Abstract
The serotonin transporter (SERT) is the primary target for selective serotonin reuptake inhibitor (SSRI) antidepressants that are thought to exert their therapeutic effects by increasing the synaptic concentration of serotonin. Consequently, probes that can be utilized to study cellular trafficking of SERT are valuable research tools. We have developed a novel ligand (IDT785) that is composed of a SERT antagonist (a tetrahydro pyridyl indole derivative) conjugated to a biotinylated poly ethylene glycol (PEG) via a phenethyl linker. This compound was determined to be biologically active and inhibited SERT-mediated reuptake of IDT307 with the half-maximal inhibitory concentration of 7.2 ± 0.3 μM. We demonstrated that IDT785 enabled quantum dot (QD) labeling of membrane SERT in transfected HEK-293 cultures that could be blocked using the high affinity serotonin reuptake inhibitor paroxetine. Molecular docking studies suggested that IDT785 might be binding to the extracellular vestibule binding site rather than the orthosteric substrate binding site, which could be attributable to the hydrophilicity of the PEG chain and the increased loss of degrees of freedom that would be required to penetrate into the orthosteric binding site. Using IDT785, we were able to study the membrane localization and membrane dynamics of YFP-SERT heterologously expressed in HEK-293 cells and demonstrated that SERT expression was enriched in the membrane edge and in thin cellular protrusions.
Highlights
5-hydroxytryptamine (5-HT, serotonin) is a monoamine neurotransmitter involved in the modulation of a myriad of functions in the brain and periphery, including mood, appetite, sleep, memory and learning
We study its ability to inhibit the human serotonin transporter (SERT) transiently expressed in HEK293T cells, examine specific labeling of hSERT and SERT fused to yellow fluorescent protein (YFP) at the N-terminus with a combination of biotinylated IDT785 streptavidin-conjugated quantum dot (QD) (SavQD), and demonstrate that hSERT surface dynamics may be tracked with IDT785-QD conjugates
As expected, hSERTexpressing HEK-293T cells incubated with 10 μM IDT307 for 15 min exhibited characteristic intracellular accumulation of IDT307 most pronounced in the mitochondria and nucleoli, whereas cells pretreated with excess IDT785 (100 μM) and 10 μM paroxetine [SERT binding affinity of ∼70 pM according to Cool et al (1990)] showed significantly reduced intracellular IDT307 accumulation
Summary
5-hydroxytryptamine (5-HT, serotonin) is a monoamine neurotransmitter involved in the modulation of a myriad of functions in the brain and periphery, including mood, appetite, sleep, memory and learning. Biocompatible colloidal semiconductor quantum dots (QDs) are an attractive alternative to dye fluorophores (Rosenthal et al, 2011) These highly fluorescent semiconductor crystals range in diameter from 2 to 10 nm, have a quantum yield that is significantly larger than many commercial dyes and a narrow, size-tunable, Gaussian emission spectrum. We have successfully developed antagonist-based QD probes to label SERT and other membrane-bound neuronal proteins, including structurally related dopamine transporter (DAT) (Kovtun et al, 2011; Chang et al, 2012b; Kovtun et al, 2015; Rosenthal, 2019; Tomlinson et al, 2019). We study its ability to inhibit the human SERT transiently expressed in HEK293T cells (hSERT), examine specific labeling of hSERT and SERT fused to yellow fluorescent protein (YFP) at the N-terminus with a combination of biotinylated IDT785 streptavidin-conjugated QDs (SavQD), and demonstrate that hSERT surface dynamics may be tracked with IDT785-QD conjugates
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