Abstract
Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.
Highlights
The serotonin transporter (SERT) mediates neuronal reuptake of serotonin and is inhibited by most antidepressants
Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT
We found a novel high affinity outwardfacing conformational state of the human SERT induced by serotonin
Summary
The serotonin transporter (SERT) mediates neuronal reuptake of serotonin and is inhibited by most antidepressants. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. Accessibility of S277C in the MTS-inert C5X background has earlier been shown to function as a good measure of conformational changes upon ligand and ion binding [31] Using this construct it has been shown that serotonin induces a more inward-facing conformation in SERT in the presence of both sodium and chloride ions [31]. The ionic requirements for this new conformational response to serotonin mirrors the ionic requirements for translocation and suggest that this high affinity outward-facing conformation is a new state in the transport cycle of serotonin, which has not been described earlier
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