title: 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.
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP) and NCCR Kidney.CH, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Liviu
authors.initials: L
authors.lastname: Vanoaica
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP) and NCCR Kidney.CH, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Alok
authors.initials: A
authors.lastname: Behera
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP) and NCCR Kidney.CH, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Simone M R
authors.initials: SM
authors.lastname: Camargo
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP) and NCCR Kidney.CH, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Forster
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP) and NCCR Kidney.CH, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland. verrey@access.uzh.ch.
authors.forename: François
authors.initials: F
authors.lastname: Verrey
mesh_terms: D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D027201:Cationic Amino Acid Transporter 1 / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
chemical_list: C120339:AhrC protein, Bacillus subtilis; D001426:Bacterial Proteins; D027201:Cationic Amino Acid Transporter 1; D012097:Repressor Proteins; D015534:Trans-Activators; D001120:Arginine
keywords: ADMA; CATs; FRET sensor; L-arginine transport; Membrane potential; SDMA