Is the Plasma Membrane Calcium Pump Regulated by the Cytoskeleton?

LAURA VANAGAS; MANGIALAVORI IRENE; CARIDE, AJ; ROSSI, R.C.; ROSSI, J.P.F.C.

(8-10 de noviembre, Rosario, Santa Fe)

Congreso; Reunión Annual de la Sociedad Argentina de Biofísica; 2006

SOCIEDAD ARGENTINA DE BIOFISICA

Transporters, receptors and channels -          Is the Plasma Membrane Calcium Pump regulated by the cytoskeleton? Vanagas, Laura, Mangialavori Irene Cecilia, Caride, Ariel, Rossi, Rolando and Rossi Juan Pablo IQUIFIB. Departamento de Química Biológica. Universidad de Buenos Aires. irenem@qb.ffyb.uba.ar     Plasma membrane calcium pump (PMCA) is an integral membrane protein that actively transports Ca2+ towards the extra cellular milieu, maintaining low intracellular Ca2+ concentrations which are necessary for efficient signaling. PMCA is activated with acidic phospholipids and regulated by protein kinases, but the main regulatory mechanism is mediated by the binding of calmodulin (CaM) to a domain located near the C-terminus of PMCA, which increases the maximum velocity and the apparent affinity for calcium of the pump. We have previously shown that specific activity of Ca2+-ATPase of erythrocyte membranes, measured at concentrations below 50 mg/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 both from erythrocyte membranes and 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. Present results show that the phenomenon can be described as an activation-inhibition behavior as a function of protein concentration which is dependent on the degree of PMCA expression. The dilution effect is present in different PMCA isoforms and in the truncated variant CT120 which lacks the segment of 15 kDa pertaining to the calmodulin binding site. A solubilized highly-purified micellar PMCA preparation shows that specific activity is constant at all the protein concentrations tested, provided that enough concentration of phosphatidylcholine was added. This fact indicates that the activation-inhibition behavior does not occur in the absence of other proteins associated with the native membrane. Taken together, these results suggest that, regardless of the particular isoform, interaction of PMCA with other proteins in the membrane is needed for the “dilution” effect. Furthermore, it is known that Cytochalasin D could promote or inhibit the polymerization of actin depending on the concentration of Mg2+ and K+ ions present in the media. Our results show that 1 mg/ml Cytochalasin D on erythrocyte membranes inhibits the activation effect at low concentration of protein in the presence of 120 mM K+. Conversely, in the presence of 0.5 mM Mg2+, Cytochalasin D activates the enzyme at high protein concentration. Altogether, these results strongly suggest that the activation-inhibition behavior is a consequence of interaction of cytoskeletal proteins with PMCA. With grants of ANPCYT, CONICET, NIH (TW06837) and UBACYT.