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

Anderluh, Andreas; Klotzsch, Enrico; Kumar, Vivek; Newman, Amy Hauck; Sitte, Harald H.; Schüetz, Gerhard J.

doi:10.1016/j.bpj.2014.11.1753

Cited by 6 publications

(10 citation statements)

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“…The fluorescent cocaine analog JHC1-64 has previously been shown to inhibit uptake in SERT, the norepinephrine transporter, and DAT (18); JHC1-64 has also been used to demonstrate internalization of DAT in CAD cells, as well as in dopaminergic neurons, and importantly, the compound did not affect trafficking of the transporter per se (10,17). To support previous findings that the fluorescent cocaine analog JHC1-64 labels and visualizes transiently expressed SERT in the nanomolar range (19,34), we incubated CAD cells transiently expressing SERT tagged at the N terminus with EGFP (EGFP-SERT) with 10 nM JHC1-64 alone or together with 1 M paroxetine, a SERT-specific blocker. Confocal live imaging of the cells showed clear EGFP fluorescence corresponding to the plasma membrane of a subset of cells, conceivably corresponding to the cells transfected with EGFP-SERT.…”

Section: Resultsmentioning

confidence: 91%

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

Rahbek-Clemmensen

Bay

Eriksen

et al. 2014

Journal of Biological Chemistry

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Background: SERT is a target for antidepressants, but little is known about its constitutive cellular trafficking properties. Results: SERT undergoes marked constitutive internalization, and internalized SERT co-localizes primarily with markers of the late endosomal/lysosomal pathway. Conclusion: SERT is primarily sorted to degradation rather than to recycling. Significance: The findings are important for our general understanding of how SERT regulates serotonin signaling.

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Section: Resultsmentioning

confidence: 91%

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

Rahbek-Clemmensen

Bay

Eriksen

et al. 2014

Journal of Biological Chemistry

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“…For the related serotonin transporter (SERT), the co‐existence of different degrees of oligomerization at the cell surface has been shown by Anderluh et al . (,b). In the case of DAT, evidence suggests its quaternization into tetramers or dimers of dimers (Hastrup et al .…”

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confidence: 99%

Functional properties of dopamine transporter oligomers after copper linking

Zhen

Reith

2017

Journal of Neurochemistry

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Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross-linked natural DAT dimers in LLC-PK cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross-linked by CuP showed normal DA uptake with DA K at ~ 0.5 μM and DA efflux with basal and amphetamine-induced DA efflux as much as control values. The cocaine analog 2β-carbomethoxy-3β-[4-fluorophenyl]-tropane (CFT) was capable to bind to copper-cross-linked DATs, albeit with an affinity more than fivefold decreased (K of CFT = 109 nM after cross-linking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer.

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“…In this regard, a recent study shows the co‐existence of different degrees of oligomerization at the cell surface for SERT (Anderluh et al . ). The distribution of oligomeric states may be defined by some unknown mechanism in the endoplasmic reticulum, and experiments tracking single SERT molecules in the endoplasmic reticulum have begun (Anderluh et al .…”

Section: Resultsmentioning

confidence: 97%

Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities

Zhen

Antonio

Cheng

et al. 2015

Journal of Neurochemistry

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Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [3H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious “mixing” experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among 5 pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [3H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer.

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

Abstract: Many RFPs are prone to form artificial puncta when labeling proteins in secretory pathway, which may severely impede their further uses in living cell 322a Monday,

Cited by 6 publications

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The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

Functional properties of dopamine transporter oligomers after copper linking

Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities

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