ISOLATION AND CHARACTERIZATION OF A STABLE CHINESE-HAMSTER OVARY CELL-LINE OVEREXPRESSING THE PLASMA-MEMBRANE CA2-ATPASE()

Stable Chinese hamster ovary (CHO) cell lines overexpressing the human plasma membrane Ca2+-ATPase (PMCA) were generated, and three independ ent cell clones were characterized in details. They ovel expressed hig h amounts of active PMCA pump (15-20 times over the amount of endogeno us PMCA) as indicated by experiments in which the formation of the pho sphoenzyme intermediate and the uptake of Ca2+ by microsomes were meas ured. Immunocytochemistry experiments coupled to the biotinylation of the pump in the intact cells indicated the correct delivery of the exp ressed pump to the plasma membrane. The expressed pump was purified by affinity chromatography on calmodulin sepharose. The PMCA of transfec ted CHO cells promoted an increase of Ca2+ into the medium, after indu ction of Ca2+ release from the internal stores by activation of a puri nergic receptor. An evident decrease of the activity of the endogenous sarcoplasmic reticulum Ca2+-ATPase pump was observed, probably relate d to the down-regulation of its expression. The cells overexpressing t he PMCA pump had delayed recovery after trypsinization and plating. Th eir doubling time was, however, the same as CHO cells.