The N terminus of monoamine transporters is a lever required for the action of amphetamines.
The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERT(T81A) and SERT(T81D), suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERT(T81A) in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERT(T81A) (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERT(T81A) were comparable with those through the wild type transporter. Both abundant Na(+) entry and accumulation of SERT(T81A) in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.
Sucic
Sonja
S
Center of Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Medical University Vienna, A-1090 Vienna, Austria.
Dallinger
Stefan
S
Zdrazil
Barbara
B
Weissensteiner
René
R
Jørgensen
Trine N
TN
Holy
Marion
M
Kudlacek
Oliver
O
Seidel
Stefan
S
Cha
Joo Hwan
JH
Gether
Ulrik
U
Newman
Amy H
AH
Ecker
Gerhard F
GF
Freissmuth
Michael
M
Sitte
Harald H
HH
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000662:Amphetamines / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics; D013912:Threonine / Q000235:genetics; D014982:Xenopus laevis
D000662:Amphetamines; D050486:Serotonin Plasma Membrane Transport Proteins; D013912:Threonine