Molecular cloning and characterization of an L-epinephrine transporter from sympathetic ganglia of the bullfrog, Rana catesbiana

Abstract

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 (V(max)) for the terminal products of catecholamine biosynthesis (L-Epi > L-NE >> DA), unlike hNET, in which a V(max) 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.