Consequences of Functional Expression of the Plasma Membrane Ca Pump Isoform 1a

doi:10.1074/jbc.271.10.5536

Consequences of Functional Expression of the Plasma Membrane Ca Pump Isoform 1a (*)

From the (1)Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201 and the
(2)Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 76235

Abstract

The plasma membrane Ca-ATPase pump (PMCA) is an integral component of the Ca 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. Ca uptake assays in a membrane preparation indicated a 3-4-fold increase in Ca pumping activity in the transfected cells, and the expressed PMCA1a showed typical dependence on Ca and calmodulin for stimulation of activity. Measurement of [Ca] and [Ca] showed that expression of PMCA1a had a profound effect on different aspects of the Ca signal. The peak increase in [Ca] evoked by ATP and/or thapsigargin was lower but the plateau phase was similar in the PMCA1a expressing cells. Accordingly, titration with ionomycin of Ca content of internal stores, measurement of Ca uptake into the thapsigargin- and oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes, Ca 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 (IP)-activated Ca channel and the rate of IP-mediated Ca release in permeable cells, without affecting the affinity of the channel for IP. On the other hand the rate of store depletion-dependent Ca and Mn influx (Ca entry) into PMCA1a expressing cells was increased by about 2.6-fold. These changes prevented estimating the rate of pump-mediated Ca efflux from changes in [Ca]. Measurement of [Ca] showed that the rate of Ca 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 Ca signaling systems to regulate the expression and activity of each component and maintain a nearly constant Ca signal.

Footnotes

↵^§ These authors contributed equally to this study.
↵* This work was supported by National Institutes of Health Grant HL 39481 and a grant-in-aid from the American Heart Association of Michigan (to T. H. K.) and by National Institutes of Health Grants DK 38938 and DK 46591 (to S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PMCA

plasma membrane Ca-ATPase

RAEC

rat aortic endothelial cell

SERCA

sarcoplasmic/endoplasmic reticulum Ca-ATPase

ER

endoplasmic reticulum

IP

inositol 1,4,5-trisphosphate

IPR

IP receptor

CRAC

Ca release-activated Ca influx

Tg

thapsigargin

[Ca]

free intracellular Ca concentration

[Ca]

free extracellular Ca concentration

SLO

streptolysin O toxin

CHO

Chinese hamster ovary.
↵²B.-F. Liu, X. Xu, R. Fridman, S. Muallem, and T. H. Kuo, unpublished data.
↵³B.-F. Liu, X. Xu, R. Fridman, S. Muallem, and T. H. Kuo, unpublished observation.
- Received October 5, 1995.
- Revision received December 27, 1995.