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

摘要: 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, fast reliable assessment its intracellular concentration changes highly desirable. Here, we report on genetically encoded Forster resonance energy transfer (FRET)-based arginine nanosensor employs repressor/activator ahrC gene from Bacillus subtilis. This new was expressed in HEK293T cells, experiments with cell lysate showed it binds high specificity K d ∼177 μM. Live imaging throughout cytoplasm displayed half maximal FRET increase at an extracellular ∼22 By expressing together SLC7A1, SLC7A2B, or SLC7A3 cationic transporters (CAT1-3), shown imported similar rate via SLC7A1 SLC7A2B slower SLC7A3. In contrast, upon withdrawal L-arginine, levels decreased SLC7A3-expressing cells compared but efflux SLC7A2B. SLC7A4 (CAT4) could not be convincingly to transport L-arginine. We also demonstrated impact membrane potential physiological concentrations symmetrical asymmetrical dimethylarginine do significantly interfere through SLC7A1. Our results demonstrate can used assess plasma real time.