Partitioning of the Serotonin Transporter into Lipid Microdomains Modulates Transport of Serotonin

Abstract

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V max ), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT. The serotonin transporter (SERT)1 is a member of a Na ϩ / Cl Ϫ -dependent family of integral membrane proteins that includes carriers for neurotransmitters, osmolytes, and nutrients (1-3). SERT, the norepinephrine transporter, and the dopamine transporter are most closely related, defining a subgroup characterized by a high degree of similarity both in sequence and pharmacological properties (4). SERT is responsible for the clearance of serotonin (5-hydroxytryptamine, 5HT) from the synaptic cleft following the release of the neurotransmitter (2).5HT is believed to accumulate inside the cell through cotransport with Na ϩ and Cl Ϫ and countertransport with K ϩ (4). SERT is an important pharmacological target for substances of abuse, such as cocaine, 3,4-methylenedioxymethamphetamine (Ecstasy), and p-chloramphetamine, and a variety of therapeutic antidepressants (1, 5, 6).Acute changes in SERT endogenous activity are likely to originate from local variations in 5HT concentration, cell surface distribution of SERT, interaction with regulatory proteins, or reversible post-translational modifications. To date SERT expression on the plasma membrane has been shown to be down-regulated by protein kinase C activation, following elevation of intracellular levels of Ca 2ϩ (7,8), or by phorbol 12-myristate 13-acetate treatment (9 -12). The SNARE protein syntaxin 1A (Syn 1A) is one of the few proteins known to modulate SERT function via protein-protein interaction by regulating the number of SERT molecules on the plasma membrane (13,14).To carry out clearance of extracellular 5HT, SERT must be inserted into the plasma membrane in an active form. This may require targeting the transport...

Selective serotonin reuptake inhibitors and raft inhibitors shorten the period of Period1-driven circadian bioluminescence rhythms in rat-1 fibroblasts

“…Results of real-timebioluminescence recording suggest that SSRI significantly shortened the period in rat-1 fibroblasts and the mouse SCN. SERT is associated with lipid microdomains, which are required for efficient serotonin transport activity (Magnani et al, 2004). Therefore, to further support our findings, SERT activity was blocked using a cholesterol chelating reagent methyl-β-cyclodextrin (β-CD) and lipid binding reagent xylazine.…”

“…SERT is an N-glycosylated integral membrane protein with 12 transmembrane regions, and has been shown to associate with lipid raft. The association with lipid raft on the cell membrane is required for an efficient 5-HT transporter activity (Magnani et al, 2004). The loss of interaction with lipid microdomains is detrimental to SERT function, possibly due to conformational changes that mainly affect the translocation of the substrate across the plasma membrane.…”

“…As the association of SERT with lipid rafts may represent a mechanism for regulating the SERT activity (Scanlon et al, 2001;Magnani et al, 2004), we further used a cholesterol chelating reagent β-CD to analyze the effects of cholesterol depletion, consequently the inhibition of SERT activity, on circadian rhythms. Our preliminary results showed that 0.5, 0.8, and 1 mM β-CD shortened the period, but cells were not able to survive in the presence of 2 mM β-CD.…”

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

“…A dependence of the uptake activity on the presence of cholesterol has been shown by Magnani et al (38). It may well be that the oligomerization is not directly based on protein-protein interaction but rather on an indirect clustering due to the local lipid environment, as was shown for glycosylphosphatidylinositol-anchored proteins (21,40).…”

“…Magnani et al have found that plasma membrane cholesterol is necessary for proper uptake activity of SERT (38). Because the presence of cholesterol has also been found to be important for the oligomerization of various membrane proteins (39,40), we wondered whether cholesterol also has an effect on the distribution of oligomeric states of SERT.…”