Manoharan Palanikumar, Radzyukevich Tatiana L, Hakim Javadi Hesamedin, Stiner Cory A, Landero Figueroa Julio A, Lingrel Jerry B, Heiny Judith A
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio;
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio; and.
Am J Physiol Cell Physiol. 2015 Dec 15;309(12):C813-22. doi: 10.1152/ajpcell.00205.2015. Epub 2015 Oct 14.
The Na(+)-K(+)-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na(+)-K(+)-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb(+) transport by the α2-Na(+)-K(+)-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na(+) affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.
骨骼肌中的钠钾ATP酶α2亚型在肌肉运动时会迅速被激活，并且在抗疲劳过程中发挥关键作用。目前尚不完全清楚刺激α2活性的急性机制。本研究旨在探讨钠钾ATP酶的调节亚基磷膜蛋白（PLM/FXYD1）的磷酸化是否在工作肌肉中对α2的急性刺激中发挥作用。与骨骼肌中缺乏α2的小鼠运动能力大幅降低不同，缺乏PLM的小鼠（PLM基因敲除小鼠）α2亚基含量正常，运动能力也正常。在野生型（WT）或PLM基因敲除小鼠中，体内神经诱发的收缩均未引起总PLM或Ser63或Ser68位点磷酸化的PLM发生变化。PLM基因敲除小鼠的离体肌肉与野生型一样能够维持收缩并抵抗疲劳。在收缩的WT和PLM基因敲除肌肉中，α2-钠钾ATP酶对铷离子的转运受到同等程度的刺激。从WT而非PLM基因敲除的骨骼肌制备的肌膜磷酸化以PLM依赖的方式刺激α1和α2的活性。这种刺激是通过增加钠离子亲和力实现的，Vmax没有显著变化，并且对α1的作用比对α2更有效。这些结果表明，PLM的磷酸化能够刺激骨骼肌中两种同工酶的活性；然而，仅收缩活动不足以诱导PLM磷酸化。重要的是，对α2的急性刺激足以支持运动并对抗疲劳，并不需要PLM或其磷酸化。
