 
act directly on these transporters include the tri- 
cyclic antidepressants, which inhibit both norepi- 
nephrine and serotonin uptake; amphetamines, 
which inhibit norepinephrine and dopamine 
transport; and cocaine, which inhibits all three 
uptake systems. The impact of these agents on 
society underscores the importance of under- 
standing the molecular basis of their actions. 
Until recently little structural information has 
been available about the proteins responsible for 
reuptake of these neurotransmitters at synapses. 
Previously we used Xenopus oocytes as a system 
in which to express four major classes of trans- 
port activity in the brain — those for catechol- 
amine, indoleamine, choline, and excitatory and 
inhibitory amino acid (L-glutamate, 7-aminobu- 
tyric acid [GABA], and glycine) transport. After 
injection of mRNA prepared from various brain 
regions, uptake of the radiolabeled transmitters 
can be measured and the ion-dependence and 
pharmacologic specificity of these transport ac- 
tivities can be assessed in single oocytes. These 
and other studies, which confirmed that the trans- 
port activities are encoded by single mRNAs, 
paved the way for expression cloning of the genes 
encoding these carriers. 
This year we utilized a mammalian cell-based 
expression system to clone a human cDNA encod- 
ing a catecholamine transporter. The transport ac- 
tivity encoded by this cDNA has the substrate- 
specificity and pharmacologic properties of a 
norepinephrine transporter, including sensitivity 
to antidepressants, amphetamine, and cocaine. 
The single polypeptide contains regions impor- 
tant for binding and transport inhibition by co- 
caine and thus provides a first structural insight 
into the family of cocaine receptors. The cDNA 
sequence predicts a protein of 617 amino acids, 
with multiple hydrophobic regions consistent 
with the presence of 1 2 membrane-spanning do- 
mains. The amino acid sequence has significant 
homology with a recently cloned GABA trans- 
porter and thus identifies a new family of brain 
transport proteins. Studies are under way to de- 
fine and characterize other members of this car- 
rier family. 
The availability of a human norepinephrine 
transporter cDNA also provides an opportunity to 
investigate the contributions of structural ele- 
ments to transporter function and regulation. 
Many tricyclic antidepressants accomplish their 
therapeutic effects by blockade of reuptake 
of norepinephrine transporters and subsequent 
elevation of synaptic neurotransmitter concen- 
trations. Delineation of the structural basis of an- 
tidepressant binding and function through muta- 
genesis studies of the cloned transporter should 
aid in the development of more-selective thera- 
peutic agents for the treatment of human depres- 
sion. The availability of the human cDNA encod- 
ing the norepinephrine carrier also offers the first 
opportunity to determine whether alterations in 
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