Real-time functional characterization of cationic amino acid transporters using a new FRET sensor

Abstract

L-arginine is a semi-essential amino acid that serves as precursor for the production of urea, nitric oxide (NO), polyamines, and other biologically important metabolites. Hence, a fast and reliable assessment of its intracellular concentration changes is highly desirable. Here, we report on a genetically encoded Förster resonance energy transfer (FRET)-based arginine nanosensor that employs the arginine repressor/activator ahrC gene from Bacillus subtilis. This new nanosensor was expressed in HEK293T cells, and experiments with cell lysate showed that it binds L-arginine with high specificity and with a K d of ∼177 μM. Live imaging experiments showed that the nanosensor was expressed throughout the cytoplasm and displayed a half maximal FRET increase at an extracellular L-arginine concentration of ∼22 μM. By expressing the nanosensor together with SLC7A1, SLC7A2B, or SLC7A3 cationic amino acid transporters (CAT1-3), it was shown that L-arginine was imported at a similar rate via SLC7A1 and SLC7A2B and slower via SLC7A3. In contrast, upon withdrawal of extracellular L-arginine, intracellular levels decreased as fast in SLC7A3-expressing cells compared with SLC7A1, but the efflux was slower via SLC7A2B. SLC7A4 (CAT4) could not be convincingly shown to transport L-arginine. We also demonstrated the impact of membrane potential on L-arginine transport and showed that physiological concentrations of symmetrical and asymmetrical dimethylarginine do not significantly interfere with L-arginine transport through SLC7A1. Our results demonstrate that the FRET nanosensor can be used to assess L-arginine transport through plasma membrane in real time.

The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery

“…To begin to understand the significance of autophagy-linked differences in cellular amino acid levels in relation to transporter trafficking during starvation, we used a Förster resonance energy transfer (FRET) sensor [ 74 ] to measure cellular L-arginine levels. Arginine is a semi-essential amino acid, requiring dietary uptake to compensate for insufficient biosynthesis.…”

“…The arginine FRET sensor is based on the product of the Bacillus subtilis ahrC gene, an arginine repressor, and reports changes in cytosolic arginine levels in the low micromolar range ( Fig. 7F ) [ 74 ]. We used confocal microscopy to measure basal FRET levels and changes in FRET over a starvation time-course in wild-type and atg5 −/− MEFs ( Fig.…”

Cationic Amino Acid Transporter-1-Mediated Arginine Uptake Is Essential for Chronic Lymphocytic Leukemia Cell Proliferation and Viability

“…Inhibition of amino acid transporters by small molecule compounds as a successful novel anti-cancer strategy (5) has already been reported for SLC1A5 (31), SLC7A5 (56), SLC7A11 (57), and SLC6A14 (58). An appropriate read-out assay for CAT-1 mediated arginine flux (59) could be used for compound library-based high-throughput screening to develop small molecule- or antibody-based inhibitors against CAT-1. These reagents could serve as potential novel cancer therapeutics for CLL and other arginine-auxotrophic tumor entities that share the non-redundant dependence on CAT-1 for arginine uptake (Figure 7).…”

Identification of novel inhibitors of the amino acid transporter B0AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

“…Here, we established the FLIPR system as a suitable assay to screen large compound libraries and to characterize amino acid transport inhibitors. Alternatively, FRET sensors can be used to detect amino acid transport (Whitfield et al, 2015;Vanoaica et al, 2016). However, these monitor amino acid accumulation through any transport system and therefore require secondary screening to ensure targeting is correct.…”