Molecular cloning and characterization of an L-epinephrine transporter from sympathetic ganglia of the bullfrog, Rana catesbiana.
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.
Apparsundaram
S
S
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Moore
K R
KR
Malone
M D
MD
Hartzell
H C
HC
Blakely
R D
RD
D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D050483:Dopamine Plasma Membrane Transport Proteins; D004837:Epinephrine / Q000378:metabolism / Q000494:pharmacology; D005728:Ganglia, Sympathetic / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011892:Rana catesbeiana; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050486:Serotonin Plasma Membrane Transport Proteins; D027981:Symporters; D014162:Transfection
D029845:Amphibian Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011994:Recombinant Proteins; C493227:SLC6A2 protein, human; C493214:SLC6A3 protein, human; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493228:Slc6a2 protein, mouse; C493226:Slc6a2 protein, rat; C493215:Slc6a3 protein, mouse; C493216:Slc6a3 protein, rat; C493231:Slc6a4 protein, mouse; C493232:Slc6a4 protein, rat; D027981:Symporters; C105261:epinephrine transporter, Rana catesbeiana; D004837:Epinephrine