Illuminating the monoamine transporters: Fluorescently labelled ligands to study dopamine, serotonin and norepinephrine transporters

Abstract

Fluorescence microscopy has revolutionized the visualization of physiological processes in live‐cell systems. With the recent innovations in super resolution microscopy, these events can be examined with high precision and accuracy. The development of fluorescently labelled small molecules has provided a significant advance in understanding the physiological relevance of targeted proteins that can now be visualized at the cellular level. One set of physiologically important target proteins are the monoamine transporters (MATs) that play an instrumental role in maintaining monoamine signalling homeostasis. Understanding the mechanisms underlying their regulation and dysregulation is fundamental to treating several neuropsychiatric conditions such as attention deficit hyperactivity disorder (ADHD), anxiety, depression and substance use disorders. Herein, we describe the rationale behind the small molecule design of fluorescently labelled ligands (FLL) either as MAT substrates or inhibitors as well as their applications to advance our understanding of this class of transporters in health and disease.

Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes

“…13−16 Elements that traditionally constitute the structure of these probes are (1) the parent ligand, which acts as the "carrier" of the probe and binds to the target; (2) the fluorescent dye, which varies depending on the application of the FLL; and (3) the linker, which tethers these two elements together. 17,18 We have previously reported several FLL to study MATs such as JHC1-064, a rhodamine-labeled fluorescent cocaine-based analogue, which has been extensively used due to its high binding affinity, slow off-rate, and lack of selectivity for all three members of this transporter family. 13,18,19 More recently, analogues of JHC1-064, DG3-80, and DG4-91 that used Janelia Fluorophores (JF549 and JF646, respectively) with a modified polyethylene glycol tether proved useful for single-particle tracking and dSTORM experiments.…”

“…Fluorescently labeled ligands (FLL) have proven highly useful as tools to study protein expression, localization, distribution, and downstream processes in distinct live cell systems, opening a venue to study endogenously expressed proteins. − Elements that traditionally constitute the structure of these probes are (1) the parent ligand, which acts as the “carrier” of the probe and binds to the target; (2) the fluorescent dye, which varies depending on the application of the FLL; and (3) the linker, which tethers these two elements together. , …”

“…We have previously reported several FLL to study MATs such as JHC1-064, a rhodamine-labeled fluorescent cocaine-based analogue, which has been extensively used due to its high binding affinity, slow off-rate, and lack of selectivity for all three members of this transporter family. ,, More recently, analogues of JHC1-064, DG3-80, and DG4-91 that used Janelia Fluorophores (JF549 and JF646, respectively) with a modified polyethylene glycol tether proved useful for single-particle tracking and dSTORM experiments. , In addition, we have expanded our fluorescent tool box by generating other MAT-selective FLL, such as VK2-83, a selective serotonin transporter (SERT) FLL based on (S)-citalopram, and more recently AC1-146, , a nisoxetine-based NET preferential probe.…”

Transmembrane transporter proteins: Capturing transport in motion

“…The review by Camacho‐Hernandez et al 11 delves into the use of fluorescently labelled ligands (FLLs) as valuable tools for visualizing the cellular distribution of transporters. The study outlines their applications in primary neuronal cell cultures and cell lines, with specific ligands targeting dopamine (DAT), norepinephrine (NET) and serotonin (SERT) transporters.…”

Welcome to Basic & Clinical Pharmacology & Toxicology 2024

“…As we embark on the new year, we invite you to explore the wealth of knowledge and exciting content our journal offers. In 2023, we published three Special Collections from focused conferences supported by the Nordic Association for the Publication of Basic and Clinical Pharmacology and Toxicology, including Adhesion G‐Protein‐coupled Receptors, 1–10 Transmembrane Transporter Proteins: Catching Transport in Motion 11–19 and Advancing Deprescribing: Learnings from the first International Conference on Deprescribing 20–31 . The second part of the latter Special Collection will appear in the January 2024 issue.…”