Wan Tina C, Dean William L
Department of Pharmacy and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Platelets. 2003 Aug;14(5):295-303. doi: 10.1080/0953710031000154713.
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.
质膜钙ATP酶（PMCA）是一种能量驱动的膜转运蛋白，可将钙泵出细胞胞质溶胶。为实现正常的细胞功能，必须维持稳定的静息钙水平和高度调节的胞质钙通量。PMCA在钙稳态中的主要功能是在细胞处于静息状态时调节稳态钙浓度。我们研究了靶向PMCA亚型4b（PMCA4b）的反义RNA稳定产生对培养的人巨核母细胞（Meg-01）细胞的影响。通过Western免疫印迹和体外ATP酶测定评估，这些细胞中PMCA-4b的表达减少了约50%。还确定这些细胞中PMCA1b的内源性表达水平低至无法通过Western免疫印迹检测到。在PMCA4b表达降低的细胞中，由PMVA4b催化的钙流出速率被抑制了约60%。然而，当肌质（内质）网ATP酶（SERCA）无功能时，挤出速率没有差异。这些细胞中细胞内钙浓度的静息水平与野生型细胞也没有区别。这些结果表明，Meg-01细胞中PMCA表达的降低得到了补偿，以维持正常的细胞内钙水平。
