Abstract
In the recent years, the toxicity of certain divalent cations has been associated with the alteration of intracellular Ca2+ homeostasis. Among other mechanisms, these cations may affect the functionality of certain Ca2+-binding proteins and/or Ca2+ pumps. The plasma membrane calcium pump (PMCA) maintains Ca2+ homeostasis in eukaryotic cells by mediating the efflux of this cation in a process coupled to ATP hydrolysis. The aim of this work was to investigate both in vitro and in cultured cells if other divalent cations (Sr2+, Ba2+, Co2+, Cd2+, Pb2+ or Be2+) could be transported by PMCA. Current results indicate that both purified and intact cell PMCA transported Sr2+ with kinetic parameters close to those of Ca2+ transport. The transport of Pb2+ and Co2+ by purified PMCA was, respectively, 50 and 75% lower than that of Ca2+, but only Co2+ was extruded by intact cells and to a very low extent. In contrast, purified PMCA—but not intact cell PMCA—transported Ba2+ at low rates and only when activated by limited proteolysis or by phosphatidylserine addition. Finally, purified PMCA did not transport Cd2+ or Be2+, although minor Be2+ transport was measured in intact cells. Moreover, Cd2+ impaired the transport of Ca2+ through various mechanisms, suggesting that PMCA may be a potential target of Cd2+-mediated toxicity. The differential capacity of PMCA to transport these divalent cations may have a key role in their detoxification, limiting their noxious effects on cell homeostasis.
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Abbreviations
- [125I]TID-PC/16:
-
1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin-3-yl)benzyl]oxy]carbonyl] nonanoyl]-sn-glycero-3-phosphocholine
- C12E10 :
-
Polyoxyethylene 10 lauryl ether
- CaM:
-
Calmodulin
- DC:
-
Divalent cation
- DMPC:
-
Dimyristoyl phosphatidylcholine
- DMSO:
-
Dimethylsulfoxide
- DTT:
-
Dithiothreitol
- EGTA:
-
Ethylene glycol tetraacetic acid
- eIOVs:
-
Erythrocyte inside-out vesicles
- ER:
-
Endoplasmic reticulum
- MOPS:
-
3-(N-morpholino)propanesulfonic acid
- PC:
-
Phosphatidylcholine
- PMCA:
-
Plasma membrane calcium pump
- PS:
-
Phosphatidylserine
- RB:
-
Reaction buffer
- SERCA:
-
Sarco(endo)plasmic reticulum Ca2+-ATPase
- SOCs:
-
Store-operated calcium channels
- TG:
-
Thapsigargin
- TLCK:
-
N-α-tosyl-l-lysine chloromethyl ketone
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Acknowledgements
This work was supported by grants of Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Secretaria de Ciencia y Técnica de la Universidad de Buenos Aires (UBACyT), Argentina. Authors are grateful to Dr. Osvaldo Rey for the generous gift of HEK293T cells.
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204_2017_2031_MOESM1_ESM.tif
Supplementary Fig. 1. Fura-2 is not a good sensor for cell Co2+ uptake. HEK293T cells were loaded with the fluorescent probe Fura-2. Cells were pre-incubated for 30 min with 1 µM TG, either in the absence (closed circles) or in the presence (open circles) of 100 µM La3+, and baseline fluorescence was recorded for 2 min. The arrow indicates the moment when 1 mM Co2+ was added to the cells and the kinetics of Fura-2 fluorescence intensity was recorded. Results are shown as the mean ± SD (n = 3) (TIFF 76 kb)
204_2017_2031_MOESM2_ESM.tif
Supplementary Fig. 2. Ca2+ increases CaM auto-fluorescence. Fluorescence emission spectra of CaM (5 µM) were determined in samples without added Ca2+ (gray solid line), or containing 150 µM Ca2+ (black solid line) or 1 mM EGTA (dashed line). Traces correspond to a representative experiment (TIFF 78 kb)
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Ferreira-Gomes, M.S., Mangialavori, I.C., Ontiveros, M.Q. et al. Selectivity of plasma membrane calcium ATPase (PMCA)-mediated extrusion of toxic divalent cations in vitro and in cultured cells. Arch Toxicol 92, 273–288 (2018). https://doi.org/10.1007/s00204-017-2031-9
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DOI: https://doi.org/10.1007/s00204-017-2031-9