The N terminus of monoamine transporters is a lever required for the action of amphetamines

TY - JOUR

T1 - The N terminus of monoamine transporters is a lever required for the action of amphetamines

AU - Sucic, Sonja

AU - Dallinger, Stefan

AU - Zdrazil, Barbara

AU - Weissensteiner, René

AU - Jørgensen, Trine N

AU - Holy, Marion

AU - Kudlacek, Oliver

AU - Seidel, Stefan

AU - Cha, Joo Hwan

AU - Gether, Ulrik

AU - Newman, Amy H

AU - Ecker, Gerhard F

AU - Freissmuth, Michael

AU - Sitte, Harald H

N1 - Keywords: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Amphetamines; Animals; Cell Membrane; Electrophysiology; Humans; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Mutation; Oocytes; Phosphorylation; Protein Conformation; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serotonin Plasma Membrane Transport Proteins; Thermodynamics; Threonine; Xenopus laevis

PY - 2010/4/2

Y1 - 2010/4/2

N2 - 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 SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation ofSERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (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 SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A 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 theNterminus 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.

AB - 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 SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation ofSERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (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 SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A 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 theNterminus 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.

U2 - 10.1074/jbc.M109.083154

DO - 10.1074/jbc.M109.083154

M3 - Journal article

C2 - 20118234

SN - 0021-9258

VL - 285

SP - 10924

EP - 10938

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 14

ER -