CONICET | Buscador de Institutos y Recursos Humanos

The store operated Ca2+ entry (SOCE) is a cell response triggered by endoplasmic reticulum Ca2+ depletion. Under this stimulus, the ORAI protein subunits in the plasma membrane aggregate to form the SOCE channels that allow the entry of extracellular Ca2+ into the cytoplasm. The SOCE response has been used in living cells to study the mechanisms of Ca2+ extrusion following the kinetics of cytoplasmic [Ca2+]. Although there have been proposed different methods to assess the experimental data, nowadays there is no consensus in the literature about how to interpret the changes in [Ca2+] kinetics and how these changes are related to alterations in different Ca2+ control mechanisms. In order to investigate the Ca2+ transport by the plasma membrane Ca2+ pump (PMCA) in living cells, we studied the [Ca2+] kinetics elicited by SOCE in conditions where PMCA is the main mechanism of Ca2+ extrusion. An HEK-293 T cell line was transfected transiently with PMCA4 and incubated in media with different Ca2+ concentrations. Cytoplasmic [Ca2+] was measured in real time by loading cells with the fluorophores Fura-2 or Fluo-4. Our results show that cytoplasmic [Ca2+] kinetics have a transient increase, followed by a slow decrease until a stationary level is reached. The cytoplasmic [Ca2+] levels depended on the extracellular Ca2+ concentration and PMCA expression. The experimental data were analyzed by means of an empirical equation that allows to describe them in terms of the balance between Ca2+ uptake and release by the cells. Then we compared this method to others found in the literature. The results were interpreted in terms of a mathematical model for the kinetics of the concentration of cytoplasmic [Ca2+]. This analysis allowed us to relate the value of parameters obtained by different methods with the PMCA Ca2+ transport activity.This work was supported by Agencia Nacional de Promoción Científica y Tecnológica PICT 2014 0065, Consejo Nacional de Investigaciones Científicas y Técnicas PIP 11220150100250CO, and Universidad de Buenos Aires Ciencia y Técnica grant 2014-2017: 20020130100254B