Plasma Membrane Calcium Pump regulation by the cytoskeleton

VANAGAS LAURA; MANGIALAVORI IRENE; ROSSI ROLANDO; ROSSI JUAN PABLO

Montevideo, Uruguay

Congreso; VI International Conferences of Biological Physics (ICBP); 2007

Plasma membrane calcium pump (PMCA) is an integral membrane protein that actively transports Ca2+ towards the extra cellular milieu. PMCA is activated with acidic phospholipids and regulated by protein kinases, but the main regulatory mechanism is mediated by the binding of calmodulin (CaM). We have previously shown that specific activity of Ca2+-ATPase of erythrocyte membranes, measured at concentrations below 50 g/ml of protein, increases steeply up to 3-5 times when the membrane protein concentration decreases from 40 µg/ml to 1 µg/ml. The activation by dilution was also observed during ATP-dependent Ca2+ uptake measurements into inside-out vesicles from Sf9 cells which over-express the enzyme, confirming that it is a property of PMCA. Dilution of the protein did not modify the activation by ATP, Ca2+ or Ca2+-calmodulin, and is present in different PMCA isoforms; however it cannot be observed when using a solubilized highly-purified micellar PMCA preparation, indicating that interaction of PMCA with other proteins in the membrane is needed for the dilution effect. Furthermore, when using 1 mg/ml Cytochalasin D on erythrocyte membranes under conditions that promote the polymerization of actin, the activation effect at low concentration of protein does not take place. (Vanagas L. et al, BBActa 1768 (2007) 1641-1649) Results in this work show that when using Cytochalasin D on erythrocyte membranes in a medium with a low ionic strength and devoid of Mg2+ and K+ salts, a condition that induce the depolymerization of actin, the dilution effect is further increased. On the other hand, when we measured Ca2+-ATPase activity of a purified PMCA preparation by continuously monitoring the release of inorganic phosphate, the addition of monomeric actin to the reaction medium activated the ATP hydrolysis by the enzyme. As far as the reaction of actin polymerization proceeds, Ca2+-ATPase activity progressively decreased. These results are consistent with the hypothesis that monomeric actin would be responsible for the activation observed at low concentrations of membranes – proposing that in diluted samples, actin exists in short filaments or as monomers - and oligomeric actin would act as an inhibitor. With grants of ANPCYT, CONICET, NIH (TW06837) and UBACYT.