Is the Plasma Membrane Calcium Pump regulated by the cytoskeleton?

L. VANAGAS; I. C. MANGIALAVORI; A. J. CARIDE; R. C. ROSSI; J. P. F. C. ROSSI

Rosario, Argentina

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

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 principal regulatory mechanism of enzymatic activity is mediated by the binding of calmodulin (CaM) to a domain located near the C-terminus of PMCA, which increase 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 decrease 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. These results indicate that the effect is dependent of the ratio PMCA/cytoskeletal proteins. Further, a solubilized highly-purified micellar PMCA preparation show that specific activity is constant alongside the protein concentration tested, provided that enough concentration of phosphatidylcholine was added. This fact indicates that the activation-inhibition behavior does not occur in the absence of cytoskeletal proteins. On the other hand, 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. Taken together, 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 and UBACYT.