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Mutations in the amino-terminus impair amphetamine-induced efflux by inducing inward-facing conformations of the serotonin transporter

Background

The serotonin transporter (SERT) is responsible for the rapid termination of neurotransmission by removing serotonin from the synaptic cleft. We have explored the functional significance of a highly conserved threonine residue, at position 81, located within the amino-terminus of SERT.

Methods and results

Our findings indicate that, although the mutated transporters are normally targeted to the plasma membrane, they exhibit marked functional defects, such as: (i) a dramatic decrease in amphetamine-induced efflux (despite retaining normal amphetamine-induced currents), (ii) a 3-fold reduction in transporter turnover numbers (indicating impaired substrate translocation) and (iii) a 4-fold decrease in inhibitor affinity (due to a declined on-rate and an enhanced off-rate). The latter suggests that the mutated SERTs have a preference for inward-facing transporter conformations, as further supported by our molecular dynamics simulation experiments. By studying several H-bond and hydrophobic interactions of the wild-type T81, compared to its mutations to alanine or aspartate, structural changes were detected in the juxtamembrane N-terminus region of SERT. The computer models demonstrate a degradation of N-terminus interactions with IL2 and IL3 (which are likely involved in the transition between inward- and outward-facing SERT conformations) and a shift of the C-terminus away from the N-terminus upon mutation. Moreover, truncation of the first 64 residues of the amino-terminus results in functional defects comparable to the sole mutation of T81.

Conclusion

Hence, alterations in the amino-terminus region of SERT induce inward-facing transporter states, causing hindrance to conformational changes required for amphetamine-stimulated release, without simultaneously obstructing the transporter's ability to operate in its channel or uptake mode of action.

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Correspondence to Harald H Sitte.

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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 ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Sucic, S., Dallinger, S., Zdrazil, B. et al. Mutations in the amino-terminus impair amphetamine-induced efflux by inducing inward-facing conformations of the serotonin transporter. BMC Pharmacol 9, A12 (2009). https://doi.org/10.1186/1471-2210-9-S2-A12

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Keywords

  • Molecular Dynamic Simulation
  • Serotonin Transporter
  • Synaptic Cleft
  • Threonine Residue
  • Turnover Number