EXPRESSION OF NEURAL GENES 
Susan G. Amara, Ph.D., Assistant Investigator 
Dr. Amara is concerned with the molecular bi- 
ology and regulation of genes encoding proteins 
with important roles in neurotransmission. Neuro- 
transmitter transporters have a central role in syn- 
aptic transmission and are the site of action for a 
wide range of clinically important drugs. Most 
recently the laboratory has pursued a molecular 
characterization of the proteins responsible for 
neurotransmitter reuptake and has developed a 
functional expression system for identifying the 
genes encoding these carriers. Other investigations 
have continued to explore the basic mechanisms 
for regulating both neuronal gene transcription 
and alternative pathways of pre-mRNA processing, 
using a neuropeptide gene family as a model 
system. 
I. Neurotransmitter Transporters. 
Despite a plethora of pharmacologically impor 
tant inhibitors of monoamine uptake (including co- 
caine, amphetamines, and the tricyclic antidepres- 
sants), little molecular detail is known about the 
proteins responsible for reuptake of neurotransmit- 
ters at these synapses. Even for acidic amino acid 
transporters, which are relatively more abundant 
in brain and have been the focus of detailed 
bioenergetic and kinetic studies, little structural in- 
formation is available, despite the significant roles 
these activities play in synaptic function and signal 
termination in the nervous system. Dr. Amara and 
her colleagues have been using microinjection of 
mRNA into Xenopus laevis oocytes to express four 
major classes of brain transport activity: catechola- 
mine, indoleamine, choline, and excitatory and in- 
hibitory amino acid transport. After injection of 
mRNA prepared from various brain regions, uptake 
of the radiolabeled transmitters can be measured in 
single oocytes and displays a pattern of regional 
distribution consistent with the known anatomical 
location of neurotransmitter-synthesizing cell bod- 
ies. Sodium dependence and pharmacologic speci- 
ficity of uptake can also be assessed in oocytes; 
these studies have confirmed that the transport ac- 
tivities induced by brain mRNA display properties 
very similar to the high-affinity, sodium-dependent 
transporters observed in brain slices and synapto- 
somal preparations. 
Studies on the L-glutamate carrier expressed from 
cerebellar mRNA have permitted the characteriza- 
tion of a transport activity from a population of 
well-defined glutamatergic neurons, the cerebellar 
granule cells. In these studies L-glutamate transport 
was observed to be sodium- and time-dependent, 
temperature sensitive, and saturable at micromolar 
substrate concentrations. In addition, cerebellar 
mRNA-induced glutamate uptake was inhibited by 
compounds known to block high-affinity uptake in 
brain slices and synaptosomes. Cerebellar forebrain 
and brain stem RNAs have been size-fractionated on 
sucrose gradients and assayed for transport of L-glu- 
tamate and GABA (7-aminobutyric acid). These 
studies reveal single, comigrating, pharmacologi- 
cally distinguishable peaks for L-glutamate and 
GABA transport, with maximal activity observed in 
fractions containing RNA in the 2.5-3.0 kb range. 
In contrast, both L-glutamate and GABA transport 
activities in forebrain appear to be encoded by 
larger mRNA species. Dihydrokainate fails to inhibit 
the cerebellar mRNA-induced L-glutamate trans- 
port, while significantly reducing forebrain and spi- 
nal cord mRNA-induced transport, supporting the 
presence of at least two forms of sodium-depen- 
dent L-glutamate transporters. Developmental stud- 
ies have demonstrated substrate- and region-spe- 
cific postnatal increases in L-glutamate, GABA, and 
glycine transport activity. The identification of the 
genes encoding these transporters will provide a 
basis for future studies on their heterogeneity and 
distribution and on the molecular mechanisms of 
transporter function and specificity; such experi- 
ments are in progress. 
II. Neuropeptide Gene Regulation. 
Previous studies on the structure and expression 
of the calcitonin/a-CGRP (calcitonin gene-related 
peptide) gene demonstrated several of its unique 
features. Alternative pre-mRNA splicing within cod- 
ing domains generates two mRNAs: calcitonin 
mRNA is made within the thyroid and a-CGRP 
mRNA is made in a variety of neuronal cell types. 
The gene consists of six exons: three upstream 
exons used in both mRNAs, a fourth exon contain- 
ing the calcitonin mRNA-specific domain and poly- 
adenylation site, and two downstream CGRP 
mRNA-specific exons followed by a second poly- 
adenylation site. Another form of CGRP, P-CGRF| is 
encoded on a distinct gene. The calcitonin/a-CGRP 
and P-CGRP genes code for two nearly identical 
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