Skip to main content

Advertisement

SpringerLink
Log in

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

  • Ion channels, receptors and transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Barbul A (1986) Arginine: biochemistry, physiology, and therapeutic implications. JPEN J Parenter Enteral Nutr 10:227–238

    Article  CAS  PubMed  Google Scholar 

  2. Behera AVL, Camargo SM, Oparija L, Surchat L, Kretz M, Balbo-Pogliano C, Lindenmeyer MT, Verrey F (2015) FRET sensors to investigate amino acid transport. Amino Acids 47:1626–1627

    Google Scholar 

  3. Belhage B, Hansen GH, Schousboe A (1993) Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA. Neuroscience 54:1019–1034

    Article  CAS  PubMed  Google Scholar 

  4. Bode-Böger SM, Scalera F, Ignarro LJ (2007) The L-arginine paradox: importance of the L-arginine/asymmetrical dimethylarginine ratio. Pharmacol Ther 114:295–306

    Article  PubMed  Google Scholar 

  5. Böger RH, Sullivan LM, Schwedhelm E, Wang TJ, Maas R, Benjamin EJ, Schulze F, Xanthakis V, Benndorf RA, Vasan RS (2009) Plasma asymmetric dimethylarginine and incidence of cardiovascular disease and death in the community. Circulation 119:1592–1600

    Article  PubMed  PubMed Central  Google Scholar 

  6. Bogner M, Ludewig U (2007) Visualization of arginine influx into plant cells using a specific FRET-sensor. J Fluoresc 17:350–360

    Article  CAS  PubMed  Google Scholar 

  7. Brosnan ME, Brosnan JT (2004) Renal arginine metabolism. J Nutr 134:2791S–2795S, discussion 2796S-2797S

    CAS  PubMed  Google Scholar 

  8. Castillo L, Chapman TE, Sanchez M, Yu YM, Burke JF, Ajami AM, Vogt J, Young VR (1993) Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets. Proc Natl Acad Sci U S A 90:7749–7753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Cherla G, Jaimes EA (2004) Role of L-arginine in the pathogenesis and treatment of renal disease. J Nutr 134:2801S–2806S, discussion 2818S-2819S

    CAS  PubMed  Google Scholar 

  10. Closs EI, Basha FZ, Habermeier A, Förstermann U (1997) Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B. Nitric Oxide 1:65–73

    Article  CAS  PubMed  Google Scholar 

  11. Closs EI, Boissel JP, Habermeier A, Rotmann A (2006) Structure and function of cationic amino acid transporters (CATs). J Membr Biol 213:67–77

    Article  CAS  PubMed  Google Scholar 

  12. Dittmer PJ, Miranda JG, Gorski JA, Palmer AE (2009) Genetically encoded sensors to elucidate spatial distribution of cellular zinc. J Biol Chem 284:16289–97

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. E-Cv L, Klinke A, Atzler D, Slocum JL, Lund N, Kielstein JT, Maas R, Schmidt-Haupt R, Pekarova M, Hellwinkel O, Tsikas D, D’Alecy LG, Lau D, Willems S, Kubala L, Ehmke H, Meinertz T, Blankenberg S, Schwedhelm E, Gadegbeku CA, Böger RH, Baldus S, Sydow K (2011) Pathogenic cycle between the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine and the leukocyte-derived hemoprotein myeloperoxidase. Circulation 124:2735–2745

    Article  Google Scholar 

  14. Fehr M, Lalonde S, Lager I, Wolff MW, Frommer WB (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J Biol Chem 278:19127–19133

    Article  CAS  PubMed  Google Scholar 

  15. Garnett JA, Baumberg S, Stockley PG, Phillips SEV (2007) Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine. Acta Crystallogr Sect F: Struct Biol Cryst Commun 63:918–921

    Article  CAS  Google Scholar 

  16. Hyla-Klekot L, Bryniarski P, Pulcer B, Ziora K, Paradysz A (2015) Dimethylarginines as risk markers of atherosclerosis and chronic kidney disease in children with nephrotic syndrome. Adv Clin Exp Med 24:307–313

    Article  PubMed  Google Scholar 

  17. Kaper T, Looger LL, Takanaga H, Platten M, Steinman L, Frommer WB (2007) Nanosensor detection of an immunoregulatory tryptophan influx/kynurenine efflux cycle. PLoS Biol 5, e257

    Article  PubMed  PubMed Central  Google Scholar 

  18. Kiechl S, Lee T, Santer P, Thompson G, Tsimikas S, Egger G, Holt DW, Willeit J, Xu Q, Mayr M (2009) Asymmetric and symmetric dimethylarginines are of similar predictive value for cardiovascular risk in the general population. Atherosclerosis 205:261–265

    Article  CAS  PubMed  Google Scholar 

  19. Kielstein JT, Salpeter SR, Bode-Boeger SM, Cooke JP, Fliser D (2006) Symmetric dimethylarginine (SDMA) as endogenous marker of renal function—a meta-analysis. Nephrol Dial Transplant 21:2446–2451

    Article  CAS  PubMed  Google Scholar 

  20. Lajer M, Tarnow L, Jorsal A, Teerlink T, Parving H-H, Rossing P (2008) Plasma concentration of asymmetric dimethylarginine (ADMA) predicts cardiovascular morbidity and mortality in type 1 diabetic patients with diabetic nephropathy. Dia Care 31:747–752

    Article  CAS  Google Scholar 

  21. Lindenburg LH, Vinkenborg JL, Oortwijn J, Aper SJA, Merkx M (2013) MagFRET: the first genetically encoded fluorescent Mg2+ sensor. PLoS ONE 8, e82009

    Article  PubMed  PubMed Central  Google Scholar 

  22. Meier C, Ristic Z, Klauser S, Verrey F (2002) Activation of system L heterodimeric amino acid exchangers by intracellular substrates. EMBO J 21:580–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Merkx M, Golynskiy MV, Lindenburg LH, Vinkenborg JL (2013) Rational design of FRET sensor proteins based on mutually exclusive domain interactions. Biochem Soc Trans 41:1201–5

    Article  CAS  PubMed  Google Scholar 

  24. Morris SM Jr (2006) Arginine: beyond protein. Am J Clin Nutr 83:508S–512S

    CAS  PubMed  Google Scholar 

  25. Mountain A, Baumberg S (1980) Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168. Mol Gen Genet 178:691–701

    Article  CAS  PubMed  Google Scholar 

  26. Mountain A, Mann NH, Munton RN, Baumberg S (1984) Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis. Mol Gen Genet 197:82–89

    Article  CAS  PubMed  Google Scholar 

  27. Napolitano L, Scalise M, Galluccio M, Pochini L, Albanese LM, Indiveri C (2015) LAT1 is the transport competent unit of the LAT1/CD98 heterodimeric amino acid transporter. Int J Biochem Cell Biol 67:25–33

    Article  CAS  PubMed  Google Scholar 

  28. Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB (2005) Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc Natl Acad Sci U S A 102:8740–5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Qin Y, Dittmer PJ, Park JG, Jansen KB, Palmer AE (2011) Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors. Proc Natl Acad Sci U S A 108:7351–7356

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schulze F, Carter AM, Schwedhelm E, Ajjan R, Maas R, von Holten R-A, Atzler D, Grant PJ, Böger RH (2010) Symmetric dimethylarginine predicts all-cause mortality following ischemic stroke. Atherosclerosis 208:518–523

    Article  CAS  PubMed  Google Scholar 

  31. Smith MC, Czaplewski L, North AK, Baumberg S, Stockley PG (1989) Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli. Mol Microbiol 3:23–28

    Article  CAS  PubMed  Google Scholar 

  32. Strobel J, Mieth M, Endress B, Auge D, König J, Fromm MF, Maas R (2012) Interaction of the cardiovascular risk marker asymmetric dimethylarginine (ADMA) with the human cationic amino acid transporter 1 (CAT1). J Mol Cell Cardiol 53:392–400

    Article  CAS  PubMed  Google Scholar 

  33. Vékony N, Wolf S, Boissel JP, Gnauert K, Closs EI (2001) Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues. Biochemistry 40:12387–12394

    Article  PubMed  Google Scholar 

  34. Verrey F, Closs EI, Wagner CA, Palacin M, Endou H, Kanai Y (2004) CATs and HATs: the SLC7 family of amino acid transporters. Pflugers Arch 447:532–542

    Article  CAS  PubMed  Google Scholar 

  35. Whitfield JH, Zhang W, Herde MK, Clifton BE, Radziejewski J, Janovjak H, Henneberger C, Jackson CJ (2015) Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction. Protein Sci 24:1412–1422

    Article  CAS  PubMed  Google Scholar 

  36. Wolf S, Janzen A, Vékony N, Martiné U, Strand D, Closs EI (2002) Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity. Biochem J 364:767–775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wu G, Morris SM Jr (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336(Pt 1):1–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Young JM, Terrin N, Wang X, Greene T, Beck GJ, Kusek JW, Collins AJ, Sarnak MJ, Menon V (2009) Asymmetric dimethylarginine and mortality in stages 3 to 4 chronic kidney disease. Clin J Am Soc Nephrol 4:1115–1120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zheng L, Godfrey DA, Waller HJ, Godfrey TG, Chen K, Sun Y (2000) Effects of high-potassium-induced depolarization on amino acid chemistry of the dorsal cochlear nucleus in rat brain slices. Neurochem Res 25:823–835

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Imaging was performed with equipment maintained by the Center for Microscopy and Image Analysis (ZMB), University of Zurich, and with the assistance and support of U. Ziegler, C. Aemisegger, and J.M.M. Melero. The authors thank Prof. Nathan W. Luedtke from Department of Chemistry for the permission to use the microplate reader and the Functional Genomics Center Zurich (FGCZ) for amino acid measurements. The laboratory of FV is supported by Swiss National Science Foundation grant 31-130471/1 and the National Centre of Competence in Research (NCCR) Kidney.CH.

Author information

Authors and Affiliations

  1. Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP) and NCCR Kidney.CH, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Liviu Vanoaica, Alok Behera, Simone M. R. Camargo, Ian C. Forster & François Verrey

Authors
  1. Liviu Vanoaica
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alok Behera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Simone M. R. Camargo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ian C. Forster
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. François Verrey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to François Verrey.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PPTX 332 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vanoaica, L., Behera, A., Camargo, S.M.R. et al. Real-time functional characterization of cationic amino acid transporters using a new FRET sensor. Pflugers Arch - Eur J Physiol 468, 563–572 (2016). https://doi.org/10.1007/s00424-015-1754-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-015-1754-9

Keywords

Search

Navigation