Skip to main content
  • Meeting abstract
  • Open access
  • Published:

The N-terminus acts as a lever to support amphetamine-induced substrate efflux by the serotonin transporter


We have previously shown that a highly conserved threonine at position 81, in the amino terminus of SERT, plays a key role in SERT function, by driving the transporter into a state that supports amphetamine-induced efflux [1]. Truncation of the first 64 amino acids or tethering the N-terminus to an additional transmembrane helix both abolish amphetamine-induced efflux by SERT [1].


Alanine scanning mutagenesis was carried out along the N-terminal region of SERT to pinpoint the residues involved in maintaining amphetamine-induced efflux. Two residues at a time were replaced by alanine using a site-directed mutagenesis kit (Quikchange kit, Stratagene). The mutants were characterised by uptake, release and binding assays; surface expression was visualised by confocal microscopy. Conformational sensitivity of the N-terminus was examined by proteolytic cleavage. Tryptic digestion of membranes prepared from SERT-expressing cells was performed under different buffer conditions, in the absence or presence of various ligands, and detected by Western blotting.


Although all mutant SERTs generated in this study were targeted to the plasma membrane, some exhibited dramatic reductions in amphetamine-induced efflux. Moreover, they were all active with respect to [3H]serotonin uptake, showing no marked changes in the affinity or velocity of substrate uptake. The reduction in efflux did not result from impaired affinity of the mutants for amphetamines (shown by [3H]imipramine binding assays). In outward-facing conformations of SERT, the N-terminus is less susceptible to proteolytic digestion, possibly because it is shielded upon accompanying structural rearrangements. In inward-facing states, it is less susceptible to cleavage only when amphetamines are bound.


The region encompassing residues 22–32 may be a pivot for the movement of the N-terminus allowing amphetamine-induced release to occur. The mutagenesis and proteolysis data are consistent with the N-terminus of SERT acting as a lever, promoting substrate release by a second moiety of the oligomer.


  1. Sucic S, Dallinger S, Zdrazil B, Weissensteiner R, Jørgensen TN, Holy M, Kudlacek O, Seidel S, Cha JH, Gether U, Newman AH, Ecker GF, Freissmuth M, Sitte HH: The N terminus of monoamine transporters is a lever required for the action of amphetamines. J Biol Chem. 2010, 285: 10924-10938. 10.1074/jbc.M109.083154.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references


This work was supported by the Austrian Science Fund (SFB35).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Michael Freissmuth.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Sucic, S., Kern, C., Sitte, H.H. et al. The N-terminus acts as a lever to support amphetamine-induced substrate efflux by the serotonin transporter. BMC Pharmacol Toxicol 13 (Suppl 1), A54 (2012).

Download citation

  • Published:

  • DOI: