CONICET | Buscador de Institutos y Recursos Humanos

Aluminium (Al3+) is a metal widely distributed in the environment. It is involved with the pathophysiology of neurodegenerative disorders, such as Parkinsonism dementia and Alzheimer?s disease. The mechanisms that have been proposed to explain the toxicity of Al3+ are linked to changes in the cellular calcium homeostasis. PMCA is a P-ATPase involved in the regulation of the calcium homeostasis. Its function is to transport Ca2+ from cytoplasm towards the extracellular medium against the electrochemical gradient modulating the cytoplasmic Ca2+ concentration. In previous works, we already showed that Al3+ irreversibly inhibits Ca2+-ATPase activity of PMCA by preventing the dephosphorylation of the pump. The aim of this work is study the effect of Al3+ on PMCA at cellular level. To characterize this effect, we measured the changes on intracellular calcium in HEK293T cells using a fluorescent probe (Fluo4-AM) in presence of different concentrations of AlCl3. Subsequently, we used lumogallion as a direct fluorescence molecular probe for aluminium. On the one hand, we characterized the entry of aluminium and lumogallion to HEK293T cells over time, measured by fluorometry. On the other hand, using confocal fluorescence microscopy, we studied the location of aluminum in the cells.Our results show that AlCl3 inhibits calcium efflux mediated by PMCA in HEK293T cells (p≤0.05). The aluminium entrance to the cell as a function of both incubation time and concentration present a saturable behavior. The lumogallion probe gets into the cell through diffusion and its entrance also depends on the incubation time and concentration. Confocal fluorescence microscopy suggests that Al3+ first interacts with the membrane lipids and afterwards is located inside the cell.This work was supported by ANCYT PICT 2014 0065, CONICET PIP 0250 and Universidad de Buenos Aires 2014-2017: 20020130100254B