The N Terminus of Monoamine Transporters Is a Lever Required for the Action of Amphetamines

Abstract

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 of SERTT81A 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 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.

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

“…We should note, however, that even though uptake and trafficking properties are preserved in Tac fusion constructs tethering of the N terminus might influence other less apparent functions; e.g. TacSERT was found to display impaired reverse transport (46). This does not preclude the use of Tac fusion constructs in trafficking studies but underlines that although the constructs might be advantageous observations should be supported by alternative strategies as also done in the present study.…”

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

“…Cells were cultured at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM; PAA Laboratories) supplemented with 10% FBS (Invitrogen) and penicillin/ streptomycin. Functional expression of the mGFP-SERT was tested by uptake experiments essentially performed as described (26). In brief, for the determination of nonspecific uptake by mGFP-SERT, we used 10 M paroxetine (preincubation time 5 min), and […”

Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release*

“…Alternatively, the phosphorylations may promote a channel-like mode of the transporter leading to rapid bursts of DA efflux (47). The conformational flexibility of the N terminus for AMPH-induced efflux might also be important; hence, tethering the N terminus of SERT to the single transmembrane domain Tac (␣-subunit of the interleukin-2 receptor) was found to strongly impair AMPH-induced efflux (48).…”