Ching Biyun, Woo Jia M, Hiong Kum C, Boo Mel V, Choo Celine Y L, Wong Wai P, Chew Shit F, Ip Yuen K
Department of Biological Sciences, National University of Singapore, Kent Ridge, Singapore, 117543, Republic of Singapore.
Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616, Republic of Singapore.
PLoS One. 2015 Mar 20;10(3):e0118352. doi: 10.1371/journal.pone.0118352. eCollection 2015.
This study aimed to obtain the coding cDNA sequences of Na+/K+-ATPase α (nkaα) isoforms from, and to quantify their mRNA expression in, the skeletal muscle (SM), the main electric organ (EO), the Hunter's EO and the Sach's EO of the electric eel, Electrophorus electricus. Four nkaα isoforms (nkaα1c1, nkaα1c2, nkaα2 and nkaα3) were obtained from the SM and the EOs of E. electricus. Based on mRNA expression levels, the major nkaα expressed in the SM and the three EOs of juvenile and adult E. electricus were nkaα1c1 and nkaα2, respectively. Molecular characterization of the deduced Nkaα1c1 and Nkaα2 sequences indicates that they probably have different affinities to Na+ and K+. Western blotting demonstrated that the protein abundance of Nkaα was barely detectable in the SM, but strongly detected in the main and Hunter's EOs and weakly in the Sach's EO of juvenile and adult E. electricus. These results corroborate the fact that the main EO and Hunter's EO have high densities of Na+ channels and produce high voltage discharges while the Sach's EO produces low voltage discharges. More importantly, there were significant differences in kinetic properties of Nka among the three EOs of juvenile E. electricus. The highest and lowest Vmax of Nka were detected in the main EO and the Sach's EO, respectively, with the Hunter's EO having a Vmax value intermediate between the two, indicating that the metabolic costs of EO discharge could be the highest in the main EO. Furthermore, the Nka from the main EO had the lowest Km (or highest affinity) for Na+ and K+ among the three EOs, suggesting that the Nka of the main EO was more effective than those of the other two EOs in maintaining intracellular Na+ and K+ homeostasis and in clearing extracellular K+ after EO discharge.
本研究旨在获取电鳗(Electrophorus electricus)骨骼肌(SM)、主发电器官(EO)、亨特氏发电器官和萨克氏发电器官中Na⁺/K⁺-ATP酶α(nkaα)亚型的编码cDNA序列,并对其mRNA表达进行定量分析。从电鳗的SM和EO中获得了四种nkaα亚型(nkaα1c1、nkaα1c2、nkaα2和nkaα3)。根据mRNA表达水平,幼年和成年电鳗的SM以及三个EO中主要表达的nkaα分别为nkaα1c1和nkaα2。推导的Nkaα1c1和Nkaα2序列的分子特征表明,它们对Na⁺和K⁺可能具有不同的亲和力。蛋白质免疫印迹法表明,在幼年和成年电鳗的SM中几乎检测不到Nkaα的蛋白质丰度,但在主发电器官和亨特氏发电器官中检测到其丰度较高,而在萨克氏发电器官中检测到的丰度较低。这些结果证实了以下事实:主发电器官和亨特氏发电器官具有高密度的Na⁺通道并产生高电压放电,而萨克氏发电器官产生低电压放电。更重要的是,幼年电鳗的三个EO中Nka的动力学特性存在显著差异。Nka的最高和最低Vmax分别在主发电器官和萨克氏发电器官中检测到,亨特氏发电器官的Vmax值介于两者之间,这表明主发电器官中EO放电的代谢成本可能最高。此外,在三个EO中,主发电器官的Nka对Na⁺和K⁺的Km最低(或亲和力最高),这表明主发电器官的Nka在维持细胞内Na⁺和K⁺稳态以及在EO放电后清除细胞外K⁺方面比其他两个EO更有效。