Abstract
Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25–10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R 2 = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.
Similar content being viewed by others
References
Aiken NR, Galey WR et al (1995) A peroxidative model of human erythrocyte intracellular Ca2+ changes with in vivo cell aging: measurement by 19F-NMR spectroscopy. Biochim Biophys Acta 1270(1):52–57
Almog B, Gamzu R et al (2005) Enhanced erythrocyte aggregation in clinically diagnosed pelvic inflammatory disease. Sex Transm Dis 32(8):484–486
Bennett V (1989) The spectrin–actin junction of erythrocyte membrane skeletons. Biochim Biophys Acta 988(1):107–121
Bennett J, Weeds A (1986) Calcium and the cytoskeleton. Br Med Bull 42(4):385–390
Brini M, Carafoli E (2009) Calcium pumps in health and disease. Physiol Rev 89(4):1341–1378
Carafoli E (1987) Intracellular calcium homeostasis. Annu Rev Biochem 56:395–433
Deman J, Bruyneel E (1973) A method for the quantitative measurement of cell aggregation. Exp Cell Res 81(2):351–359
Dodge JT, Mitchell C et al (1963) The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100:119–130
Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463(7280):485–492
Fowler V, Taylor DL (1980) Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium. J Cell Biol 85(2):361–376
Fusman G, Mardi T et al (2002) Red blood cell adhesiveness/aggregation, C-reactive protein, fibrinogen, and erythrocyte sedimentation rate in healthy adults and in those with atherosclerotic risk factors. Am J Cardiol 90(5):561–563
Goldin Y, Tulshinski T et al (2007) Rheological consequences of acute infections: the rheodifference between viral and bacterial infections. Clin Hemorheol Microcirc 36(2):111–119
Jaiswal JK (2001) Calcium—how and why? J Biosci 26(3):357–363
Kabaso D, Shlomovitz R et al (2010) Curling and local shape changes of red blood cell membranes driven by cytoskeletal reorganization. Biophys J 99(3):808–816
Lew VL, Tiffert T (2007) Is invasion efficiency in malaria controlled by pre-invasion events? Trends Parasitol 23(10):481–484
Liu F, Mizukami H et al (2005) Calcium-dependent human erythrocyte cytoskeleton stability analysis through atomic force microscopy. J Struct Biol 150(2):200–210
Luna EJ, Hitt AL (1992) Cytoskeleton–plasma membrane interactions. Science 258(5084):955–964
Mostafavi E, Nakhjavani M et al (2013) Protective role of calcium ion against stress-induced osmotic fragility of red blood cells in patients with type 2 diabetes mellitus. Clin Hemorheol Microcirc 53(3):239–245
O’Brien ET, Salmon ED et al (1997) How calcium causes microtubule depolymerization. Cell Motil Cytoskeleton 36(2):125–135
Rivas FV, O’Keefe JP et al (2004) Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration. Mol Cell Biol 24(4):1628–1639
Rosado JA, Sage SO (2000) The actin cytoskeleton in store-mediated calcium entry. J Physiol 526(Pt 2):221–229
Schmidt C, Kosche E et al (1995) Arachidonic acid metabolism and intracellular calcium concentration in inflammatory bowel disease. Eur J Gastroenterol Hepatol 7(9):865–869
Sharshun Y, Brill S et al (2003) Inflammation at a glance: erythrocyte adhesiveness/aggregation test to reveal the presence of inflammation in people with atherothrombosis. Heart Dis 5(3):182–183
Takakuwa Y, Mohandas N (1988) Modulation of erythrocyte membrane material properties by Ca2+ and calmodulin. Implications for their role in regulation of skeletal protein interactions. J Clin Invest 82(2):394–400
Verkhratsky A (2005) Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol Rev 85(1):201–279
von Tempelhoff GF, Nieman F et al (2000) Association between blood rheology, thrombosis and cancer survival in patients with gynecologic malignancy. Clin Hemorheol Microcirc 22(2):107–130
Wehrens XH, Lehnart SE et al (2005) Intracellular calcium release and cardiac disease. Annu Rev Physiol 67:69–98
Williams RJ (2006) The evolution of calcium biochemistry. Biochim Biophys Acta 1763(11):1139–1146
Yamawaki S, Kagaya A et al (1998) Intracellular calcium signaling systems in the pathophysiology of affective disorders. Life Sci 62(17–18):1665–1670
Yu R, Hinkle PM (2000) Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators. J Biol Chem 275(31):23648–23653
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mostafavi, E., Nargesi, A.A., Ghazizadeh, Z. et al. The Degree of Resistance of Erythrocyte Membrane Cytoskeletal Proteins to Supra-Physiologic Concentrations of Calcium: An In Vitro Study. J Membrane Biol 247, 695–701 (2014). https://doi.org/10.1007/s00232-014-9689-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00232-014-9689-1