Expression cloning of a human norepinephrine transporter

Neurotransmitter transporters terminate synaptic signalling by a rapid reaccumulation of neurotransmitter into presynaptic terminals in the central and peripheral nervous system. Uptake systems for the biogenic amines are the initial site of action for therapeutic antidepressants and drugs such as cocaine and the amphetamines. To isolate a clone for a catecholamine transporter, a mammalian cell expression cloning strategy was developed, based on the hypothesis that a single RNA species would suffice to reconstitute transport activity. COS-1 cells transfected with cDNA libraries derived from the SK-N-SH human neuroblastoma cell line were assessed for their ability to accumulate ({dollar}sp{lcub}125{rcub}{dollar}I) meta-iodobenzylguanidine, a molecule related to norepinephrine and carried by its transporter. Plasmid DNA was isolated from transporter-bearing transfectants and this enriched pool was subdivided to obtain a single clone. Norepinephrine uptake was examined in HeLa cells following transfection with the isolated clone (pNET), and found to be sodium-dependent and sensitive to a number of inhibitors including antidepressants, amphetamine, cocaine, and mazindol. The rank order of potency and the absolute K{dollar}sb{lcub}rm i{rcub}{dollar} values for the inhibitors examined were comparable to those obtained in synaptosomal and tissue studies of norepinephrine uptake. The molecule was modeled with 11-13 membrane spanning regions based on hydrophobicity analysis. Translation of the cDNA indicated primary amino acid sequence identity of 46% with the recently cloned GABA transporter, identifying a new gene family for neurotransmitter transporters. The transporter gene was localized to chromosome 16 by PCR. Northern blot analysis indicated the presence of a 3.6 and a 5.8 kb species in select tissues and brain regions. The two species were demonstrated to differ near their 3{dollar}spprime{dollar} ends. The gene for an apparent transporter isoform was also isolated ({dollar}Delta{dollar}NET) which has an in-frame deletion that removes the putative 5th membrane spanning region. While incapable of transporting monoamines when transfected alone into HeLa cells, {dollar}Delta{dollar}NET appears able to augment transport of norepinephrine when co-transfected with pNET. In summary, the gene for a functional human norepinephrine transporter has been isolated with the appropriate pharmacology, kinetics and regional message distribution for such a transporter.