Consequences of functional expression of the plasma membrane Ca2+ pump isoform 1a

The plasma membrane Ca2+-ATPase pump (PMCA) is an integral component of the Ca2+ signaling system which participates in signal transduction during agonist stimulated cell activation, To better understand the physiological function of the pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter, The cell lines selected after transfection with PMCA1a construct, expressed 3-4-fold increased pump protein which was mostly targeted to the plasma membrane as indicated by immunoperoxidase staining, Ca2+ uptake assays in a membrane preparation indicated a 3-4-fold increase in Ca2+ pumping activity in the transfected cells, and the expressed PMCA1a showed typical dependence on Ca2+ and calmodulin for stimulation of activity, Measure ment of [Ca2+](i) and [Ca2+](out) showed that expression of PMCA1a had a profound effect on different aspects of the Ca2+ signal, The peak increase in [Ca2+](i) evoked by ATP and/or thapsigargin was lower but the plateau phase was similar in the PMCA1a expressing cells, Accordingly, titration with ionomycin of Ca2+ content of internal stores, measurement of Ca2+ uptake into the thapsigargin- and oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes, Ca2+ uptake into streptolysin O-permeabilized cells, and analysis of SERCA mRNA and protein, showed that expression and activity of the SERCA pump was down-regulated in cells expressing PMCA1a pump, Expression of PMCA1a also down-regulated expression of the inositol 1,4,5-trisphosphate (IP3)-activated Ca2+ channel and the rate of IP3-mediated Ca2+ release in permeable cells, without affecting the affinity of the channel for IP3. On the other hand the rate of store depletion-dependent Ca2+ and Mn2+ influx (Ca2+ entry) into PMCA1a expressing cells was increased by about 2.6-fold, These changes prevented estimating the rate of pump mediated Ca2+ efflux from changes in [Ca2+](i). Measurement of [Ca2+](out) showed that the rate of Ca2+ efflux in cells expressing PMCA1a was about 1.45-fold higher than Neo controls, despite the 4-fold increase in the amount of functional pump protein, The overall study points to the flexibility, interdependence, and adaptability of the different components of the Ca2+ signaling systems to regulate the expression and activity of each component and maintain a nearly constant Ca2+ signal.