Toustrup-Jensen Mads S, Holm Rikke, Einholm Anja Pernille, Schack Vivien Rodacker, Morth J Preben, Nissen Poul, Andersen Jens Peter, Vilsen Bente
Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, DK-8000 Aarhus C, Denmark.
J Biol Chem. 2009 Jul 10;284(28):18715-25. doi: 10.1074/jbc.M109.015099. Epub 2009 May 5.
The Na(+),K(+)-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na(+) sites, as well as E(1)-E(2) conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr(1017) and Tyr(1018), as well as putative interaction partners, Arg(935) in the loop between transmembrane segments M8 and M9 and Lys(768) in transmembrane segment M5, as crucial to Na(+) activation of phosphorylation of E(1), a partial reaction reflecting Na(+) interaction on the cytoplasmic side of the membrane. Tyr(1017), Tyr(1018), and Arg(935) are furthermore indispensable to Na(+) interaction on the extracellular side of the membrane, as revealed by inability of high Na(+) concentrations to drive the transition from E(1)P to E(2)P backwards toward E(1)P and inhibit Na(+)-ATPase activity in mutants. Lys(768) is not important for Na(+) binding from the external side of the membrane but is involved in stabilization of the E(2) form. These data demonstrate that the C terminus controls Na(+) affinity on both sides of the membrane and suggest that Arg(935) constitutes an important link between the C terminus and the third Na(+) site, involving an arginine-pi stacking interaction between Arg(935) and the C-terminal tyrosines. Lys(768) may interact preferentially with the C terminus in E(1) and E(1)P forms and with the loop between transmembrane segments M6 and M7 in E(2) and E(2)P forms.
钠钾ATP酶的C末端位于跨膜片段之间的独特位置，似乎参与了一个相互作用网络。我们研究了该区域氨基酸替代以及不同长度C末端缺失的功能后果。已应用分别揭示突变对面向内部和外部的钠位点以及E1-E2构象变化影响的检测方法。结果表明，两个末端酪氨酸Tyr(1017)和Tyr(1018)，以及假定的相互作用伙伴，跨膜片段M8和M9之间环中的Arg(935)和跨膜片段M5中的Lys(768)，对于E1磷酸化的钠激活至关重要，E1磷酸化是反映膜细胞质侧钠相互作用的部分反应。此外，Tyr(1017)、Tyr(1018)和Arg(935)对于膜细胞外侧的钠相互作用不可或缺，这是通过高钠浓度无法驱动从E1P向E2P反向转变为E1P并抑制突变体中的钠钾ATP酶活性所揭示的。Lys(768)对于从膜外侧结合钠并不重要，但参与E2形式的稳定。这些数据表明C末端控制膜两侧的钠亲和力，并表明Arg(935)构成C末端与第三个钠位点之间的重要联系，涉及Arg(935)与C末端酪氨酸之间的精氨酸-π堆积相互作用。Lys(768)可能在E1和E1P形式中优先与C末端相互作用，而在E2和E2P形式中与跨膜片段M6和M7之间的环相互作用。
