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. Measurement 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.
Liu
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BF
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Xu
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X
Fridman
R
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Muallem
S
S
Kuo
T H
TH
D000818:Animals; D001011:Aorta; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D002999:Clone Cells; D003587:Cytomegalovirus / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D006706:Homeostasis; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D015398:Signal Transduction; D014162:Transfection
D007527:Isoenzymes; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D002118:Calcium