Control of serotonin transporter phosphorylation by conformational state

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 cGMPdependent 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.(1) reported a rare missense mutation in the coding region of serotonin transporter (SERT) in two unrelated families. Within these families, individuals with the mutation (Ile425Val, or I425V) were more strongly affected with several psychiatric disorders, including obsessive-compulsive disorder and autism spectrum disorders. Subsequently, additional families with this mutation and others in SERT have been identified (2-8).In transfected cells, the I425V mutation led to enhanced 5-HT transport relative to WT SERT (5, 9). The enhanced transport was similar to the up-regulation of 5-HT transport that had been observed in rat basophilic leukemia (RBL-2H3) cells on stimulation of adenosine A3 receptors with subsequent NO formation and cGMP production (10). Results with heterologously expressed SERT suggest that the I425V mutation interferes with this pathway either by directly increasing basal SERT phosphorylation or by inhibiting SERT dephosphorylation (11).Mutation of serine and threonine residues on the cytoplasmic surface of SERT led to the identification of Thr276 as a potential cGMP-dependent phosphorylation site (12). From homology models based on prokaryotic and eukaryotic homologs, this residue is located near the cytoplasmic end of transmembrane helix 5 (TM5), a location that agrees with cysteine scanning accessibility studies (13) that also have shown this position to be poorly accessible to aqueous reagents. The unlikely location of a phosphorylation site in a transmembrane helix, and the inaccessibility of this position, cast some doubt on the proposal that Thr276 is the site of cGMP-stimulated phosphorylat...

Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter

“…Computational modeling predicts that the conformational equilibrium of DAT is shifted towards occluded-intermediate (OC) and IF states in the presence of substrates ( Cheng and Bahar, 2015 ). This prediction is supported by the experimental evidence in studies of SERT ( Zhang et al, 2016 ). We found that oligomerization and endocytosis of DAT in AIM-100-treated cells were significantly decreased by saturating concentrations (20–100 µM) of DAT substrates, dopamine (DA) and amphetamine (Amph) ( Figure 6 ).…”

A structural model of the human serotonin transporter in an outward-occluded state

“…To facilitate 5HT reuptake, the transporter needs to undergo distinct conformational changes. In principle, these changes expose the substrate binding site(s) to one side of the membrane at a time, according to the so-called alternatingaccess mechanism [9], support for which has been provided by structural modeling and biochemical experiments [10][11][12], and recently also by X-ray and cryo-EM crystallography [13][14][15]. Taken together, the hSERT transport cycle can be represented roughly as shown in Fig 1, albeit with the caveat that several important details remain elusive.…”

The role of transmembrane segment 5 (TM5) in Na2 release and the conformational transition of neurotransmitter:sodium symporters toward the inward-open state

“…In contrast, serotonin, in the presence of its symported ions Na ϩ and Cl Ϫ , enhanced accessibility at these positions, consistent with this region contributing to the substrate permeation pathway. Notably, there is strong evidence for regulation of SERT function through phosphorylation of Thr-276 (30), which aligns with Gly-171 in MhsT, adjacent to unwound Arg-174 -Met-180 in MhsT U . In the outward-facing SERT structure, this residue is located on the back side of the helical TM5i-IL2 region with its side chain forming an H-bond with the backbone of Ser-269 (13), in a position and orientation that would seem rather inaccessible to kinases.…”