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Volume 17, Number 8,
Issue of April 15, 1997
pp. 2691-2702
Copyright ©1997 Society for Neuroscience
Molecular Cloning and Characterization of an
L-Epinephrine Transporter from Sympathetic Ganglia of the
Bullfrog, Rana catesbiana
Received Dec. 5, 1996; revised Jan. 28, 1997; accepted Jan. 31, 1997.
Subramaniam Apparsundaram1,
Kimberly R. Moore2,
M. D. Malone1,
H. Criss Hartzell2, and
Randy D. Blakely1
1 Department of Pharmacology and Center for
Molecular Neuroscience, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232, and 2 Department of Anatomy
and Cell Biology, Emory University School of Medicine, Atlanta, Georgia
30322
Chemical signaling by dopamine (DA) and
L-norepinephrine (L-NE) at synapses is
terminated by uptake via specialized presynaptic transport proteins
encoded by the DA transporter (DAT) and L-NE transporter
(NET) genes, respectively. In some vertebrate neurons, particularly the
sympathetic neurons of amphibians, L-NE is converted to
L-epinephrine (L-Epi, adrenaline) and released
as the primary neurotransmitter. Although evidence exists for a
molecularly distinct L-Epi transporter (ET) in the
vertebrate brain and peripheral nervous system, a transporter
specialized for extracellular L-Epi clearance has yet to be
identified. To pursue this issue, we cloned transporter cDNAs from
bullfrog (Rana catesbiana) paravertebral sympathetic
ganglia and characterized functional properties via heterologous
expression in non-neuronal cells. A cDNA of 2514 bp (fET) was
identified for which the cognate 3.1 kb mRNA is highly enriched in frog
sympathetic ganglia. Sequence analysis of the fET cDNA reveals
an open reading frame coding for a protein of 630 amino acids. Inferred
fET protein sequence bears 75, 66, and 48% amino acid identity
with human NET, DAT, and the 5-hydroxytryptamine transporter (SERT),
respectively. Transfection of fET confers Na+- and
Cl -dependent catecholamine uptake in HeLa cells. Uptake
of [3H]-L-NE by fET is
inhibited by catecholamines in a stereospecific manner.
L-Epi and DA inhibit fET-mediated
[3H]-L-NE uptake more potently than
they inhibit [3H]-L-NE uptake by human
NET (hNET), whereas L-NE exhibits equivalent potency
between the two carriers. Moreover, fET exhibits a greater maximal velocity (Vmax) for the terminal
products of catecholamine biosynthesis (L-Epi > L-NE DA), unlike hNET, in which a
Vmax rank order of L-NE > DA > L-Epi is observed. fET-mediated transport of catecholamines is sensitive to cocaine and tricyclic
antidepressants, with antagonist potencies significantly correlated
with hNET inhibitor sensitivity. Amino acid conservation and divergence
of fET with mammalian catecholamine transporters help define
residues likely to be involved in catecholamine recognition and
translocation as well as blockade by selective reuptake inhibitors.
Key words:
frog;
catecholamine;
epinephrine;
neurotransmitter;
antidepressant;
transporter
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