Molecular Cloning and Characterization of anl-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 (f ET) was identified for which the cognate 3.1 kb mRNA is highly enriched in frog sympathetic ganglia. Sequence analysis of the f ET cDNA reveals an open reading frame coding for a protein of 630 amino acids. Inferred f ET protein sequence bears 75, 66, and 48% amino acid identity with human NET, DAT, and the 5-hydroxytryptamine transporter (SERT), respectively. Transfection of f ET confers Na ϩ -and Cl Ϫ -dependent catecholamine uptake in HeLa cells. Uptake of [ 3 H]-L-NE by f ET is inhibited by catecholamines in a stereospecific manner. L-Epi and DA inhibit f ET-mediated [3 H]-L-NE uptake more potently than they inhibit [ 3 H]-L-NE uptake by human NET (hNET), whereas L-NE exhibits equivalent potency between the two carriers. Moreover, f ET exhibits a greater maximal velocity (V max ) for the terminal products of catecholamine biosynthesis (L-Epi Ͼ L-NE Ͼ Ͼ DA), unlike hNET, in which a V max rank order of L-NE Ͼ DA Ͼ L-Epi is observed. f ET-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 f ET with mammalian catecholamine transporters help define residues likely to be involved in catecholamine recognition and translocation as well as blockade by selective reuptake inhibitors.

Ancestry of neuronal monoamine transporters in the Metazoa

“…Earthworm OAT, like many other transporters in the MAT clade, has considerable affinity for DA. This is not unusual, in that fish NET (Roubert et al, 2001) and frog ET (Apparsundaram et al, 1997) have higher affinity for DA than their nominal substrates, and arthropod OAT (Gallant et al, 2003) and mammalian NET (Roubert et al, 2001) have approximately equal affinity for DA and their nominal substrates. Kinetic data from other cloned MATs imply that a high affinity for DA does not necessarily mean that a transporter's primary role in situ is in DA re-uptake.…”

“…The fully sequenced genomes of animals representative of major bilaterian phyla, such as those of the flatworm, fruitfly, nematode and human, all contain serotonin transporters (SERTs) and dopamine transporters (DATs) (Pörzgen et al, 2001;Caveney and Donly, 2002). Other monoamine transporters expressed by monoaminergic neurons include an OA/TA transporter (OAT) associated with arthropod octopaminergic pathways (Malutan et al, 2002;Donly and Caveney, 2005) and the norepinephrine/epinephrine transporter (NET) associated with chordate adrenergic pathways (Apparsundaram et al, 1997;Roubert et al, 2001). These monoamine transporters are all metazoan members of the solute-linked carrier (SLC) family 6 [or alternatively sodium:neurotransmitter symporter (SNF) family] of Na + -dependent nutrient transporters that are expressed in both prokaryotes (Androutsellis-Theotokis et al, 2003;Yamashita et al, 2005) and eukaryotes (Chen et al, 2004;Boudko et al, 2005b).…”

Adrenaline and Noradrenaline: Partners and Actors in the Same Play"

“…The transporters show a similar configuration of intracellular and extracellular loops or related regions that contain phosphorylation and glycosylation sites and intracellular located amino-and carboxylterminal residues (Eisenhofer, 2001). Dopamine is cleared by dopamine transporter whilst NA and ADR are cleared by NA transporter (NET) (Figure 3) (Trendelenburg, 1988, Apparsundaram et al, 1997, Eisenhofer, 2001). These plasma membrane transporters have higher substrate specificity than vesicular transporters and may be viewed as targets ("receptors") for several drugs such as cocaine (NET) or fluoxetine (5-HT transporter) (Bönisch andBrüss, 2006, Capela et al, 2008), but also MDMA (both) (Capela et al, 2009).…”

“…In amphibians, an ADR specific transporter has been identified, with characteristics distinct from those of dopamine transporter or NET (Apparsundaram et al, 1997). In mammals, the presence of an ADR specific transporter was not yet clarified, however in situ hybridization studies indicate the absence of both dopamine transporter and NET messenger ribonucleic acid (mRNA) in ADR-synthesizing neurons in the brainstem of adult rats (Lorang et al, 1994).…”

“…In mammals, the presence of an ADR specific transporter was not yet clarified, however in situ hybridization studies indicate the absence of both dopamine transporter and NET messenger ribonucleic acid (mRNA) in ADR-synthesizing neurons in the brainstem of adult rats (Lorang et al, 1994). Thus, as ADR is also cleared at these sites, these terminals might have a distinct catecholamine transporter from dopamine transporter or NET; as an alternative, ADR may act as an endocrine regulator that does not require rapid reuptake in those neurons (Lorang et al, 1994, Apparsundaram et al, 1997.…”