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

Anderluh, Andreas; Klotzsch, Enrico; Reismann, Alexander W.A.F.; Brameshuber, Mario; Kudlacek, Oliver; Newman, Amy Hauck; Sitte, Harald H.; Schütz, Gerhard J.

doi:10.1074/jbc.m113.531632

Cited by 63 publications

(62 citation statements)

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“…We used the ‘Thinning out clusters while conserving stoichiometry' (TOCCSL) technique previously established in our lab7252627 to determine the oligomeric state of mGFP–SERT. TOCCSL extends fluorescence recovery after photobleaching (FRAP) to the level of single-molecule fluorescence microscopy (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To discriminate between stable association and rapid subunit exchange, we used the previously established method of repetitive TOCCSL runs7 (Fig. 2a).…”

Section: Resultsmentioning

confidence: 99%

“…Using single-molecule fluorescence microscopy, we have previously discovered that SERT forms a broad distribution of assemblies ranging from monomers up to pentamers7. The homo-association at the plasma membrane did not depend on SERT surface density and was stable at least over 10 min.…”

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confidence: 97%

“…Biochemical and spectroscopic studies have reported that SERT is present as oligomeric complexes at the plasma membrane56789. Likewise, oligomerization of a number of other NSS family members has been described10.…”

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confidence: 99%

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Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

et al. 2017

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The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by singlemolecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP 2 ). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

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Section: Resultsmentioning

confidence: 99%

“…To discriminate between stable association and rapid subunit exchange, we used the previously established method of repetitive TOCCSL runs7 (Fig. 2a).…”

Section: Resultsmentioning

confidence: 99%

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confidence: 97%

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confidence: 99%

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Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

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“…It is interesting that although there is substantial evidence for oligomerization of both DAT and SERT [28], the crystal structures, to date, have failed to capture the oligomeric forms [29–31]. This might be due to the formation of relatively weakly associated oligomers unable to withstand the rigours of the extraction and isolation processes required for structural studies.…”

Section: The Leut Superfamilymentioning

confidence: 99%

Transporter oligomerization: form and function

Alguel

Cameron

Diallinas

et al. 2016

Biochemical Society Transactions

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Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. Many transporters exist as oligomers in the membrane. Depending on the individual transport protein, oligomerization can have roles in membrane trafficking, function, regulation and turnover. For example, our recent studies on UapA, a nucleobase ascorbate transporter, from Aspergillus nidulans, have revealed both that dimerization of this protein is essential for correct trafficking to the membrane and the structural basis of how one UapA protomer can affect the function of the closely associated adjacent protomer. Here, we review the roles of oligomerization in many particularly well-studied transporters and transporter families.

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Amine Transporters

Reith¹

2020

Encyclopedia of Life Sciences

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Amine transporters in plasma membranes of nerve cells mediate the transport of dopamine, (nor)adrenaline and serotonin. These transporters are members of the larger Na + ‐ and Cl − ‐dependent neurotransmitter transporter family. All three transporters have been cloned, and the bacterial leucine transporter was the first prototypical structure available; this was followed by crystals of the drosophila dopamine transporter and the human serotonin transporter. Amine transporters are implicated in psychiatric disorders, such as attention‐deficit/hyperactivity disorder, schizophrenia, Parkinson disease, suicidal and aggressive‐impulsive behaviour, and are involved in affective disorders including depression. Amine transporters are important targets for psychostimulant drugs as well as antidepressants. Key Concepts Amine transporters in plasma membranes of nerve cells mediate the transport of dopamine, noradrenaline, adrenaline and serotonin through cotransport with Na + towards the cytosol where the Na + concentration is low. Amine uptake is best described by the alternating access model; accordingly, the transporter protein moves back and forth between outward‐ and inward‐facing conformations. Amine transporters are organised in multimeric structures, that is, oligomers consisting of varying numbers of protomers. How oligomerisation affects the function of each protomer is not fully understood. In addition to a primary central binding site for substrate and drugs, amine transporters have an extracellular vestibule, accommodating with appreciable flexibility varying small‐molecule allosteric ligands. Transporter polymorphisms and variants have been associated with increased risk for psychiatric diseases; a number of DAT variants, one‐point mutations, are known to cause parkinsonian symptoms starting at infancy in a hereditary recessive fashion. In addition to allosteric modulators, atypical inhibitors and releasers targeting amine transporters are subject of intense research.

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

Cited by 63 publications

References 50 publications

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

Transporter oligomerization: form and function

Amine Transporters

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