CONICET | Buscador de Institutos y Recursos Humanos

We have previously shown that purified actin can directly bind to hPMCA4b and exert a dual modulation on its Ca2+-ATPase activity: on one side F-actin inhibits PMCA [1] while short actin oligomers may be responsible for PMCA activation [2]. These studies required to be performed using purified proteins given the nature of the biophysical and biochemical approaches used. In order to assess whether this functional interaction may be of physiological relevance, we decided to characterize this phenomenon in the context of a living cell by monitoring in real-time the changes in the cytosolic calcium levels ([Ca2+]CYT). We tested the influence of drugs that change the actin and microtubules polymerization state on the activity and membrane expression of the PMCA transiently expressed in human embryonic kidney (HEK293) cells. We found that disrupting the actin cytoskeleton with cytochalasin D significantly increased PMCA-mediated Ca2+ extrusion (~50-100%) at the time that pre-treatment with the F-actin stabilizing agent jasplakinolide (JAS, 1μM) caused its full inhibition. These results are in full agreement with our previous in vitro observations. When the microtubule network was disrupted by pretreatment of the cells with colchicine, we observed a significantly decrease in PMCA activity (~40-60% inhibition) in agreement with the previously reported role of acetylated tubulin on the calcium pump [3]. In any of these cases we observe a difference in the level of expression of the pump at the cell surface. Taken together, these data demonstrate that our and other?s previous observations on the in vitro effect of the actin and tubulin cytoskeleton on PMCA activity is also evident in a living cell model. References:[1] Vanagas et al. Cell Biochem Biophys 66:187-198, 2013.[2] Dalghi et al. J Biol Chem 288:23380-23393, 2013.[3] Monesterolo et al. FEBS J. 275:3567-3579, 2008