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A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens

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Abstract

Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity.

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Data availability

All data generated in this study are included in the manuscript. Raw values from individual trials are available upon request.

References

  • Akerboom J, Chen TW, Wardill TJ, Tian L, Marvin JS, Mutlu S, Calderon NC, Esposti F, Borghuis BG, Sun XR, Gordus A, Orger MB, Portugues R, Engert F, Macklin JJ, Filosa A, Aggarwal A, Kerr RA, Takagi R, Kracun S, Shigetomi E, Khakh BS, Baier H, Lagnado L, Wang SS, Bargmann CI, Kimmel BE, Jayaraman V, Svoboda K, Kim DS, Schreiter ER, Looger LL (2012) Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci 32:13819–13840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berridge MJ (2016) The inositol trisphosphate/calcium signaling pathway in health and disease. Physiol Rev 96:1261–1296

    Article  CAS  PubMed  Google Scholar 

  • Bogdanova A, Makhro A, Wang J, Lipp P, Kaestner L (2013) Calcium in red blood cells-a perilous balance. Int J Mol Sci 14:9848–9872

    Article  PubMed  PubMed Central  Google Scholar 

  • Bootman MD, Allman S, Rietdorf K, Bultynck G (2018) Deleterious effects of calcium indicators within cells; an inconvenient truth. Cell Calcium 73:82–87

    Article  CAS  PubMed  Google Scholar 

  • Clapham DE (2007) Calcium signaling. Cell 131:1047–1058

    Article  CAS  PubMed  Google Scholar 

  • Desai SA, Schlesinger PH, Krogstad DJ (1991) Physiologic rate of carrier-mediated Ca2+ entry matches active extrusion in human erythrocytes. J Gen Physiol 98:349–364

    Article  CAS  PubMed  Google Scholar 

  • Duan P, Li S, Ai N, Hu L, Welsh WJ, You G (2012) Potent inhibitors of human organic anion transporters 1 and 3 from clinical drug libraries: discovery and molecular characterization. Mol Pharm 9:3340–3346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Elies J, Yanez M, Pereira TMC, Gil-Longo J, MacDougall DA, Campos-Toimil M (2020) An update to calcium binding proteins. Adv Exp Med Biol 1131:183–213

    Article  CAS  PubMed  Google Scholar 

  • Eraly SA, Bush KT, Sampogna RV, Bhatnagar V, Nigam SK (2004) The molecular pharmacology of organic anion transporters: from DNA to FDA? Mol Pharmacol 65:479–487

    Article  CAS  PubMed  Google Scholar 

  • Fox JT, Myung K (2012) Cell-based high-throughput screens for the discovery of chemotherapeutic agents. Oncotarget 3:581–585

    Article  PubMed  PubMed Central  Google Scholar 

  • Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201:477–482

    Article  CAS  PubMed  Google Scholar 

  • Hammes A, Oberdorf-Maass S, Jenatschke S, Pelzer T, Maass A, Gollnick F, Meyer R, Afflerbach J, Neyses L (1996) Expression of the plasma membrane Ca2+-ATPase in myogenic cells. J Biol Chem 271:30816–30822

    Article  CAS  PubMed  Google Scholar 

  • Hayashi H, Miyata H (1994) Fluorescence imaging of intracellular Ca2+. J Pharmacol Toxicol Methods 31:1–10

    Article  CAS  PubMed  Google Scholar 

  • Karlish SJ, Beauge LA, Glynn IM (1979) Vanadate inhibits (Na+ + K+)ATPase by blocking a conformational change of the unphosphorylated form. Nature 282:333–335

    Article  CAS  PubMed  Google Scholar 

  • Krebs J (2015) The plethora of PMCA isoforms: alternative splicing and differential expression. Biochim Biophys Acta 1853:2018–2024

    Article  CAS  PubMed  Google Scholar 

  • Kubitscheck U, Pratsch L, Passow H, Peters R (1995) Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging. Biophys J 69:30–41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kushwaha AK, Apolis L, Ito D, Desai SA (2018) Increased Ca++ uptake by erythrocytes infected with malaria parasites: evidence for exported proteins and novel inhibitors. Cell Microbiol 20:e12853

    Article  PubMed  PubMed Central  Google Scholar 

  • Lew VL, Tsien RY, Miner C, Bookchin RM (1982) Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator. Nature 298:478–481

    Article  CAS  PubMed  Google Scholar 

  • Lew VL, Daw N, Perdomo D, Etzion Z, Bookchin RM, Tiffert T (2003) Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells. Blood 102:4206–4213

    Article  CAS  PubMed  Google Scholar 

  • Nigam SK, Bush KT, Martovetsky G, Ahn SY, Liu HC, Richard E, Bhatnagar V, Wu W (2015) The organic anion transporter (OAT) family: a systems biology perspective. Physiol Rev 95:83–123

    Article  PubMed  PubMed Central  Google Scholar 

  • Nisar S, Torres M, Thiam A, Pouvelle B, Rosier F, Gallardo F, Ka O, Mbengue B, Diallo RN, Brosseau L, Spicuglia S, Dieye A, Marquet S, Rihet P (2022) Identification of ATP2B4 regulatory element containing functional genetic variants associated with severe malaria. Int J Mol Sci 23:4849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pande J, Szewczyk MM, Grover AK (2011) Allosteric inhibitors of plasma membrane Ca pumps: invention and applications of caloxins. World J Biol Chem 2:39–47

    Article  PubMed  PubMed Central  Google Scholar 

  • Paredes RM, Etzler JC, Watts LT, Zheng W, Lechleiter JD (2008) Chemical calcium indicators. Methods 46:143–151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Patton C, Thompson S, Epel D (2004) Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium 35:427–431

    Article  CAS  PubMed  Google Scholar 

  • Pretorius E, du Plooy JN, Bester J (2016) A comprehensive review on eryptosis. Cell Physiol Biochem 39:1977–2000

    Article  CAS  PubMed  Google Scholar 

  • Reed PW, Lardy HA (1972) A23187: a divalent cation ionophore. J Biol Chem 247:6970–6977

    Article  CAS  PubMed  Google Scholar 

  • Ronquist G, Rudolphi O, Engstrom I, Waldenstrom A (2001) Familial phosphofructokinase deficiency is associated with a disturbed calcium homeostasis in erythrocytes. J Intern Med 249:85–95

    Article  CAS  PubMed  Google Scholar 

  • Rossi JP, Garrahan PJ, Rega AF (1981) Vanadate inhibition of active Ca2+ transport across human red cell membranes. Biochim Biophys Acta 648:145–150

    Article  CAS  PubMed  Google Scholar 

  • Schatzmann HJ, Luterbacher S, Stieger J, Wuthrich A (1986) Red blood cell calcium pump and its inhibition by vanadate and lanthanum. J Cardiovasc Pharmacol 8(Suppl 8):S33-37

    Article  PubMed  Google Scholar 

  • Simonsen LO, Gomme J, Lew VL (1982) Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells. Biochim Biophys Acta 692:431–440

    Article  CAS  PubMed  Google Scholar 

  • Strehler EE, James P, Fischer R, Heim R, Vorherr T, Filoteo AG, Penniston JT, Carafoli E (1990) Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane. J Biol Chem 265:2835–2842

    Article  CAS  PubMed  Google Scholar 

  • Strehler EE, Caride AJ, Filoteo AG, Xiong Y, Penniston JT, Enyedi A (2007) Plasma membrane Ca2+ ATPases as dynamic regulators of cellular calcium handling. Ann NY Acad Sci 1099:226–236

    Article  CAS  PubMed  Google Scholar 

  • Takuwa N, Zhou W, Takuwa Y (1995) Calcium, calmodulin and cell cycle progression. Cell Signal 7:93–104

    Article  CAS  PubMed  Google Scholar 

  • Tiffert T, Lew VL (2001) Kinetics of inhibition of the plasma membrane calcium pump by vanadate in intact human red cells. Cell Calcium 30:337–342

    Article  CAS  PubMed  Google Scholar 

  • Tiffert T, Garcia-Sancho J, Lew VL (1984) Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta 773:143–156

    Article  CAS  PubMed  Google Scholar 

  • Timmann C, Thye T, Vens M, Evans J, May J, Ehmen C, Sievertsen J, Muntau B, Ruge G, Loag W, Ansong D, Antwi S, Asafo-Adjei E, Nguah SB, Kwakye KO, Akoto AO, Sylverken J, Brendel M, Schuldt K, Loley C, Franke A, Meyer CG, Agbenyega T, Ziegler A, Horstmann RD (2012) Genome-wide association study indicates two novel resistance loci for severe malaria. Nature 489:443–446

    Article  CAS  PubMed  Google Scholar 

  • Tsien RY, Pozzan T, Rink TJ (1982) Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol 94:325–334

    Article  CAS  PubMed  Google Scholar 

  • Weiss E, Rees DC, Gibson JS (2011) Role of calcium in phosphatidylserine externalisation in red blood cells from sickle cell patients. Anemia 2011:379894

    Article  PubMed  Google Scholar 

  • Wesseling MC, Wagner-Britz L, Boukhdoud F, Asanidze S, Nguyen DB, Kaestner L, Bernhardt I (2016) Measurements of intracellular Ca2+ content and phosphatidylserine exposure in human red blood cells: methodological issues. Cell Physiol Biochem 38:2414–2425

    Article  CAS  PubMed  Google Scholar 

  • Zipprer EM, Neggers M, Kushwaha A, Rayavara K, Desai SA (2014) A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes. Malar J 13:184

    Article  PubMed  PubMed Central  Google Scholar 

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Funding

This study was supported by the Intramural Research Program of National Institutes of Health, National Institute of Allergy and Infectious Diseases. The authors have no competing interests to declare.

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Authors and Affiliations

  1. Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA

    Jeremiah N. Sims, EJun Yun, Jonathan Chu, Mansoor A. Siddiqui & Sanjay A. Desai

  2. Institute of Protein Design, Medical Scientist Training Program, Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA

    Jeremiah N. Sims

  3. Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

    EJun Yun

Authors
  1. Jeremiah N. Sims
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  2. EJun Yun
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  3. Jonathan Chu
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  4. Mansoor A. Siddiqui
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  5. Sanjay A. Desai
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Contributions

JS and SAD devised the study. JS, EY, and JC designed and performed experiments with guidance from MS and SAD. All authors analyzed data. SAD wrote the paper with input from all authors.

Corresponding author

Correspondence to Sanjay A. Desai.

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Sims, J.N., Yun, E., Chu, J. et al. A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens. Eur Biophys J 52, 101–110 (2023). https://doi.org/10.1007/s00249-022-01623-y

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  • DOI: https://doi.org/10.1007/s00249-022-01623-y

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