A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression

Abstract

We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3ʹ-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.