The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of the serotonin transporter. Although recent crystal structures have identified several potential cholesterol binding sites it is unclear whether any of these potential cholesterol sites are occupieed by cholesterol and functionally relevant. In the present study we focus on the conserved Cholesterol Site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5 and TM7. By molecular dynamics simulations we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation and vice versa mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity and turn-over rates. Taken together we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.

The serotonin transporter (SERT) belongs to the SLC6 family of sodium-and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast twohybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.

Background: Psilocybin is a serotonergic psychedelic found in “magic mushrooms” with a putative therapeutic potential for treatment-resistant depression, anxiety, obsessive-compulsive disorder, and addiction. In rodents, psilocybin acutely induces plasticity-related immediate early genes in cortical tissue; however, studies into the effects on subcortical regions, of different doses, and the subsequent translation of corresponding proteins are lacking. Methods: We examined the acute effects of a single administration of psilocybin (0.5–20 mg/kg) on the expression of selected genes in the prefrontal cortex and hippocampus. In total, 46 target genes and eight reference genes were assessed using real-time quantitative polymerase chain reaction. Corresponding protein levels of the three most commonly regulated genes were assessed using Western blotting. Results: In the prefrontal cortex, psilocybin increased the expression of Cebpb, c-Fos, Dups1, Fosb, Junb, Iκβ-α, Nr4a1, P11, Psd95, and Sgk1, and decreased the expression of Clk1. In the hippocampus, psilocybin strongly increased the expression of Arrdc2, Dusp1, Iκβ-α, and Sgk1 in a dose-dependent manner, and decreased the expression of Arc, Clk1, Egr2, and Ptgs2. Protein levels of Sgk1, Dusp1, and Iκβ-α showed only partial agreement with transcriptional patterns, stressing the importance of assessing downstream translation when investigating rapid gene responses. Conclusion: The present study demonstrates that psilocybin rapidly induces gene expression related to neuroplasticity, biased towards the prefrontal cortex, compared to the hippocampus. Our findings provide further evidence for the rapid plasticity-promoting effects of psilocybin.

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low-(5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [gamma-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effect on autophosphorylation (30 +/- 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 +/- 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 microM to 80 microM) for ATP of IRK from high-glucose-treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells after acute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)