Research Centre, Centre hospitalier de l'Université de Montréal (CRCHUM) - Technopôle Angus, 2901 Rachel East, Montreal, QC H1W 4A4, Canada.
Can J Physiol Pharmacol. 2012 Jan;90(1):83-8. doi: 10.1139/y11-113. Epub 2011 Dec 21.
Na(+),K(+)-ATPase is a heterodimer consisting of catalytic α1-α4 and regulatory β1-β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na(+),K(+)-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K(+) ((86)Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na(+),K(+)-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of (86)Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of (86)Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na(+),K(+)-ATPase subunit mRNA and immunoreactive protein does not contribute to Na(+)/K(+) pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na(+),K(+)-ATPase on Na(+)/K(+) pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.
钠钾-ATP 酶是一种由催化亚基 α1-α4 和调节亚基 β1-β3 组成的异二聚体。最近，我们报道了用哇巴因抗性 α1R-钠钾-ATP 酶转染，可使仅表达哇巴因敏感的 α1S-同工型的肾上皮 C7-MDCK 细胞免受哇巴因的细胞毒性作用。为了探讨 α2 亚基在哇巴因的离子转运和细胞毒性作用中的作用，我们比较了哇巴因对稳定转染 α1R-和 α2R-钠钾-ATP 酶的 C7-MDCK 细胞中 K+((86)Rb)内流和哇巴因处理细胞存活的影响。转染细胞中的 α2RmRNA 比 α1RmRNA 丰富约 8 倍，而免疫反应性 α2R 蛋白含量比内源性 α1S 蛋白低 5 倍。浓度为 10µmol/L 的哇巴因可完全抑制对照和 α2R 转染细胞中 (86)Rb 的内流，而在 α1R 转染细胞中，(86)Rb 内流的最大抑制作用发生在 1000µmol/L 哇巴因。与对照和 α2R 转染细胞暴露于 3µmol/L 哇巴因时大量死亡形成对比，α1R 细胞在孵育 24 小时后，用 1000µmol/L 哇巴因处理后仍存活。因此，我们的结果表明，与 α1R 不同，α2R-Na(+),K(+)-ATP 酶亚基 mRNA 和免疫反应性蛋白的存在并不有助于钠钾泵的活性，也不能使 C7-MDCK 细胞免受哇巴因的细胞毒性作用。我们的结果还表明，转染的 α2-Na(+),K(+)-ATP 酶对钠钾泵活性和细胞存活没有影响，这可能归因于其翻译效率低和（或）递送至肾上皮细胞的质膜。
