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 syn apses is terminated by uptake via specialized presynaptic transport pr oteins encoded by the DA transporter (DAT) and L-NE transporter (NET) genes, respectively. In some vertebrate neurons, particularly the symp athetic neurons of amphibians, L-NE is converted to L-epinephrine (L-E pi, 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 spec ialized for extracellular L-Epi clearance has yet to be identified. To pursue this issue, we cloned transporter cDNAs from bullfrog (Rana ca tesbiana) paravertebral sympathetic ganglia and characterized function al properties via heterologous expression in non-neuronal cells. A cDN A 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 am ino acids. Inferred fET protein sequence bears 75, 66, and 48% amino a cid identity with human NET, DAT, and the 5-hydroxytryptamine transpor ter (SERT), respectively. Transfection of fET confers Na+- and Cl--dep endent catecholamine uptake in HeLa cells. Uptake of [H-3]-L-NE by fET is inhibited by catecholamines in a stereospecific manner. L-Epi and DA inhibit fET-mediated [H-3]-L-NE uptake more potently than they inhi bit [H-3]-L-NE uptake by human NET (hNET), whereas L-NE exhibits equiv alent potency between the two carriers. Moreover, FET exhibits a great er maximal velocity (V-max) for the terminal products of catecholamine biosynthesis (L-Epi >L-NE much greater than DA), unlike hNET, in whic h a V-max rank order of L-NE >DA >L-Epi is observed. fET-mediated tran sport of catecholamines is sensitive to cocaine and tricyclic antidepr essants, with antagonist potencies significantly correlated with hNET inhibitor sensitivity. Amino acid conservation and divergence of fET w ith mammalian catecholamine transporters help define residues likely t o be involved in catecholamine recognition and translocation as well a s blockade by selective reuptake inhibitors.