Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking,stability, and activity

The function of the human norepinephrine transporter (hNET) depends on its presence at the cell surface. A role for the hNET C-terminus in trafficking the transporter to the surface has been suggested by the report of a bovine NET C-terminal splice variant that accumulates within heterologous host cells, and a human variant homolog has also been reported. We examined the relevance of the C-terminus of hNET to trafficking and function using transfected LLC-PK1 cells. The intracellular and surface expression of NET proteins was evaluated by Western blots, and their functional capacities were assessed using transport assays. We found that the C-terminal residues encoded by hNET 1a enable the efficient maturation and surface expression of hNET and therefore critically impact transporter activity. Alternative splicing causes the retention of immature hNETs within the cell, whereas introduced C-terminal deletions result in significant degradation. The loss of the terminal isoleucine alone (Delta617-hNET) is sufficient to cause the degradation of hNET, an effect that can be mimicked by nonconservative point mutations at the terminal position. The phenotype of Delta617-hNET is recapitulated in neuronal SK-N-MC cells, but is significantly less severe in HEK-293 cells, suggesting a role for host cell factors in enabling the biosynthetic progression of wild-type hNET. Additional proximal residues may act at other steps to affect the expression of the fully mature protein on the cell surface (Q608A) and to more directly affect transporter activity (F609A). Together our studies document a critical contribution of the hNET C-terminus to transporter trafficking, stability, and function.