Decreased PMCA4b expression has no effect on calcium homeostasis in Meg-01 cells

Abstract

PMCA (plasma membrane calcium ATPase) is an energy-driven membrane transporter that pumps calcium out of the cell cytosol. Stable resting calcium and highly regulated cytosolic calcium fluxes must be maintained for proper cellular function. The primary function of PMCA in calcium homeostasis is to regulate the steady-state calcium concentration while cells are still at rest. We examined the effects of stable production of antisense RNAs targeted to the PMCA subtype 4b (PMCA4b) on cultured human megakaryoblastic (Meg-01) cells. The expression of PMCA-4b in these cells was diminished by approximately 50% as assessed by Western immunoblotting and in vitro ATPase assay. It was also determined that endogenous expression of PMCA1b in these cells was at a level such as that it can not be detected by Western immunoblotting. The rate of calcium efflux catalyzed by PMVA4b was inhibited in cells with decreased PMCA4b expression by approximately 60%. However, there was no difference in extrusion rate when sarco(endo)plasmic reticular ATPases (SERCA) were not functional. The resting levels of intracellular calcium concentrations in these cells were also not distinguishable from those of wild-type cells. These results suggest that a decrease in PMCA expression in Meg-01 cells is compensated to maintain normal intracellular calcium levels.