Department of Pharmacology and Toxicology, University of Lausanne, Lausanne 1005, Switzerland.
J Biol Chem. 2011 May 27;286(21):18562-72. doi: 10.1074/jbc.M110.184101. Epub 2011 Mar 30.
The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.
FXYD 蛋白家族的七个成员与 Na(+)-K(+)泵结合并调节其活性。我们研究了 FXYD 蛋白中的保守半胱氨酸是否易发生谷胱甘肽化，以及这种反应性是否会影响心肌细胞和非洲爪蟾卵母细胞中的 Na(+)-K(+)泵功能。通过免疫印迹从生物素-GSH 加载的细胞中的链霉亲和素沉淀或通过 GSH 抗体检测谷胱甘肽化。用重组 FXYD 蛋白孵育心肌细胞导致天然 FXYD1 的竞争性置换。用全细胞和双电极电压钳技术分别测量心肌细胞和非洲爪蟾卵母细胞的泵电流。心肌细胞中的天然 FXYD1 和卵母细胞中表达的 FXYD1 易发生谷胱甘肽化。突变鉴定出反应性的细胞质末端的特定半胱氨酸。其反应性取决于侧翼碱性氨基酸。在之前的研究中，我们报道了氧化信号诱导的 Na(+)-K(+)泵β(1)亚基谷胱甘肽化导致泵抑制。在本研究中，我们发现通过将心肌细胞暴露于外源性野生型 FXYD3 可以逆转β(1)亚基谷胱甘肽化和泵抑制。无半胱氨酸的 FXYD3 衍生物没有作用。在卵母细胞中表达的野生型和突变 FXYD 蛋白也得到了类似的结果。FXYD1(-/-)小鼠心肌中的β(1)亚基谷胱甘肽化增加。总之，Na(+)-K(+)泵调节依赖于两种特定鉴定的半胱氨酸的反应性，这两种半胱氨酸位于多聚体 Na(+)-K(+)泵复合物的不同组成部分上。通过促进β(1)亚基的去谷胱甘肽化，FXYD 蛋白逆转 Na(+)-K(+)泵的氧化抑制作用，并在其调节中发挥动态作用。
