Measurement of plasma membrane Ca2+ pump activity following cytoplasmic Ca2+ kinetics

SAFFIOTI NA; RINALDI D.; DALGHI M; ROSSI RC; ROSSI JPFC; FERREIRA GOMES MS

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

The store-operated Ca2+ entry (SOCE) is a cell response triggered by endoplasmicreticulum Ca2+ depletion. Under this stimulus, the ORAI subunits in the plasmamembrane aggregate to form the SOCE channels that allow extracellular Ca2+ toentry into the cytoplasm. The SOCE response has been used in living cells tostudy the mechanisms of Ca2+ extrusion following the kinetics of cytoplasmicCa2+. Although there have been proposed different methods to assess theexperimental data, nowadays there is no consensus in the literature about how tointerpret the changes in Ca2+ kinetics and how these changes are related toalterations in different Ca2+ control mechanisms. In order to investigate the Ca2+transport by the plasma membrane Ca2+ pump (PMCA) in living cells, we studiedthe Ca2+ kinetics elicited by SOCE in conditions where PMCA is the mainmechanism of Ca2+ extrusion. HEK-293T cell line was transfected transiently withPMCA4 and incubated in mediums with different Ca2+ concentrations.Cytoplasmic Ca2+ was measured in real time by loading cells with fluorescentCa2+ indicators. Our results showed that cytoplasmic Ca 2+ kinetics had atransient increase, followed by a slow decrease until a stationary level wasreached. The cytoplasmic Ca2+ levels depended on the extracellular Ca 2+concentration and PMCA expression. The experimental data were analyzed bymeans of an empirical equation that allows describing the data in terms of thebalance between Ca2+ uptake and release by the cells. Then we compared thismethod to others found in the literature. At the same time, the results wereinterpreted in terms of a mathematical model of cytoplasmic Ca2+ kinetics. Thisanalysis allowed us to relate the value of parameters obtained by differentmethods with the PMCA Ca2+ transport activity.