CONSEQUENCES OF FUNCTIONAL EXPRESSION OF THE PLASMA-MEMBRANE CA2+ PUMP ISOFORM 1A

The plasma membrane Ca2+-ATPase pump (PMCA) is an integral component o f the Ca2+ signaling system which participates in signal transduction during agonist stimulated cell activation, To better understand the ph ysiological function of the pump, isoform 1a (PMCA1a) was over-express ed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter, The cell lines select ed after transfection with PMCA1a construct, expressed 3-4-fold increa sed pump protein which was mostly targeted to the plasma membrane as i ndicated by immunoperoxidase staining, Ca2+ uptake assays in a membran e preparation indicated a 3-4-fold increase in Ca2+ pumping activity i n the transfected cells, and the expressed PMCA1a showed typical depen dence on Ca2+ and calmodulin for stimulation of activity, Measure ment of [Ca2+](i) and [Ca2+](out) showed that expression of PMCA1a had a p rofound effect on different aspects of the Ca2+ signal, The peak incre ase in [Ca2+](i) evoked by ATP and/or thapsigargin was lower but the p lateau phase was similar in the PMCA1a expressing cells, Accordingly, titration with ionomycin of Ca2+ content of internal stores, measureme nt of Ca2+ uptake into the thapsigargin- and oxalate-sensitive pool (e ndoplasmic reticulum) of isolated microsomes, Ca2+ uptake into strepto lysin O-permeabilized cells, and analysis of SERCA mRNA and protein, s howed that expression and activity of the SERCA pump was down-regulate d in cells expressing PMCA1a pump, Expression of PMCA1a also down-regu lated expression of the inositol 1,4,5-trisphosphate (IP3)-activated C a2+ channel and the rate of IP3-mediated Ca2+ release in permeable cel ls, without affecting the affinity of the channel for IP3. On the othe r 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+ eff lux 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 o f 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 eac h component and maintain a nearly constant Ca2+ signal.