Calcium transport by the Plasma Membrane Ca2+ pump (PMCA)

VIGIL MAXIMILIANO ANGEL; SAFFIOTI NICOLÁS; RINALDI, DEBORA; MANGIALAVORI IRENE; ROLANDO C ROSSI; ROSSI JUAN PABLO F. C.; FERREIRA GOMES, MARIELA

Congreso; 20th IUPAB Congress, 45th Annual SBBf Meeting, and 50th Annual SBBq Meeting; 2021

Calcium (Ca2+) is an important second messenger that participates in many cellular activities. Cells display different mechanisms to maintain low levels of cytoplasmic Ca2+ concentration ([Ca2+]cyt) (100-200 nM) needed to regulate its targets with optimal effectiveness. These mechanisms include transport systems at the plasma membrane level (e.g. Ca2+-ATPases -PMCAs-, Na+/Ca2+ exchanger) and at certain intracellular organelle membranes (e.g. the sarcoplasmic reticulum Ca2+ ATPase -SERCA-).The aim of this study was to investigate the Ca2+ transport by the plasma membrane Ca2+ pump (PMCA) in living cells. HEK-293T cell line was transfected transiently with PMCA4 and [Ca2+]cyt was measured in real-time by loading cells with the fluorescent probe Fluo-4. [Ca2+]cyt kinetics were examined by studying the alterations in [Ca2+]cyt generated by Ca2+ released from the sarcoplasmic reticulum (ER), and by extracellular Ca2+ entry through store-operated Ca2+ channels (SOCs). Finally, the results were interpreted in terms of a mathematical model of [Ca2+]cyt kinetics and the parameters were obtained. The typical experiment of [Ca2+]cyt kinetics showed two phases: (a) transient elevation in [Ca2+]cyt generated by the addition of 1 µM thapsigargin, a SERCA inhibitor that causes Ca2+ released from ER and (b) [Ca2+]cyt increase generated by the addition of 4 mM Ca2+ to the external medium which that induced the SOC activity. In both phases, [Ca2+]cyt reached a maximum and then its stationary level was reestablished. Overexpression of hPMCA4 led to a significant decrease in the global [Ca2+]cyt at all times after the stimulus. On the other hand, the results were interpreted in terms of a mathematical model of [Ca2+]cyt kinetics suggesting that Ca2+ transport by PMCA must increase slowly after [Ca2+]cyt increase to explain the [Ca2+]cyt kinetics. In conclusion, the model built in this study is a useful tool to analyze the PMCA Ca2+ transport activity in living cells.Keywords: Ca2+-signaling, Ca2+ homeostasis, Fluo 4, SERCA, SOCS, Calmodulin, Mathematical modelFunding: ANPCYT, CONICET, UBA