New Fluorescent Substrate Enables Quantitative and High-Throughput Examination of Vesicular Monoamine Transporter 2 (VMAT2)

Abstract

Vesicular Monoamine Transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent Km of FFN206 at VMAT2 was found to be 1.16 ± 0.10 µM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z'-factor of approximately 0.7–0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture.

Structure-Activity Relationships of Substituted Cathinones, with Transporter Binding, Uptake, and Release

“…For example, substrates induce inward sodium currents in HEK cells expressing the hNET or hDAT, thereby elevating intracellular sodium to drive reverse transport (Galli et al, 1995;Sitte et al, 1998). However, as HEK cells do not have synaptic vesicles (Hu et al, 2013), release via vesicle fusion is not operational in our model system. These potentially depolarizing transporter-mediated substrate-induced sodium currents (Sonders et al, 1997;Sitte et al, 1998) cause reverse transport or release of neurotransmitter.…”

The vesicular monoamine transporter 2: An underexplored pharmacological target

“…Therefore, we hypothesized in our paper that a non-pH-sensitive FFN that was taken up in cell culture would be the ideal reagent for a high throughput screen for VMAT2 function. Recently, the Sames and Sulzer groups developed a novel FFN (FFN206) that is taken up in cell culture and is not pH-dependent (131). This dye localizes specifically to VMAT2-positive compartments and does not localize to the mitochondria, reducing non-specific noise in the assay.…”

“…This specificity precludes the need for high content imaging and allows the fluorescence to be read by plate reader. The authors demonstrate that FFN206 allows for high throughput analysis with a Z-factor of 0.7–0.8 (131). …”

Importance of Substrate-Coupled Proton Antiport by the Vesicular Monoamine Transporter in the Actions of Amphetamines in Drosophila Brain

“…The fluorescent VMAT2-specific substrate, FFN206 [12], selectively labeled presynaptic DA nerve terminals throughout the brain. Specificity of dVMAT-dependent FFN206 labeling was confirmed following acute treatment with VMAT2 inhibitors including reserpine or the novel dihydrotetrabenazine derivative, (+)-CYY477 which completely blocked labeling.…”