Inhibition of the plasma membrane calcium pump by aluminium in vitro and in living cells

MARILINA DE SAUTU; MARIELA FERREIRA-GOMES; NICOLAS ANDRÉS SAFFIOTI; DEBORA RINALDI; ROLANDO ROSSI; JUAN PABLO FC ROSSI; IRENE CECILIA MANGIALAVORI

Congreso; XLII Congreso da Sociedade Brasileira de Biofisica.; 2017

Mechanism of molecular aluminium inhibition on Ca2+- ATPases and its effect on the intracellular homeostasis of calcium De Sautu, M., Saffioti, N., Ferreira-Gomes, M., Rinaldi D. Rossi RC Rossi, JPFC and Mangialavori, IC. IQUIFIB- Dr. Paladini -Departamento de Química Biológica, Universidad de Buenos AiresAluminum (Al3+) is a chemically highly reactive metal that is widely distributed in the environment. Al3+ is involved with the pathophysiology of neurodegenerative disorders, such as Parkinsonism dementia and Alzheimer?s disease. Several mechanisms explain its neurotoxicity, for example, damage to the glycolytic metabolism, lipid peroxidation leading increased free radicals, protein modifications and changes in the cellular calcium homeostasis. The aim of this work was to study the effect of Al3+ on intracellular calcium concentration ([Ca2+])i and moreover the molecular inhibitory mechanism of Al3+ on Ca2+-ATPases like the plasma membrane (PMCA) and the sarcoplasmic reticulum (SERCA). For these purposes, we purified these pump and, on the one hand we characterized the effect of Al3+ on enzymatic activity and parcial reactions and, on the other, we study its effect on trasmenbrane and citoplasmàtic domains by use of proteases accesibility, fluorescent probes and hidrophobic phtolabeling. In addiction, we study the calcium eflux with fluorecent probes and the location of Al3+ in HEK293T cells using fluorescence microscopy.Our results show that: (1) Al3+ inhibits calcium efflux mediated by PMCA in HEK293 cells, suggesting that aluminum inhibits PMCA in vivo; (2) Studies using fluorescence microscopy suggest that Al3+ could be located in the closeness of PMCA; (3) Al3+ inhibits Ca2+-ATPase activity of PMCA and SERCA with similar apparent affinity; (4) Al3+ increases the phosphorylated intermediate (EP) of PMCA while it has not effect on SERCA suggesting that they are inhibited in different intermediaries of the cycle. (5) In PMCA, structural studies suggest that Al3+ fixed the pump in a intermediate conformation between E1P and E2PThis work shows a possible toxic effect of Al3+ on cells and proposes for the first time a different inhibitory mechanism of action for Al3+ that involves intermediates of the ATP hydrolysis by these two Ca2+-transport ATPases. Key Words: Calcium ATPases, Aluminium, inhibition