Control of serotonin transporter phosphorylation by conformational state.
Yuan-Wei Zhang, Benjamin E Turk, Gary Rudnick
Proceedings of the National Academy of Sciences of the United States of America May 17, 2016 DOI: 10.1073/pnas.1603282113 via PubMed
Summary
AI-generated from the abstractThe serotonin transporter (SERT) clears serotonin from synapses, and mutations in human SERT are linked to psychiatric disorders and autism. Phosphorylation at a specific site, threonine 276 near the cytoplasmic end of transmembrane helix 5, regulates SERT activity. Agents that stabilize the outward-open conformation of SERT, such as sodium ions and cocaine, decrease phosphorylation, while agents that stabilize the inward-open conformation, such as serotonin and ibogaine, increase phosphorylation. The opposing effects of cocaine and ibogaine are reversible by an excess of the other inhibitor. These findings suggest that serotonin transport itself activates a regulatory mechanism, possibly involving unwinding of the helix to allow phosphorylation.
Study at a glance
| Characteristics | Experimental study Peer reviewed |
|---|---|
| Population | HeLa cells expressing SERT and intact rat basophilic leukemia cells |
| Interventions | cocaine ibogaine serotonin |
| Topics | Ibogaine Serotonin |
| Keywords | Phosphorylation Serotonin transporter Conformational regulation |
| Citations | 53 |
| Key finding | Phosphorylation of SERT at threonine 276 is decreased by outward-open conformation stabilizers (Na+, cocaine) and increased by inward-open conformation stabilizers (5-HT, ibogaine), consistent with regulation activated by serotonin transport. |
Abstract
Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMP-dependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na(+) and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na(+) and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation.