Research reportThe rat norepinephrine transporter: molecular cloning from PC12 cells and functional expression
Introduction
Neural transmission is rapidly terminated by reuptake of the neurotransmitter into presynaptic nerve terminals by the action of neurotransmitter transporters. Reuptake of released catecholamines (dopamine, norepinephrine) by high affinity, saturable, sodium- and chloride-dependent transport processes has been studied over a period of more than three decades [2]. Many studies concerning the high affinity norepinephrine transporter (NET) have taken advantage of the rat pheochromocytoma cell line PC12, known to express the rat NET (rNET) 3, 10, 14, 16, 20. The NET is a therapeutic target for tricyclic antidepressant drugs, such as desipramine, and a target for psychostimulants such as amphetamines. In PC12 cells this transporter sometimes is designated as a `dopamine transporter' since these cells contain predominantly dopamine in their storage vesicles and since tritiated dopamine was used as a substrate for uptake studies [32].
NETs were biochemically and pharmacologically characterized (for review see Ref. [13]) before molecular cloning revealed the primary structure of the human NET (hNET) [28]and the bovine NET (bNET) [24]. These transporters belong to the family of sodium- and chloride-dependent neurotransmitter transporters which share some typical structural features (for review see Ref. [1]and [2]). Hydropathy analysis predicts twelve potential transmembrane domains (TMs) and N- and C-termini located intracellularly. A large extracellular loop, containing potential sites for N-glycosylation is located between TM3 and TM4 and potential sites for phosphorylation by protein kinases are found at intracellular domains.
We here report the molecular cloning and characterization of a cDNA that was derived from PC12 cells and encodes the rNET. The rNET shows the structural and functional properties expected of a NET. Prior to this study, some putative, partial fragments of the rNET sequence had been published for use in in situ hybridization experiments, mRNA quantification and regulation studies 7, 25, 29, 31. All of these fragments were found to be parts of our cloned rNET cDNA.
Section snippets
Materials
PC12 (ATCC No. CRL 1721) and HEK293 (ATCC No. CRL 1573) cells were purchased from the American Type Culture Collection. DMEM and HAM′s F12 media were obtained from Sigma and RPMI 1640 was from Gibco/BRL. Cell culture flasks and 12-well plates were purchased from Falcon. Fetal calf serum, horse serum and TRIzol® reagent were from Gibco/BRL. Primers for PCR and sequencing were synthesized by MWG-Biotech. Avian myeloma virus (AMV) reverse transcriptase and the fmol® DNA cycle sequencing system
Results and discussion
The sequence of the rNET cDNA was elucidated by 5′- and 3′-RACE with PC12 cell first strand cDNA as template, starting with an antisense primer derived from a partial, potential rNET sequence [7]and a primer derived from the hNET sequence [28], as described in Section 2.2. Subsequent cycle sequencing of overlapping PCR products revealed the complete rNET sequence. Finally, full-length cDNA was produced by PCR amplification based on the new rNET sequence with the exception that the ATG start
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 400) and by a grant from the University of Queensland Round Three Quality Funds. We thank Petra Breiden for technical assistance, and we wish to express our appreciation to Upjohn for the donation of U-0521.
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