Regulation of plasma membrane calcium ATPase (PMCA) by actin cytoskeleton.

VIGIL, M.; PICCO, ME; RINALDI, D.E; ROSSI, R.C; REY, O; ROSSI, J.P.; FERREIRA GOMES, M

Virtual

Congreso; XLIX REUNION ANUAL SAB 2021; 2021

Sociedad Argentina de Biofisica

Plasma membrane calcium ATPase (PMCA) is a calmodulin modulated ATPase responsible for maintaining low levels of intracellular Ca2+ in most eukaryotic cells. Our group has already shown that purified actin can induce a double modulation of the activity of the isoform hPMCAb: F-actin inhibits PMCA while short activated oligomers can contribute to the activation of the PMCA. These studies should be performed with purified proteins depending on the nature of the biophysical and biochemical approaches used. On the other hand, in human HEK293 cells overexpressing PMCA2w/b isoforms, after actin depolymerization by cytochalasin D (CytD) treatment, significantly increased PMCA2-mediated Ca2+ extrusion and when F-actin is stabilized with the help of jasplakinolide, the activity of PMCA2w/b was completely abolished.In order to assess whether the functional interaction between the hPMCA isoform and the actin cytoskeleton may be of physiological relevance, we decided to further characterize it in the context of a living cell by monitoring in realtime the changes in the actin polymerization and cytosolic Ca2+concentration ([Ca2+ ]cyt). For this, hPMCA isoform was transiently expressed in HEK293T cells. The dynamics of [Ca2+]cyt was performed using the fluorescent probe Fluo and studying the alterations in [Ca2+]cyt generated by Ca2+ release from the endoplasmic reticulum (ER), and by extracellular Ca2+ entry through store-operated Ca2+ channels. The dynamics of actin polymerization were performed transiently expressing LifeActRuby. Results showed that the alteration of actin polymerization by the CytD treatment significantly increased the hPMCA activity (202%). On the other hand, in the absence of CytD, the dynamics of actin polymerization was unchanged after stimulation by TG, whereas after stimulation by Ca2+, a reorganization of actin was observed. This reorganization occurred at the same time as hPMCA increased its activity, suggesting that hPMCA is activated by depolymerization of actin in the cell.This work was supported by ANPCYT, CONICET, UBA.