Abstract
The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule 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 (PIP2). 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.
Highlights
The human serotonin transporter mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling
We have recently found that the functional activity of serotonin transporter (SERT) and the dopamine transporter (DAT) was influenced by PIP2 binding[20,21]: upon enzymatic depletion of PIP2 or mutation of identified PIP2 binding sites, amphetamine-induced substrate efflux was markedly reduced whereas uptake rates were essentially unaffected
Plasma membrane-localized Monomeric GFP (mGFP)–SERT was recorded at the bottom cell membrane via total internal reflection fluorescence (TIRF) microscopy
Summary
The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. The human serotonin transporter (SERT) is a 12-pass transmembrane protein targeted to presynaptic nerve terminals and belongs to the neurotransmitter: sodium symporter (NSS) or solute carrier 6 (SLC6) family[1]. These transmembrane proteins mediate the high-affinity uptake of neurotransmitters from the synaptic cleft and are, of pivotal importance for synaptic signal transmission by terminating chemical signal transduction between neurons. We have recently found that the functional activity of SERT and the dopamine transporter (DAT) was influenced by PIP2 binding[20,21]: upon enzymatic depletion of PIP2 or mutation of identified PIP2 binding sites, amphetamine-induced substrate efflux was markedly reduced whereas uptake rates were essentially unaffected. While the oligomeric configuration does not seem to influence neurotransmitter uptake[11], amphetamineinduced neurotransmitter release has been shown to rely on the quaternary arrangement[15]
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