FIGURE 6.
A, Western blotting and densitometric analysis of the Lys mutants of the PMCA2 z/b isoform overexpressed in CHO cells. 20 μg of crude membrane proteins from transfected CHO cells, prepared by a freeze and thaw method, were separated by SDS-PAGE as described under “Experimental Procedures” and stained with polyclonal antibody 2N. The control lane corresponds to nontransfected cells (CHO). The other lanes correspond to cells transfected with the WT or mutant variants of the PMCA2 pump. The panel also shows the immunocytochemistry analysis of the transfected CHO cells. The immunostaining was carried out with the 2N antibody and revealed with the secondary antibody Alexa Fluor 594. B, monitoring of cytosolic [Ca2+] in CHO cells transfected with cytAEQ and co-transfected with cytAEQ and the PMCA2zb_K336A, PMCA2zb_K338A, PMCA2zb_K344A, PMCA2zb_K347A, or PMCA2zb_4M, alternatively. The histograms show the means ± S.D. of [Ca2+]c peaks and of the half-time decays from the peaks. The traces are representative of at least 12 independent experiments. *, p < 0.01 calculated with respect to PMCA2zb_wt (CHO cells transfected with wt PMCA2 z/b pump).