Dominant Negative Isoform of Rat Norepinephrine Transporter Produced by Alternative RNA Splicing

Tetsurou Ikeda,Chieko Mitsuhata,Tomoyuki Sato,Toshihiro Dohi,Shigeo Kitayama,Katsuya Morita

doi:10.1074/jbc.274.16.10731

Abstract

We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3'-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a gamma-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission.

Highlights

The norepinephrine transporter (NET)1 at presynaptic nerve terminals mediates the uptake of released norepinephrine, resulting in the rapid termination of synaptic transmission and thereby controlling the fine tuning of neuronal activities
Cloning and Expression of Splicing Variants of Rat NET— From 5Ј- and 3Ј-rapid amplification of cDNA ends (RACE) and subsequent nested polymerase chain reaction (PCR), we obtained from 5Ј-RACE two positive groups of clones of different sizes and one positive pool of several clones from 3Ј-RACE
Because we failed to amplify the full-length cDNA from MarathonReadyTM cDNA used in RACE, the full-length cDNA of rat NET was cloned by PCR with reverse transcribed mRNA from rat brain and PC12 cells

Summary

Introduction

The norepinephrine transporter (NET)1 at presynaptic nerve terminals mediates the uptake of released norepinephrine, resulting in the rapid termination of synaptic transmission and thereby controlling the fine tuning of neuronal activities. To gain further understanding of the importance of these transporters in monoaminergic nervous system functions, we isolated and characterized cDNAs encoding rat NET that displayed RNA splice variants at the 3Ј-region including both coding and noncoding regions.

Results

Conclusion